阅读说明：本技术 一种高蔗果三糖含量的低聚果糖的制备方法 (Preparation method of fructo-oligosaccharide with high kestose content ) 是由 李克文 贾慧慧 孔刘娟 栾庆民 张莉 熊小兰 高蕾蕾 张倩 韩薇 徐虹 尹郑 于 2019-11-01 设计创作，主要内容包括：本发明涉及一种高蔗果三糖含量的低聚果糖的制备方法,所述制备方法包括步骤：(1)将离子液体和果糖基转移酶混合；(2)配制质量分数55～56％的蔗糖溶液；(3)向蔗糖溶液加入离子液体和果糖基转移酶混合液后,再经脱色、过滤、膜过滤、色谱分离、浓缩,制备得到高蔗果三糖含量的液体低聚果糖。本发明制备低聚果糖的过程中,可以离子液体为果糖基转移酶催化反应介质,并辅以补加果糖促进蔗果三糖生成,以达到提高蔗糖转化法制备低聚果糖中蔗果三糖含量和总低聚果糖含量的目的。(The invention relates to a preparation method of fructo-oligosaccharide with high kestose content, which comprises the following steps: (1) mixing the ionic liquid with fructosyltransferase; (2) preparing a sucrose solution with the mass fraction of 55-56%; (3) adding the mixed solution of ionic liquid and fructosyltransferase into the sucrose solution, and then carrying out decolorization, filtration, membrane filtration, chromatographic separation and concentration to prepare the liquid fructo-oligosaccharide with high content of kestose. In the process of preparing the fructo-oligosaccharide, the ionic liquid can be used as a fructosyltransferase catalytic reaction medium, and fructose is supplemented to promote the generation of kestose so as to achieve the purpose of improving the content of kestose and the total content of fructo-oligosaccharide in the fructo-oligosaccharide prepared by a sucrose conversion method.)

1. A preparation method of fructo-oligosaccharide with high kestose content comprises the following steps:

(1) mixing the ionic liquid and the fructosyltransferase according to the mass ratio of (0.7-1) to 2, and standing and balancing;

(2) preparing a sucrose solution with mass fraction of 55-56% in a reaction container, and then adjusting the pH value of the sucrose solution to 5.5-5.8;

(3) placing the reaction container in a water bath for heating at a constant temperature of 45-52 ℃, adding a balanced ionic liquid and fructosyltransferase mixed solution after the temperature of the sucrose solution is balanced, continuously stirring for saccharification reaction for 11-14 h, adding a fructose solution for continuous saccharification reaction for 30-35 h, deactivating enzyme and stopping the saccharification reaction; and (3) decoloring, filtering, membrane filtering, chromatographic separation and concentrating to obtain the liquid fructo-oligosaccharide with high kestose content.

2. The method according to claim 1, wherein in the step (1), the ionic liquid is a 1-butyl-3-methylimidazolium hexafluorophosphate solution.

3. The method according to claim 1, wherein the fructosyltransferase in step (1) is an enzyme dedicated for producing fructooligosaccharides from bowling Bio-Ltd, having an enzyme activity of 3000U, an optimum reaction temperature of 52 to 55 ℃ and a reaction pH of 5.5 to 6.0.

4. The preparation method according to claim 1, wherein in the step (1), the static equilibrium condition is 6-8 h at 4 ℃.

5. The method according to claim 1, wherein in the step (2), the pH adjusting agent for adjusting the pH of the sucrose solution is a 0.1M disodium hydrogenphosphate-citric acid buffer.

6. The method according to claim 1, wherein the fructosyltransferase is added in an amount of 3 to 6U/g on a dry basis of sucrose in step (3).

7. The preparation method according to claim 1, wherein in the step (3), the mass fraction of the added fructose solution is 10%, and the addition amount of fructose on a dry basis is 1-2% of the mass of sucrose on a dry basis.

8. The preparation method according to claim 1, wherein in the step (3), the membrane filtration is nanofiltration membrane filtration with the thickness of 200-300 nm, and the pressure is 30-35 bar.

9. The process according to claim 1, wherein in the step (3), the saccharification conversion rate of fructooligosaccharide is 59-65%, kestose accounts for 87-93% of fructooligosaccharide, kestose accounts for 78-83% of total sugar after FOS90 liquid chromatography, and FOS yield of chromatographic separation is more than 97%.

The technical field is as follows:

the invention relates to a preparation method of fructo-oligosaccharide with high kestose content, belonging to the technical synthesis field of fructo-oligosaccharide.

Background art:

fructooligosaccharides (FOS) are a mixture of kestose (GF2), nystose (GF3), nystose (GF4), and the like, which are composed of sucrose and 1 to 3 fructosyl groups bonded to the fructosyl group in the sucrose via a beta-2-1 glycosidic bond. The fructo-oligosaccharide has good health promotion effect, is nontoxic, is suitable for diabetes patients, and has effects of regulating lipid metabolism, promoting calcium absorption, and reducing serum cholesterol. At present, two methods for industrially producing fructo-oligosaccharide are mainly used, namely a sucrose conversion method, wherein the product fructo-oligosaccharide is CFn type; secondly, preparing by utilizing inulase to directionally hydrolyze inulin, wherein the product fructo-oligosaccharide is a mixed type of CFn and Fn. The fructooligosaccharide contains relatively low molecular weight kestose component, which can be metabolized and digested by beneficial bacteria in human large intestine due to low polymerization degree, has good water retention property, and can be used as proliferation factor of beneficial bacteria such as Bacillus bifidus to improve disease resistance and immunity of human body.

Chinese patent document CN104928332A discloses a preparation method of high-purity fructo-oligosaccharide, which comprises the following steps: (1) hydrolyzing chicory by inulase to obtain a primary fructo-oligosaccharide solution; (2) carrying out decoloration, debitterizing and desalting treatment on the primary fructo-oligosaccharide solution to obtain a secondary fructo-oligosaccharide solution; (3) adding beta-D-fructosyltransferase into the secondary fructo-oligosaccharide solution, and reacting for 1-8 h at 20-35 ℃ to obtain a tertiary fructo-oligosaccharide solution; (4) and (4) treating the tertiary fructo-oligosaccharide mixed solution obtained in the step (3) by using a nanofiltration membrane, and collecting filtrate to obtain a fructo-oligosaccharide solution with the purity of more than 99%. Although the purity of the prepared fructo-oligosaccharide solution is high, the content of kestose in the fructo-oligosaccharide is low.

In conclusion, the prior art lacks a method for preparing fructo-oligosaccharide, which can improve the content of kestose in the fructo-oligosaccharide and has high purity and high yield.

The invention content is as follows:

aiming at the defects of the prior art, the invention provides a preparation method of fructo-oligosaccharide with high kestose content. The ionic liquid is used as an enzyme catalysis reaction medium to be mixed and saccharified with fructosyltransferase, and fructose is supplemented in the process to promote the generation of kestose, so that fructooligosaccharide with high kestose content is prepared, and the integral saccharification conversion rate is improved.

The technical scheme of the invention is as follows:

a preparation method of fructo-oligosaccharide with high kestose content comprises the following steps:

(1) mixing the ionic liquid and the fructosyltransferase according to the mass ratio of (0.7-1) to 2, and standing and balancing;

(2) preparing a sucrose solution with mass fraction of 55-56% in a reaction container, and then adjusting the pH value of the sucrose solution to 5.5-5.8;

(3) placing the reaction container in a water bath for heating at a constant temperature of 45-52 ℃, adding a balanced ionic liquid and fructosyltransferase mixed solution after the temperature of the sucrose solution is balanced, continuously stirring for saccharification reaction for 11-14 h, adding a fructose solution for continuous saccharification reaction for 30-35 h, deactivating enzyme and stopping the saccharification reaction; and (3) decoloring, filtering, membrane filtering, chromatographic separation and concentrating to obtain the liquid fructo-oligosaccharide with high kestose content.

Preferably, in step (1), the ionic liquid is 1-butyl-3-methylimidazole hexafluorophosphate solution.

According to the invention, in the step (1), the fructosyltransferase is an enzyme special for producing fructo-oligosaccharide of bowling biological limited, the enzyme activity is 3000U, the optimal reaction temperature is 52-55 ℃, and the reaction pH is 5.5-6.0.

Preferably, in the step (1), the standing and balancing conditions are 6-8 h at 4 ℃.

Preferably, in step (2), the pH regulator for regulating the pH of the sucrose solution is 0.1M disodium hydrogen phosphate-citric acid buffer.

Preferably, in step (3), the fructosyltransferase is added in an amount of 3 to 6U/g sucrose dry basis.

According to the invention, in the step (3), the mass fraction of the added fructose solution is 10%, and the addition amount of the fructose on a dry basis is 1-2% of the mass of the sucrose on a dry basis.

According to the invention, in the step (3), the membrane filtration is nanofiltration membrane filtration of 200-300 nm, and the pressure is 30-35 bar.

According to the invention, in the step (3), the saccharification conversion rate of the fructo-oligosaccharide is 59-65%, the kestose accounts for 87-93% of the fructo-oligosaccharide, the kestose accounts for 78-83% of the total sugar after FOS90 liquid chromatography, and the FOS yield of chromatographic separation is more than 97%.

The invention has the following beneficial effects:

(1) the added ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate is low-temperature molten salt with a unique structure, is used as a catalytic reaction medium of the fructosyltransferase to be mixed with the enzyme, and can enable the fructosyltransferase to be suspended in the ionic liquid, so that the thermal stability, the operation controllability, the stereoselectivity and the enantioselectivity of the fructosyltransferase are improved, the inactivation rate of the enzyme is greatly reduced, and the reaction in a saccharification process is facilitated.

(2) In the invention, because glucose can inhibit the conversion of the fructo-oligosaccharide in the reaction of converting sucrose into the fructo-oligosaccharide, the fructose solution is supplemented in the saccharification process under the equilibrium condition of the saccharification reaction formula, so that the conversion of the fructo-oligosaccharide is stimulated, and the generation of kestose is promoted. Meanwhile, the membrane filtration step is added, so that most of ionic liquid can be removed, and then high-temperature vacuum concentration is assisted, so that the ionic liquid can be basically and completely removed.

(3) In the process of preparing the fructo-oligosaccharide, the ionic liquid can be used as a fructosyltransferase catalytic reaction medium, and fructose is supplemented to promote the generation of kestose so as to achieve the purpose of improving the content of kestose and the total content of fructo-oligosaccharide in the fructo-oligosaccharide prepared by a sucrose conversion method. The conversion rate of fructo-oligosaccharide prepared by the method is 59-65%, kestose accounts for 87-93% of fructo-oligosaccharide, kestose accounts for 78-83% of total sugar after FOS90 liquid chromatographic separation, and the FOS yield of chromatographic separation is more than 97%. The fructo-oligosaccharide with high content of kestose prepared by the invention has higher water retention and digestion of human intestinal probiotics than the common fructo-oligosaccharide.

Drawings

FIG. 1 is a line graph showing the enzyme activity of fructosyl transferase in test example 2.

The specific implementation mode is as follows:

the technical solution of the present invention is further illustrated by the following specific examples, but the scope of the present invention is not limited thereto.