Preparation method and application of nystose
By using sucrose and fructose transferase in the enzyme-catalyzed transformation reaction and adjusting the process parameters, the purity and yield of Naiss sugar were successfully improved, and the problems of low yield and poor purity in the existing technology were solved, and efficient and economical Naiss sugar preparation was achieved.
Patent Information
- Application Number
- CN202510039072.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-10
- Publication Date
- 2025-04-11
AI Technical Summary
The existing method of producing Nais sugar catalyzed through enzymes has defects such as low yield and poor purity, which is difficult to meet the market's demand for high-quality and high-yield Nais sugar.
Using sucrose as the substrate, the transformation reaction is catalyzed by fructose transferase, combined with the appropriate pH value, temperature, fructose supplementation timing and filtration process, Neiss sugar content reaches more than 75%.
It achieves high purity (≥75%) and high yield of Nais sugar, meets the needs of industrial large-scale applications, and reduces production costs.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of oligosaccharides, and specifically relates to a preparation method of nesitose and an application thereof. Background Art
[0002] Depression is a syndrome characterized by significant and persistent low mood. With the intensification of social competition and the increase of many stress factors, depression has become more common every year. Currently, depression ranks fourth among the world's disabling diseases, and by 2024 it will become the second largest disease after heart disease.
[0003] At present, there are many Western antidepressant drugs used in clinical practice. The efficacy of these drugs has been confirmed through clinical treatment, but these drugs also have disadvantages such as large side effects, high prices, drug dependence, and poor quality of life. Studies have found that nesitose (cane tetraose) can not only promote gastrointestinal health and control blood sugar, but also has significant effects in treating depression. However, the content of nesitose in natural plants is extremely low. For example, the content of nesitose in Morinda officinalis is only less than one thousandth of the dry matter content. The process of extracting nesitose from plants is cumbersome, the yield is very low, the cost is extremely high, and the impurity composition is complex, and even contains a small amount of low-toxic non-functional ingredients. Therefore, nesitose is generally converted by enzyme catalysis using sucrose or inulin as substrates.
[0004] Since the current method of producing nesbitz by enzyme catalysis still has defects such as low yield and poor purity, the present invention hopes to propose a method for scalable production of high-purity nesbitz to meet the urgent market demand for nesbitz. Summary of the invention
[0005] The present invention aims to solve at least one of the problems existing in the above-mentioned prior art. To this end, the present invention provides a preparation method of nysose and its application. The production of nysose by the preparation method can make the content of the obtained nysose reach more than 75%, and can be industrialized and applied on a large scale, thereby meeting the urgent market demand for high-quality and high-yield nysose.
[0006] The present invention provides a method for preparing nesitose, comprising the following steps:
[0007] (1) using sucrose as a substrate and dissolving it in water to obtain a sucrose aqueous solution with a concentration of 50%-60% (w / v);
[0008] (2) adding fructosyltransferase to the sucrose aqueous solution, adjusting the pH value to 5.5-5.9 and the reaction temperature to 57-62° C. to carry out a conversion reaction; the amount of the fructosyltransferase added is 3000-5000 U per kilogram of sucrose;
[0009] (3) When the glucose content in the reaction system is ≥15% (dry basis), add fructose accounting for 25%-30% of the mass of sucrose, and add water to make its DS value (soluble solid content) reach 40%-50%, and continue the conversion reaction;
[0010] (4) When the conversion reaction is carried out for 14-17 hours, boil to obtain a reserve solution;
[0011] (5) decolorizing and filtering the reserve solution to obtain a separated solution;
[0012] (6) The separated liquid is adsorbed by 7-10 groups of polystyrene sulfonate cationic resin chromatographic columns connected in series, then eluted with hydrochloric acid, and spray-dried to obtain nesitose.
[0013] The present invention uses sucrose as a substrate to transform and prepare nesose. By setting key processes and adjusting production process parameters, after continuous exploration and research, the content of the prepared nesose (kestose) is finally made to reach more than 75%, and the needs of large-scale production are met.
[0014] For example, when the enzyme activity of fructosyltransferase is too low, the conversion rate of oligofructose will be seriously affected. When the fructosyltransferase is added too high, the content of glucose and fructose will be too high, and the yield of the target conversion substance - nastose (nectetose) will be reduced. Even part of nastose will be hydrolyzed into monosaccharides and disaccharides such as glucose, fructose, sucrose, and fructose.
[0015] When the sucrose aqueous solution is acidic or alkaline, the activity of fructosyltransferase is seriously affected, and the production of nastose (nestose) is reduced or even not synthesized. Similarly, when the conversion reaction temperature is low, the activity of the enzyme deteriorates and the conversion reaction is incomplete; and when the conversion temperature is too high, the activity of the enzyme deteriorates or even inactivates, and the conversion reaction is incomplete or no conversion reaction occurs. In the present invention, acid-base adjustment can be performed by adjusting the acidity with citric acid and the alkalinity with sodium hydroxide aqueous solution.
[0016] When the conversion reaction time is between 14-17h, the conversion rate of nastose is the highest. Insufficient reaction time will lead to incomplete reaction and low conversion rate. When the reaction time is too long, after the conversion rate of nastose (nastose) reaches the highest value, some of the fructose and glucose groups in the oligofructose will be rapidly hydrolyzed into monosaccharides, thereby reducing the conversion rate of nastose in the reaction.
[0017] Since fructosyltransferase continuously hydrolyzes fructose in sucrose and combines it with sucrose, disaccharide, triose, tetraose, etc. to form oligofructose, free glucose monosaccharide increases, sucrose content decreases, and direct and indirect supply of fructose is in short supply. The shortage of fructose directly affects the synthesis of tetraose. Therefore, the present invention controls the supplementation of fructose at the right time, which will be beneficial to the synthesis of tetraose. The timing of adding fructose also has a great influence on the conversion rate. Adding it too early will interfere with the conversion reaction of fructosyltransferase, and the conversion rate of tetraose will be reduced; adding it too late will stop the enzymatic reaction, and the added fructose will not participate in the reaction. It was verified through repeated experiments that when the glucose content in the reaction system is ≥15%, it is most appropriate to add fructose, and the conversion rate of nesose (tetraose) is the highest.
[0018] Finally, the reserve solution is passed through a polypropylene filter bag, a hollow cellulose membrane selective interception, and a polystyrene sulfonate cationic resin chromatographic column to separate different polysaccharides in a gradient manner, so as to obtain neistose (kestose) with a concentration of more than 75%. If the number of polystyrene sulfonate cationic resin chromatographic columns is further increased, although the separation degree of gradient separation can be increased, this method is not cost-effective from the perspective of cost and efficiency. The present invention comprehensively considers multiple angles and estimates that 7-10 groups of chromatographic columns can meet actual needs.
[0019] Preferably, the decolorization in step (5) is decolorization using activated carbon.
[0020] Preferably, the filtering method in step (5) is: filtering with a polypropylene filter bag and then filtering with a hollow cellulose membrane.
[0021] More preferably, the pore size of the polypropylene filter bag is 0.2 μm.
[0022] More preferably, the filtration using the hollow cellulose membrane in step (5) is performed no less than twice.
[0023] More preferably, the molecular weight cut-off of the hollow cellulose membrane is 200-1500 Da.
[0024] Further preferably, the molecular weight cut-off of the hollow cellulose membrane is 500-1000Da.
[0025] Preferably, the concentration of the hydrochloric acid in step (6) is 0.1-0.5 mol / L.
[0026] Preferably, the elution rate in step (6) is 3-6 L / min.
[0027] The present invention also provides the use of the preparation method of nysitose in the preparation of food or medicine. The nysitose prepared by the preparation method of the present invention can be used to produce health food for regulating body functions and preventing and treating depression, and can also be used to produce antidepressant drugs.
[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0029] The method for preparing nesose proposed in the present invention uses sucrose as a substrate, and through the setting of key processes and the adjustment of production process parameters (such as substrate concentration, the amount of fructosyltransferase added, the pH value and temperature of the conversion reaction, the timing and amount of fructose addition, the conversion reaction time, the filtering and adsorption elution conditions), after continuous exploration and research, the content of the prepared nesose (cane fructose tetraose) is finally made to reach more than 75%. In addition, the method for preparing nesose also has the advantages of high yield, simple process, and controllable cost, and can realize quantitative production, which can meet the market demand for nesose raw materials. DETAILED DESCRIPTION
[0030] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.
[0031] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.
[0032] Example 1
[0033] This embodiment provides a method for preparing nesitose, comprising the following steps:
[0034] (1) Take 50 kg of sucrose and add it to purified water. After dissolving, adjust the volume to 100 liters so that the concentration of the sucrose aqueous solution is 50% (w / v).
[0035] (2) Add fructosyltransferase to the sucrose aqueous solution, the amount of fructosyltransferase added is 4000 U / kg sucrose (dry basis), that is, add 200000 U. Adjust the pH of the solution to 5.7, set the reaction temperature to 58°C, and carry out the conversion reaction.
[0036] (3) When the glucose content in the reaction system is ≥15%, 14 kg of fructose is added, and purified water is added to make its DS value (DS refers to the soluble solid content, which represents the concentration of sugars in the reaction system in the present invention) reach 50%, and the reaction is continued at a constant temperature of 58°C.
[0037] (4) When the conversion reaction time reaches 16 h, boil at 100° C. for 15 min to inactivate the fructosyltransferase, thereby obtaining a reserve solution.
[0038] (5) The reserve solution is sequentially decolorized by activated carbon, coarsely filtered through a 0.2 μm polypropylene filter bag, filtered once through a hollow cellulose membrane (molecular weight cutoff ≥ 1000 Da molecules, retaining the permeate), and filtered twice through a hollow cellulose membrane (molecular weight cutoff ≥ 500 Da molecules, retaining the retentate), and the secondary retentate is collected.
[0039] (6) Dilute the DS value of the retentate to 30% with pure water, thereby reducing its viscosity and increasing its fluidity. Adsorb through eight groups of polystyrene sulfonate cationic resin chromatographic columns (each group has a diameter of 20 cm and a column height of 200 cm), and then elute with 0.5 mol / L hydrochloric acid at a rate of 5 L / min. Recover the eluate in batches and sections, and then spray-dry the recovered liquid in batches to volatilize the water and hydrochloric acid components to obtain nesitose (cane tetraose).
[0040] Example 2
[0041] This embodiment provides a method for preparing nesitose, comprising the following steps:
[0042] (1) Take 60 kg of sucrose and add it to purified water. After dissolving, adjust the volume to 100 liters so that the concentration of the sucrose aqueous solution is 60% (w / v).
[0043] (2) Add fructosyltransferase to the sucrose aqueous solution, the amount of fructosyltransferase added is 3000 U / kg sucrose (dry basis), that is, 180000 U. Adjust the pH of the solution to 5.5, set the reaction temperature to 60°C, and carry out the conversion reaction.
[0044] (3) When the glucose content in the reaction system is ≥15%, add 18 kg of fructose and pure water to make the DS value reach 50%, and continue the reaction at a constant temperature of 58°C.
[0045] (4) When the conversion reaction time reaches 16 h, boil at 100° C. for 15 min to inactivate the fructosyltransferase, thereby obtaining a reserve solution.
[0046] (5) The reserve solution is sequentially decolorized by activated carbon, coarsely filtered through a 0.2 μm polypropylene filter bag, filtered once through a hollow cellulose membrane (molecular weight cutoff ≥ 1000 Da molecules, retaining the permeate), and filtered twice through a hollow cellulose membrane (molecular weight cutoff ≥ 500 Da molecules, retaining the retentate), and the secondary retentate is collected.
[0047] (6) Dilute the DS value of the retentate to 30% with pure water, thereby reducing its viscosity and increasing its fluidity. Then, the retentate is adsorbed through nine groups of polystyrene sulfonate cationic resin chromatographic columns (each group has a diameter of 20 cm and a column height of 200 cm), and eluted with 0.4 mol / L hydrochloric acid at a rate of 5 L / min. The eluate is recovered in batches and then spray-dried in batches to volatilize the water and hydrochloric acid components to obtain nesitose (cane tetraose).
[0048] Comparative Example 1
[0049] Compared with Example 1, the preparation steps and process parameters of nesitose in this comparative example are basically the same, the only difference is that the amount of sucrose added is 40 kg, and the concentration of the obtained sucrose aqueous solution is 40% (w / v).
[0050] Comparative Example 2
[0051] Compared with Example 1, the preparation steps and process parameters of nesitose in this comparative example are basically the same, and the only difference is that the added amount of fructosyltransferase is 2000U / kg sucrose (dry basis), that is, 100000U is added.
[0052] Comparative Example 3
[0053] Compared with Example 1, the preparation steps and process parameters of nesitose in this comparative example are basically the same, and the only difference is that in step (2), the pH value of the solution is adjusted to 5.3.
[0054] Comparative Example 4
[0055] Compared with Example 1, the preparation steps and process parameters of nesitose in this comparative example are basically the same, with the only difference being that the reaction temperature in step (2) is set to 55°C.
[0056] Comparative Example 5
[0057] Compared with Example 1, the preparation steps and process parameters of nesitose in this comparative example are basically the same, and the only difference is that in step (3), when the glucose content in the reaction system is ≥12%, fructose is added.
[0058] Comparative Example 6
[0059] Compared with Example 1, the preparation steps and process parameters of nesitose in this comparative example are basically the same, with the only difference being that 10 kg of fructose is added in step (3).
[0060] Comparative Example 7
[0061] Compared with Example 1, the preparation steps and process parameters of nesitose in this comparative example are basically the same, with the only difference being that in step (4), when the conversion reaction time reaches 18 hours, boiling at 100° C. for 15 minutes.
[0062] Comparative Example 8
[0063] Compared with Example 1, the preparation steps and process parameters of nastose in this comparative example are basically the same, with the only difference being that in step (5), adsorption is carried out using five groups of polystyrene sulfonate cationic resin chromatographic columns (each group with a diameter of 20 cm and a column height of 200 cm) connected in series.
[0064] Test results
[0065] The preparation methods of nysose in Example 1 and Comparative Examples 1-8 were used for testing, and the conversion rate and purity results of the obtained nysose are shown in Table 1.
[0066] Table 1 Nesbittose conversion rate and purity results
[0067]
[0068] As can be seen from Table 1, compared with the nysose obtained in Comparative Examples 1-8, the preparation method of Example 1 can produce a nysose product with a purity of up to 77.8% (≥75%), with significant results. The above results show that the concentration of the sucrose aqueous solution in the reaction system, the amount of fructosyltransferase added, the pH value and temperature of the conversion reaction, the timing and amount of fructose addition, the conversion reaction time, etc. are all important factors affecting the reaction conversion rate; in addition, the filtration process and the number of chromatographic column groups and the elution conditions are the main factors affecting the purity of nysose. It is through continuous exploration and improvement that the present invention obtains a reasonable preparation process and related reaction conditions, and finally obtains a method for preparing a high-purity nysose (purity ≥75%) product that can be industrially applied.
[0069] The embodiments of the present application are described in detail above, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present application.
Claims
1. A method for preparing nesitose, characterized in that: The following steps are involved: (1) using sucrose as a substrate and dissolving it in water to obtain a sucrose aqueous solution with a concentration of 50%-60% (w / v); (2) adding fructosyltransferase to the sucrose aqueous solution, adjusting the pH value to 5.5-5.9 and the reaction temperature to 57-62° C. to carry out a conversion reaction; the amount of the fructosyltransferase added is 3000-5000 U per kilogram of sucrose; (3) When the glucose content in the reaction system is ≥15% (dry basis), add fructose accounting for 25%-30% of the mass of sucrose, and add water to make the DS value reach 40%-50%, and continue the conversion reaction; (4) When the conversion reaction is carried out for 14-17 hours, boil to obtain a reserve solution; (5) decolorizing and filtering the reserve solution to obtain a separated solution; (6) The separated liquid is adsorbed by 7-10 groups of polystyrene sulfonate cationic resin chromatographic columns connected in series, then eluted with hydrochloric acid, and spray-dried to obtain nesitose.
2. The preparation method according to claim 1, characterized in that: The decolorization in step (5) is activated carbon decolorization.
3. The preparation method according to claim 1, characterized in that: The filtering method in step (5) is: filtering with a polypropylene filter bag and then filtering with a hollow cellulose membrane.
4. The preparation method according to claim 3, characterized in that: The pore size of the polypropylene filter bag is 0.2 μm.
5. The preparation method according to claim 3, characterized in that: In step (5), the filtration is performed using the hollow cellulose membrane for no less than 2 times.
6. The preparation method according to claim 3, characterized in that: The molecular weight cut-off of the hollow cellulose membrane is 200-1500Da.
7. The preparation method according to claim 6, characterized in that: The molecular weight cut-off of the hollow cellulose membrane is 500-1000Da.
8. The preparation method according to claim 1, characterized in that: The concentration of the hydrochloric acid in step (6) is 0.1-0.5 mol / L.
9. The preparation method according to claim 1, characterized in that: The elution rate in step (6) is 3-6 L / min.
10. Use of the method for preparing nesitose according to any one of claims 1 to 9 in preparing food or medicine.