A method for preparing oligofructose

Abstract

The present invention provides a method for preparing oligofructose, and belongs to the technical field of oligofructose synthesis. The method of the present invention comprises the following steps: (1) preparing a reaction stock solution; (2) adding fructosyl transferase to the reaction stock solution and performing a constant temperature water bath reaction; during the reaction, adding sugar solution to continue the reaction to obtain a first process saccharification solution; (3) filtering the first process saccharification solution using a semipermeable membrane to obtain a retentate and a filtrate; (4) performing a constant temperature water bath reaction on the filtrate to obtain a second process saccharification solution, and then mixing the second process saccharification solution with the retentate to obtain a crude oligofructose solution; (5) preparing a fructose powder after decolorizing, desalting, chromatographic separation, evaporation concentration, and drying the crude oligofructose solution; the reaction time is shortened, the utilization rate of fructose is increased, the generation of saccharide in the system is promoted, the content of saccharide in the fructose is increased, and the water activity of the fructose is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of oligofructose synthesis, and in particular to a method for preparing oligofructose. Background Art

[0002] Fructooligosaccharides (FOS), also known as fructooligosaccharides, are a mixture of sucrose trisaccharide (GF2), sucrose tetrasaccharide (GF3) and sucrose pentasaccharide (GF4) formed by sucrose molecules combined with 1 to 3 fructose molecules by β-(1→2) glycosidic bonds, with a molecular formula of GF-Fn. Fructooligosaccharides have the physiological functions of both prebiotics and dietary fiber, and are suitable for use by diabetics. They can lower cholesterol, improve lipid metabolism, and proliferate beneficial bacteria in the body, and have good health effects. At present, the production of fructooligosaccharides in industry mainly uses sucrose as raw material, and is produced by enzymatic conversion using fructosyltransferase (FTase) or β-furanosidase (FFase). Studies have shown that sucrose trisaccharide in fructooligosaccharides is more effective in promoting the proliferation of probiotics than other fructooligosaccharides with different polymerization degrees. It has a low degree of polymerization and is more easily absorbed and utilized in the human intestine, but its low content and difficulty in separation and purification have always been the main obstacles to production in industry.

[0003] Chinese patent document CN104928332A discloses a method for preparing high-purity oligofructose, which comprises the following steps: (1) hydrolyzing chicory with inulinase to obtain a primary oligofructose solution; (2) decolorizing, debittering and desalting the primary oligofructose solution in step (1) to obtain a secondary oligofructose solution; (3) adding β-D-fructosyltransferase to the secondary oligofructose solution in step (2), reacting at 20-35°C for 1-8h to obtain a tertiary oligofructose solution; (4) treating the tertiary oligofructose mixed solution obtained in step (3) with a nanofiltration membrane, collecting the filtrate, and obtaining an oligofructose solution with a purity greater than 99%. Although the oligofructose in the invention has a high purity, the degree of polymerization is in the range of 4-6, the obtained sugarcane trisaccharide content is low, and the water activity is high, the product is easy to absorb moisture and is not conducive to preservation and extending the shelf life.

[0004] Chinese patent document CN110669808A discloses a method for preparing oligofructose with a high content of sucrose trisaccharide, which comprises the following steps: (1) mixing an ionic liquid and a fructosyltransferase in a mass ratio of (0.7-1):2 and allowing the mixture to stand for equilibrium; (2) preparing a sucrose solution with a mass fraction of 55-56% in a reaction vessel, and then adjusting the pH of the sucrose solution to 5.5-5.8; (3) placing the reaction vessel in a water bath at a constant temperature of 45-52°C, and after the temperature of the sucrose solution is balanced, adding the balanced ionic liquid and fructosyltransferase mixture, stirring the saccharification reaction for 11-14 hours, then adding the fructose solution to continue the saccharification reaction for 30-35 hours, and then inactivating the enzyme to terminate the saccharification reaction; and preparing liquid oligofructose with a high content of sucrose trisaccharide through decolorization, filtration, membrane filtration, chromatographic separation, and concentration, wherein the sucrose trisaccharide accounts for 87-93% of the oligofructose, and the yield of oligofructose separated by chromatography is above 97%. Although the sucrose content is high in the invention, the overall reaction time is relatively long, and the yield of oligofructose and the sucrose content can be further improved.

[0005] In summary, there is currently a lack of a technology that can further increase the yield of oligofructose and the content of sucrose trisaccharide while saccharifying and polymerizing in a shorter time and obtain oligofructose with low water activity. Summary of the invention

[0006] In view of this, the present invention aims to provide a method for preparing oligofructose; the present invention uses Fe 2+ Ions are used as heat-stable protective agents for fructosyltransferase. During the saccharification process, the temperature and time are controlled for segmented saccharification, and sucrose solution is added. During the saccharification process, a semipermeable membrane device is added to collect saccharides. While shortening the reaction time, the utilization rate of fructose is increased, the formation of saccharides in the system is promoted, the content of saccharides in oligofructose is increased, and the water activity of oligofructose is reduced.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing oligofructose, comprising the following steps:

[0009] (1) Add Fe to sucrose solution 2+ Then the pH is adjusted to obtain a reaction stock solution;

[0010] (2) adding fructosyltransferase to the reaction stock solution and performing a constant temperature water bath reaction; when the sucrose mass fraction in the system is 35-40%, adding sucrose solution until the sucrose mass fraction is 50-55%, and then continuing the reaction for 10-12 hours to obtain a first process saccharification solution;

[0011] (3) filtering the first process saccharification liquid through a semipermeable membrane with a molecular weight cutoff of 350-500 Da to obtain a retentate and a filtrate;

[0012] (4) subjecting the filtrate to a constant temperature water bath reaction to obtain a second-stage saccharification liquid, and then mixing the second-stage saccharification liquid with the retentate to obtain a crude fructo-oligosaccharide liquid;

[0013] (5) adding activated carbon to the crude fructo-oligosaccharide solution, decolorizing and filtering to obtain a first filtrate;

[0014] (6) Desalting the first filtrate using a strong acid cation resin D001 and a weak base anion resin D301P to obtain a second filtrate;

[0015] (7) subjecting the second filtrate to chromatographic separation, evaporation concentration, and drying to prepare oligofructose powder;

[0016] In step (2), the reaction temperature of the constant temperature water bath is 60-65°C, and the reaction time is 5-7h;

[0017] In step (4), the water bath reaction temperature is 40-45° C., and the reaction time is 4-6 h.

[0018] Preferably, the mass fraction of the sucrose solution in step (1) is 55-60%.

[0019] Preferably, in step (1), Fe 2+ The amount added is 45~55mM.

[0020] Preferably, in step (1), the pH is adjusted to 5.5-6.0.

[0021] Preferably, the amount of fructosyltransferase added in step (2) is 6-8 U / g.

[0022] Preferably, the amount of activated carbon added in step (5) is 1.0-2.0% of the dry weight of the crude fructo-oligosaccharide liquid.

[0023] Preferably, the decolorization temperature in step (5) is 55-60°C and the decolorization time is 15-20 minutes.

[0024] Preferably, in the desalination process of step (6), the flow temperature of the first filtrate is 35-40° C., the flow rate is 1-3 BV / h, and the discharge pH is 5.0-6.0.

[0025] Beneficial technical effects:

[0026] In the process of preparing oligofructose by the present invention, the original problems of long reaction time, low content of sucrose trisaccharide, low water activity of oligofructose, easy moisture absorption and the like are overcome. The content of sucrose trisaccharide in the prepared oligofructose is ≥95%, the conversion rate of oligofructose is 65-70%, the proportion of fructose residue is ≤2%, and the water activity of oligofructose is 0.83-0.86 (based on the concentration of 50%). The oligofructose prepared by the present invention contains a high content of sucrose trisaccharide, has a better intestinal probiotic proliferation effect, and has a low water activity, which reduces the moisture absorption and prolongs the shelf life of the product. DETAILED DESCRIPTION

[0027] The present invention provides a method for preparing oligofructose, comprising the following steps:

[0028] (1) Add Fe to sucrose solution 2+ Then the pH is adjusted to obtain a reaction stock solution;

[0029] (2) adding fructosyltransferase to the reaction stock solution and performing a constant temperature water bath reaction; when the sucrose mass fraction in the system is 35-40%, adding sucrose solution until the sucrose mass fraction is 50-55%, and then continuing the reaction for 10-12 hours to obtain a first process saccharification solution;

[0030] (3) filtering the first process saccharification liquid through a semipermeable membrane with a molecular weight cutoff of 350-500 Da to obtain a retentate and a filtrate;

[0031] (4) subjecting the filtrate to a constant temperature water bath reaction to obtain a second-stage saccharification liquid, and then mixing the second-stage saccharification liquid with the retentate to obtain a crude fructo-oligosaccharide liquid;

[0032] (5) adding activated carbon to the crude fructo-oligosaccharide solution, decolorizing and filtering to obtain a first filtrate;

[0033] (6) Desalting the first filtrate using a strong acid cation resin D001 and a weak base anion resin D301P to obtain a second filtrate;

[0034] (7) The second filtrate is subjected to chromatographic separation, evaporation concentration and drying to prepare oligofructose powder.

[0035] The present invention adds Fe to the sucrose solution 2+ Then, the pH was adjusted to obtain a reaction stock solution.

[0036] In the present invention, the mass fraction of the sucrose solution is preferably 55-60%, such as 55%, 56%, 57%, 58%, 59%, 60%, preferably a range value with any of the above values ​​as the upper or lower limit.

[0037] In the present invention, the Fe 2+The added amount is preferably 45-55mM, more preferably 48-53mM, such as 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, 55mM, preferably a range value with any of the above values ​​as the upper or lower limit.

[0038] In the present invention, metal ions Fe that can protect the thermal stability of the enzyme are added to the sucrose solution before the reaction. 2+ After the reaction temperature is subsequently increased, the enzymatic activity of fructosyltransferase under high temperature conditions can be effectively protected, which helps to accelerate the hydrolysis rate of sucrose in the first process, shorten the enzyme reaction time, and improve production efficiency.

[0039] In the present invention, the adjusted pH is preferably 5.5-6.0, such as 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, preferably a range value with any of the above values ​​as the upper or lower limit.

[0040] The present invention adds fructosyltransferase to the reaction stock solution and then conducts a constant temperature water bath reaction; when the mass fraction of sucrose in the system is 35-40%, sucrose solution is added until the mass fraction of sucrose is 50-55%, and then the reaction is continued for 10-12 hours to obtain the first process saccharification liquid. Since the hydrolysis reaction is preferentially carried out at a higher temperature and a higher sucrose concentration, the present invention adds sucrose solution in the first process to increase the substrate concentration, which can further promote the hydrolysis reaction, synthesize more low-polymerization sucrose trisaccharides for the second process, reserve small molecule sugars, further increase the content of sucrose trisaccharides, and improve the quality of oligofructose.

[0041] In the present invention, the added amount of the fructosyltransferase is preferably 6-8 U / g, such as 6 U / g, 7 U / g, 8 U / g, preferably a range value with any of the above values ​​as the upper or lower limit.

[0042] In the present invention, the constant temperature water bath reaction temperature is preferably 60-65° C., more preferably 63° C.; the reaction time is preferably 5-7 h, more preferably 6 h.

[0043] The present invention uses a semipermeable membrane with a molecular weight cutoff of 350-500Da to filter the saccharified liquid of the first process to obtain a retentate and a filtrate. The present invention adds a semipermeable membrane that can intercept sucrose trisaccharide before the polymerization reaction of the second process, and the molecular weight of sucrose trisaccharide is 504.44, and the molecular weight of sucrose is 342.3, so a 350-500Da semipermeable membrane is selected for filtration, while molecules such as sucrose and fructose are ensured to pass through, so that the sucrose trisaccharide polymerized to a small extent in the first process does not enter the second process, and the generation of large molecular weight sucrose tetrasaccharide and sucrose pentasaccharide in the second process is reduced, and the content of sucrose trisaccharide is significantly increased.

[0044] The invention conducts a constant temperature water bath reaction on the filtrate to obtain a second process saccharification liquid, and then mixes the second process saccharification liquid with the intercepted liquid to obtain a crude fructo-oligosaccharide liquid.

[0045] In the present invention, the water bath reaction temperature is preferably 40-45° C., more preferably 43° C.; the reaction time is preferably 4-6 h, more preferably 5 h.

[0046] The present invention divides the production of oligofructose by utilizing sucrose with fructosyltransferase into two processes for the first time. In the first process, the hydrolysis activity of the fructosyltransferase is controlled to be greater than the polymerization activity by increasing the temperature, which is beneficial to rapid hydrolysis and accumulation of small molecule sugars. In the second process, the polymerization activity of the fructosyltransferase is controlled to be greater than the hydrolysis activity by decreasing the temperature, which is convenient for rapid polymerization. By shortening the polymerization time of the second process, the polymerization degree of a large amount of oligofructose is controlled at a relatively low level. In addition, decreasing the temperature can inhibit the fructosyltransferase from hydrolyzing the synthesized oligofructose products, thereby improving the yield.

[0047] Furthermore, the present invention produces oligofructose during the saccharification reaction. The water activity of oligofructose will decrease under high temperature conditions. At the same time, the two-step reaction process can significantly consume fructose molecules in the system. Fructose itself has significant hygroscopicity. The amount of fructose residue in the system is small, which can reduce the hygroscopicity of oligofructose and extend the shelf life of the product.

[0048] The present invention adds activated carbon to the crude fructo-oligosaccharide solution to obtain a first filtrate through decolorization and filtration;

[0049] In the present invention, the added amount of the activated carbon is preferably 1.0-2.0% of the dry weight of the crude fructo-oligosaccharide liquid, and more preferably 1.2-1.8%.

[0050] In the present invention, the intermediate bleaching temperature is preferably 55-60° C., and the bleaching time is preferably 15-20 min.

[0051] In the present invention, the first filtrate is desalted using a strong acid cation resin D001 and a weak base anion resin D301P to obtain a second filtrate;

[0052] In the present invention, the flow temperature of the first filtrate in the desalination process is preferably 35-40° C., the flow rate is preferably 1-3 BV / h, and the discharge pH is preferably 5.0-6.0.

[0053] The second filtrate is subjected to chromatographic separation, evaporation concentration and drying to prepare oligofructose powder.

[0054] In the present invention, chromatographic separation, evaporative concentration and drying can be carried out by methods in the prior art.

[0055] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0056] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0057] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0058] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0059] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0060] The "room temperature" and "normal temperature" mentioned in the present invention are all calculated as 25±2°C unless otherwise specified.

[0061] The sources of raw materials used in the following examples of the present invention are: the present invention can use conventional β-fructosyltransferase in the art;

[0062] In the embodiments of the present invention, the fructosyltransferase is the β-fructosyltransferase prepared by Chinese patent CN104130950A.

[0063] The others were obtained from commercial sources.

[0064] The various detection methods in the present invention are as follows:

[0065] Fructosyltransferase activity assay:

[0066] The enzyme activity determination method was followed in accordance with QB / T 5357-2018.

[0067] Water activity detection method:

[0068] According to the determination method of water activity in GB 5009.238-2016 "Determination of water activity of foods".

[0069] Example 1

[0070] A method for preparing oligofructose comprises the following steps:

[0071] (1) Prepare a 56% sucrose solution in a reaction vessel and add 50 mM Fe 2+ , and then adjust the pH of the sucrose solution to 5.8.

[0072] (2) After adding fructosyltransferase at a rate of 7 U / g to the sucrose solution in step (1), the solution was heated in a constant temperature water bath at 63° C. and the reaction was continued for 6 h until the sucrose mass fraction reached 38%. Sucrose solution was further added until the sucrose mass fraction reached 53%, and the reaction was continued for 11 h to obtain the first process saccharification solution.

[0073] (3) The first process saccharification liquid in step (2) is filtered through a semipermeable membrane with a molecular weight cutoff of 500 Da to obtain a retentate and a filtrate. The filtrate is reacted in a water bath at 43° C. for 5 h to obtain a second process saccharification liquid. The retentate is mixed to obtain a crude fructo-oligosaccharide liquid.

[0074] (4) Add activated carbon at a dry weight ratio of 1.5% to the crude fructo-oligosaccharide solution of step (3), heat to 58° C. for 18 min, and decolorize and filter.

[0075] (5) The crude liquid after decolorization in step (4) is desalted using strong acid cation resin D001 and weak base anion resin D301P, with a feed temperature of 37° C., a feed speed of 2 BV / h, and a discharge pH of 5.5.

[0076] (6) The crude liquid after decolorization and desalination in step (6) is subjected to chromatographic separation, evaporation concentration and drying to prepare oligofructose powder.

[0077] Example 2

[0078] A method for preparing oligofructose comprises the following steps:

[0079] (1) Prepare a 55% sucrose solution in a reaction vessel and add 45 mM Fe 2+ , and then adjust the pH of the sucrose solution to 5.5.

[0080] (2) After adding fructosyltransferase at a rate of 6 U / g to the sucrose solution in step (1), the solution was heated in a constant temperature water bath at 60° C. and the reaction was continued for 5 h until the sucrose mass fraction reached 35%. Sucrose solution was added until the sucrose mass fraction reached 50%, and the reaction was continued for 10 h to obtain the first process saccharification solution.

[0081] (3) The first process saccharification liquid in step (2) is filtered through a semipermeable membrane with a molecular weight cutoff of 400 Da to obtain a retentate and a filtrate. The filtrate is reacted in a water bath at 40° C. for 4 h to obtain a second process saccharification liquid. The retentate is mixed to obtain a crude fructo-oligosaccharide liquid.

[0082] (4) Add activated carbon at a dry weight of 1.0% to the crude fructo-oligosaccharide solution of step (3), heat to 55° C. for 15 min, and decolorize and filter.

[0083] (5) The crude liquid after decolorization in step (4) is desalted using strong acid cation resin D001 and weak base anion resin D301P, with a feed temperature of 35° C., a feed speed of 1 BV / h, and a discharge pH of 5.0.

[0084] (6) The crude liquid after decolorization and desalination in step (5) is subjected to chromatographic separation, evaporation concentration and drying to prepare oligofructose powder.

[0085] Example 3

[0086] A method for preparing oligofructose comprises the following steps:

[0087] (1) Prepare a 60% sucrose solution in a reaction vessel and add 55 mM Fe 2+ , and then adjust the pH of the sucrose solution to 6.0.

[0088] (2) After adding 8 U / g of fructosyltransferase to the sucrose solution in step (1), the solution was heated in a constant temperature water bath at 65° C. and the reaction was continued for 7 hours until the sucrose mass fraction reached 40%. Sucrose solution was added until the sucrose mass fraction reached 55%, and the reaction was continued for 12 hours to obtain the first process saccharification solution.

[0089] (3) The first process saccharification liquid in step (2) is filtered through a semipermeable membrane with a molecular weight cutoff of 450Da to obtain a retentate and a filtrate. The filtrate is reacted in a water bath at 45°C for 6 hours to obtain a second process saccharification liquid. The retentate is mixed to obtain a crude fructo-oligosaccharide liquid.

[0090] (4) Add activated carbon at a dry weight ratio of 2.0% to the crude fructo-oligosaccharide solution of step (3), heat to 60° C. for 20 min, and decolorize and filter.

[0091] (5) The crude liquid after decolorization in step (4) is desalted using strong acid cation resin D001 and weak base anion resin D301P, with a feed temperature of 40°C, a feed speed of 3BV / h, and a discharge pH of 6.0.

[0092] (6) The crude liquid after decolorization and desalination in step (5) is subjected to chromatographic separation, evaporation concentration and drying to prepare oligofructose powder.

[0093] Comparative Example 1

[0094] The specific implementation of this comparative example is basically the same as that of Example 1, except that no sucrose solution is added in step (2).

[0095] Comparative Example 2

[0096] The specific implementation of this comparative example is basically the same as that of Example 1, except that: Fe is not added in step (1). 2 + .

[0097] Comparative Example 3

[0098] The specific implementation of this comparative example is basically the same as that of Example 1, except that the water bath temperature in step (3) is the same as that in step (2).

[0099] Comparative Example 4

[0100] The specific implementation of this comparative example is basically the same as that of Example 1, except that: in step (3), a semipermeable membrane with a molecular weight cutoff of 350-500 Da is not used for filtration.

[0101] Test Example 1

[0102] The specific steps of enzyme activity determination are as follows: add high concentration sucrose substrate to acetic acid-sodium acetate buffer (pH 5.0) to prepare a 55% sucrose concentration solution, add fructosyltransferase, and measure the hydrolytic enzyme activity with the amount of free glucose (one molecule of glucose released must be hydrolyzed by one molecule of sucrose), and measure the polymerase activity with the amount of synthesized oligofructose. The enzyme activity determination results are expressed as relative enzyme activity. The test results are shown in Table 1.

[0103] Table 1 Enzyme activities in hydrolysis and polymerization reactions

[0104] Hydrolase activity (%) Polymerase activity (%) Example 1 Phase 1 95 75 Phase II 75 90 Example 2 Phase 1 93 77 Phase II 72 88 Example 3 Phase 1 97 76 Phase II 78 89 Comparative Example 1 Phase 1 90 75 Phase II 73 85 Comparative Example 2 Phase 1 70 65 Phase II 64 62 Comparative Example 3 Phase 1 95 78 Phase II 90 75 Comparative Example 4 Phase 1 95 75 Phase II 76 89

[0105] As shown in Table 1, the hydrolase activity and polymerase activity in Comparative Example 2 are lower than those in Example 1, which indicates that Fe 2+The reaction activity of fructosyltransferase under high temperature conditions can be significantly protected; in Comparative Example 3, the fructosyltransferase hydrolase activity in the two stages is higher than the polymerase activity, indicating that the hydrolysis effect of fructosyltransferase can be significantly improved under high temperature conditions, resulting in an increase in the amount of fructose residues and a decrease in the conversion rate of oligofructose; low temperature conditions can effectively enhance the polymerase activity and improve the yield.

[0106] Test Example 2

[0107] The oligofructose conversion rate, the content of sucrose trisaccharide in oligofructose, the proportion of fructose residue, and the water activity of Implementation 1 and Comparative Examples 1 to 4 were tested, and the test results are shown in Table 2.

[0108] Table 2 Parameters test table

[0109] Conversion rate of oligofructose (%) Kestose content in oligofructose (%) Fructose residue percentage (%) Water activity (based on 50% concentration) Example 1 70 96 0.5 0.84 Example 2 66 95 1.8 0.86 Example 3 68 95 0.6 0.85 Comparative Example 1 55 90 3.8 0.90 Comparative Example 2 40 80 20 0.93 Comparative Example 3 50 80 8 0.90 Comparative Example 4 60 70 5 0.90

[0110] As shown in Table 2, the conversion rate of oligofructose in Comparative Example 1 is much lower than that in Example 1, which means that when sucrose is added during the saccharification process, more fructose is hydrolyzed in the first process, and sucrose quickly combines with fructose to produce sucrose, which has a significant effect on the conversion rate of oligofructose and the content of sucrose; the conversion rate of oligofructose and the content of sucrose in oligofructose in Comparative Example 2 are lower than those in Example 1, and the proportion of residual fructose and the water activity are higher than those in Example 1, which means that the enzyme stabilizer Fe 2+ The addition of can well protect the activity of fructosyltransferase, well consume the fructose after hydrolysis, reduce the water activity of the product, ensure the smooth and efficient progress of the overall saccharification reaction, and improve the various indicators of oligofructose; the oligofructose conversion rate and the saccharide content in oligofructose in comparative example 3 are also lower than those in embodiment 1, which means that the continuous high temperature will always be in the first process, and the hydrolysis will not stop, and the large amount of fructose residue will lead to a low conversion rate of oligofructose; the saccharide content in oligofructose in comparative example 4 is lower than that in embodiment 1, which means that the addition of semipermeable membrane interception before the second process can well increase the content of saccharide and reduce the production of oligofructose with high polymerization degree in the subsequent polymerization process.

[0111] In summary, the present invention prepares oligofructose, introduces metal ions that protect the thermal stability of enzymes, controls the reaction temperature and time so that hydrolysis and polymerization are carried out in stages, and uses a semipermeable membrane for filtration. Not only is the reaction rapid, the product has a low water activity and good moisture absorption resistance, but the oligofructose yield and the cane trisaccharide content can also be increased, providing new technical support for high-quality oligofructose.

[0112] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing oligofructose, characterized in that: The following steps are involved: (1) Add Fe to sucrose solution 2+ Then the pH is adjusted to obtain a reaction stock solution; (2) adding fructosyltransferase to the reaction stock solution and performing a constant temperature water bath reaction; when the mass fraction of sucrose in the system is 35-40%, adding sucrose solution until the mass fraction of sucrose is 50-55%, and then continuing the reaction for 10-12 hours to obtain a first process saccharification solution; (3) filtering the first process saccharification liquid through a semipermeable membrane with a molecular weight cutoff of 350 to 500 Da to obtain a retentate and a filtrate; (4) subjecting the filtrate to a constant temperature water bath reaction to obtain a second-stage saccharification liquid, and then mixing the second-stage saccharification liquid with the retentate to obtain a crude fructo-oligosaccharide liquid; (5) adding activated carbon to the crude fructo-oligosaccharide solution, decolorizing and filtering to obtain a first filtrate; (6) desalting the first filtrate using a strong acid cation resin D001 and a weak base anion resin D301P to obtain a second filtrate; (7) subjecting the second filtrate to chromatographic separation, evaporation concentration, and drying to prepare oligofructose powder; In the step (1), Fe 2+ The amount added is 45-55mM; The fructosyltransferase in step (2) is the β-fructosyltransferase prepared by Chinese patent CN104130950A; In step (2), the reaction temperature of the thermostatic water bath is 60-65° C., and the reaction time is 5-7 h; In step (4), the water bath reaction temperature is 40-45° C., and the reaction time is 4-6 hours.

2. The preparation method according to claim 1, characterized in that: The mass fraction of the sucrose solution in step (1) is 55-60%.

3. The preparation method according to claim 1, characterized in that: In the step (1), the pH is adjusted to 5.5-6.

0.

4. The preparation method according to claim 1, characterized in that: The amount of fructosyltransferase added in step (2) is 6-8 U / g.

5. The preparation method according to claim 1, characterized in that: The amount of activated carbon added in step (5) is 1.0-2.0% of the dry weight of the crude fructo-oligosaccharide liquid.

6. The preparation method according to claim 1, characterized in that: In the step (5), the decolorization temperature is 55-60° C. and the decolorization time is 15-20 min.

7. The preparation method according to claim 1, characterized in that: In the desalination process of step (6), the flow temperature of the first filtrate is 35-40° C., the flow rate is 1-3 BV / h, and the discharge pH is 5.0-6.0.