Preparation method of D-allose
By employing steps such as centrifugation, decolorization, resin column desalting, and ethanol crystallization, combined with recombinant ribose-5-phosphate isomerase conversion, the problems of low safety, high cost, and low purity in the preparation of D-allose in existing technologies have been solved, achieving safe and low-cost preparation of high-purity D-allose.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHUCHENG HAOTIAN PHARMA CO LTD
- Filing Date
- 2026-01-26
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies for preparing D-allose suffer from low safety, high cost, and low purity.
The method employs centrifugation, decolorization, resin column desalting, separation using calcium-type cation exchange resin column, and ethanol crystallization, combined with recombinant ribose-5-phosphate isomerase conversion. This avoids the use of toxic solvents, optimizes process conditions, uses food-grade resin and conventional reagents, controls the crystallization process, and improves purity.
It achieves high safety, low cost, and produces D-allose with a purity of up to 99%. The process is easy to implement industrially and reduces energy and material consumption.
Smart Images
Figure CN122011055A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of D-allose production technology, and specifically to a method for preparing D-allose. Background Technology
[0002] Allose, also known as D-allose, is an aldose, a rare monosaccharide, and the C-3 epimer of glucose. It is soluble in water but almost insoluble in methanol. It is known to be extremely rare in nature, primarily isolated from the leaves of African shrubs. Recent studies have shown that the rare sugar D-allose has important applications in food, health products, and medicine, and its wide range of physiological functions has made it a research hotspot among rare sugars. D-allose itself has the effect of inhibiting the proliferation of cancer cells, which means that there is hope for the development of new drugs without side effects.
[0003] Chinese patent CN108473991A discloses a strain that produces allose from fructose and a method for producing allose using the strain. The allose conversion rate in this patent is about 12-15%, and the product contains allulose heterosaccharides, which increases the separation cost.
[0004] Chinese patent CN112521429B discloses a method for extracting D-allose from nut shells using microwave radiation. The process is complicated and requires the use of microwave radiation, posing certain safety hazards. Furthermore, the elution solution used during separation is ether, which is harmful to the human body.
[0005] Chinese patent CN102839184A discloses a recombinant ribose-5-phosphate isomerase and its application. This recombinant enzyme catalyzes the ketaldehyde isomerization reaction between D-allulose and D-allose to produce D-allose. After the reaction reaches equilibrium, the conversion rate can reach 31%. However, it only stays in the upstream stage and does not mention the subsequent extraction, separation and crystallization.
[0006] Therefore, finding a safe, low-cost method to produce high-purity D-allose is of particular importance. Summary of the Invention
[0007] The technical problem to be solved by the present invention is to provide a method for preparing D-allose that is safe, low in cost, and produces D-allose with high purity, in order to address the shortcomings of the existing technology.
[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:
[0009] A method for preparing D-allose includes the following steps:
[0010] A: The D-allose conversion solution was centrifuged and decolorized, and the decolorized solution was collected.
[0011] B: The decolorizing solution is desalted by passing it through a cation exchange resin column and an anion exchange resin column, respectively, to obtain a desalted solution;
[0012] C: Concentrate the desalting solution to a solid content of 50-60% w / w to obtain concentrated solution I;
[0013] D: Concentrate I was separated using a chromatographic column packed with calcium-type cation exchange resin. After the separated solution was concentrated, concentrate II was obtained.
[0014] E: Add 0.5 times the volume of ethanol to concentrated solution II, stir thoroughly, add seed crystals, and then cool to 40°C at a rate of 2-3°C / h, while controlling the stirring speed at 30-80 rpm. Then add 1.0 times the volume of ethanol to concentrated solution II, keep warm for 0.5-1h, and then cool to 10-15°C at a rate of 3-5°C / h to stop crystallization. Filter, wash with ethanol to obtain white crystals, and dry to obtain D-allose with a purity >99%.
[0015] Preferably, the D-allose conversion solution in step A is prepared by adding whole-cell wet bacterial cells containing recombinant ribose-5-phosphate isomerase, MnCl2, and phosphate buffer to allulose solution and converting it at 60-80°C. The whole-cell wet bacterial cells containing recombinant ribose-5-phosphate isomerase are selected from the recombinant ribose-5-phosphate isomerase with the amino acid sequence SEQ ID NO: 2 in Chinese patent CN102839184A.
[0016] Preferably, the concentration of allulose solution is 600-700 g / L, the amount of whole-cell wet bacterial cells of recombinant ribose-5-phosphate isomerase is 50-100 g / L, the amount of MnCl2 is 0.5-1 mM, the amount of phosphate buffer is 20-50 mM, and the pH of phosphate buffer is 7-8.
[0017] Preferably, in step A, the centrifugation speed is 5000-6000 rpm. The bacterial cells collected by centrifugation can be resuspended and washed with 50 mM phosphate buffer and then reused for the transformation of D-allose, so as to achieve multiple batches of transformation.
[0018] In step A, add 3-5% v / v activated carbon and 1-2% v / v diatomaceous earth to the supernatant of the centrifuged liquid, decolorize at 60-80℃ for 30-50 minutes, filter, and collect the decolorized liquid. The decolorization process removes impurities such as pigments, proteins, and macromolecular organic matter from the liquid.
[0019] In step B, the flow rate through the cation exchange resin column and the anion exchange resin column is 1-3 BV, and the conductivity of the resulting desalting solution is <50 μS / cm.
[0020] Among them, SQ-605 is selected as the cation exchange resin and SQD-936 is selected as the anion exchange resin.
[0021] Before use, cation exchange resin columns should be soaked in 4-5% wt hydrochloric acid for 1-2 hours, then washed with deionized water until neutral. Before use, anion exchange resin columns should be soaked in 4-5% wt sodium hydroxide for 1-2 hours, then washed with deionized water until neutral.
[0022] The concentration conditions for the desalting solution in step C and the separation solution in step D are both a temperature of 50-70℃ and a vacuum degree of <-0.09Mpa.
[0023] In step D, the height-to-diameter ratio of the chromatographic column is >10. After the resin is packed, 50°C water is passed through the column jacket to maintain the resin temperature. 10% v / v of the concentrated solution I is injected into the column at a flow rate of 1-1.5 BV / h. After the injection is complete, the column is eluted with 50°C deionized water. Collection begins when the effluent contains solids. During collection, samples are taken for monitoring. Collection is stopped when the solids content of the effluent reaches 3.8-4.0% w / w, yielding the separated solution. The purity of D-aloose in this collected solution is greater than 98%. The column is then eluted with deionized water until the solids content is zero, and the concentrated solution is reinjected for separation.
[0024] The chromatographic column used was LS-4850.
[0025] In step E, the amount of seed crystals added accounts for 0.5-1.5% w / w of the solids in concentrate II.
[0026] In step E, after adding the seed crystals to the liquid, the mixture is sonicated at 20-40 kHz for 30 minutes to promote the uniform formation of crystal nuclei. Then, the mixture is cooled and crystallized. D-allose is not easy to crystallize, but ultrasonic vibration can accelerate the precipitation of crystals and shorten the crystallization time.
[0027] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:
[0028] 1. This invention avoids the use of toxic organic solvents such as ether throughout the entire process, primarily using water, ethanol, food-grade resin, and conventional reagents. The process conditions are mild, easy to implement industrially, and highly safe. By optimizing each unit operation, energy and material consumption are reduced, resulting in an overall production cost significantly lower than existing technologies.
[0029] 2. The wet bacterial cells (containing recombinant ribose-5-phosphate isomerase) collected by centrifugation in step A can be reused multiple times for the transformation reaction after washing with buffer. This design not only improves enzyme utilization and reduces enzyme preparation costs, but also allows the transformation process to be carried out continuously in multiple batches, thus improving production economics.
[0030] 3. Activated carbon and diatomaceous earth are used for synergistic decolorization, which effectively removes pigments, proteins and macromolecular organic matter from the conversion solution at 60-80℃, improves the transmittance and purity of the liquid, and avoids impurities interfering with the subsequent resin separation and crystallization process.
[0031] 4. In step B, the solution is desalted sequentially by passing it through cation exchange resin (SQ-605) and anion exchange resin (SQD-936), which reduces the conductivity of the desalting solution to <50 μS / cm. This greatly reduces the contamination and burden of inorganic ions on the subsequent chromatographic separation resins, extends the service life of the chromatographic column, and improves the separation efficiency.
[0032] 5. Step D employs a calcium-type cation exchange resin column (LS-4850) with a height-to-diameter ratio >10, and separation is performed at a constant temperature of 50℃. By controlling the feed volume, flow rate, and collection endpoint (stopping when the solid content reaches 3.8-4.0% w / w), a separation solution with a D-allose purity >98% can be stably obtained. This step effectively removes allulose and other impurities and is a key purification step.
[0033] 6. In step E, after adding ethanol to the concentrate, seed crystals are added first, followed by ultrasonic treatment at 20-40 kHz for 30 min to promote uniform crystal nucleus formation, solving the problem of D-allose's difficulty in crystallization and shortening the crystallization induction period. Subsequently, a staged cooling method (slow at first, then fast) and step-by-step ethanol replenishment are used to allow the crystals to grow slowly and orderly, reducing impurity entrapment. Finally, white crystals are obtained by filtration and washing with ethanol, with a product purity >99%, good crystal morphology, and improved product quality. Attached Figure Description
[0034] Figure 1 This is the liquid chromatogram of D-allose in Example 1 of the present invention. Detailed Implementation
[0035] The present invention will be further illustrated below with reference to the embodiments. Example 1
[0036] 1. Take 2L of allulose solution with a concentration of 600g / L, add whole-cell wet bacterial cells containing recombinant ribose-5-phosphate isomerase at a concentration of 50g / L, add 0.5mM MnCl2, and incubate at 60℃ for 6h in 20mM phosphate buffer (pH 7) to obtain a conversion solution containing D-allose with a conversion rate of 35%.
[0037] 2. The conversion solution is centrifuged, and 2L of the supernatant and bacterial cells are collected separately. The centrifugation speed is 5000rpm. After collection, the bacterial cells are resuspended and washed with 50mM phosphate buffer and can be reused in step 1 for transformation again to achieve multiple batches of transformation.
[0038] 3. Add 3% v / v activated carbon and 1% v / v diatomaceous earth to the supernatant after centrifugation, decolorize at 60℃ for 50 min, filter, and collect the decolorized liquid.
[0039] 4. The decolorizing solution is first passed through a cation exchange resin column, and then through an anion exchange resin column for desalting, yielding 3.5 L of desalting solution. The column flow rate is 1 BV, and the conductivity of the desalting solution is <50 μS / cm. The cation exchange resin is model SQ-605, and the anion exchange resin is model SQD-936. Before use, the cation exchange resin is soaked in 4% wt hydrochloric acid for 2 hours, and then washed with deionized water until neutral. Before use, the anion exchange resin is soaked in 4% wt sodium hydroxide for 2 hours, and then washed with deionized water until neutral.
[0040] 5. Concentrate the desalting solution to a solid content of 50% w / w to obtain concentrated solution I. Concentration conditions: temperature 50℃, vacuum degree < -0.09MPa, and keep concentrated solution I at 50-60℃ for later use.
[0041] 6. Concentrate I was separated using a chromatographic column packed with calcium-type cation exchange resin. The high-content fraction of D-aloose was collected for later use. The resin type was LS-4850.
[0042] The chromatographic column was selected with a height-to-diameter ratio >10. After the resin was packed, 50°C water was passed through the column jacket to maintain the resin temperature. A 10% v / v concentrate (I) was injected into the column at a flow rate of 1 BV / h. After injection, the column was eluted with 50°C deionized water. Collection began when the effluent contained solids, and samples were taken for monitoring during collection. Collection was stopped when the solids content in the effluent reached 3.8%. The column was then eluted with deionized water until the solids content was zero, and the concentrate (I) was reinjected for separation.
[0043] 7. The collected liquid was concentrated to a solid content of 50% w / w under the conditions of 50℃ temperature and vacuum ≤ -0.09MPa to obtain concentrated liquid II.
[0044] 8. When 0.7 L of concentrated solution II is heated to 50 °C, 0.5 times the volume of the solution in ethanol is added. After stirring and mixing thoroughly, 0.5% w / w seed crystals of the concentrated solution solids are added. After adding the seed crystals, low-frequency ultrasound (20 kHz) is applied for 30 min to promote uniform formation of crystal nuclei. Then, the temperature is lowered to 40 °C at a rate of 2 °C / h, and the stirring speed is controlled at 30 rpm. Then, 1.0 times the volume of concentrated solution II in ethanol is added, and the temperature is maintained for 0.5 h. Then, the temperature is lowered to 15 °C at a rate of 3 °C / h to stop crystallization. After filtration and washing with ethanol, white crystals are obtained. After drying, 298 g of D-allose is obtained, with a yield of 70.95% and a purity of 99.6%. Example 2
[0045] 1. Take 2L of allulose solution with a concentration of 650g / L, add whole-cell wet bacterial cells containing recombinant ribose-5-phosphate isomerase at a concentration of 80g / L, add 0.8mM MnCl2, and incubate at 70℃ for 5h in 35mM phosphate buffer (pH 8) to obtain a conversion solution containing D-allose with a conversion rate of 35%.
[0046] 2. The conversion solution is centrifuged, and 2L of the supernatant and bacterial cells are collected separately. The centrifugation speed is 5500rpm. After collection, the bacterial cells are resuspended and washed with 50mM phosphate buffer and can be reused in step 1 for transformation again to achieve multiple batches of transformation.
[0047] 3. Add 4% v / v activated carbon and 1.5% v / v diatomaceous earth to the supernatant after centrifugation, decolorize at 70℃ for 40 min, filter, and collect the decolorized liquid.
[0048] 4. The decolorizing solution is first passed through a cation exchange resin column, and then through an anion exchange resin column for desalting, yielding 4 L of desalting solution. The column flow rate is 2 BV, and the conductivity of the desalting solution is <50 μS / cm. The cation exchange resin is model SQ-605, and the anion exchange resin is model SQD-936. Before use, the cation exchange resin is soaked in 4.5% wt hydrochloric acid for 1.5 h, and then washed with deionized water until neutral. Before use, the anion exchange resin is soaked in 4.5% wt sodium hydroxide for 1.5 h, and then washed with deionized water until neutral.
[0049] 5. Concentrate the desalting solution to a solid content of 55% w / w to obtain concentrated solution I. Concentration conditions: temperature 60℃, vacuum degree < -0.09MPa, and keep concentrated solution I at 50-60℃ for later use.
[0050] 6. Concentrate I was separated using a chromatographic column packed with calcium-type cation exchange resin. The high-content fraction of D-aloose was collected for later use. The resin type was LS-4850.
[0051] The chromatographic column was selected with a height-to-diameter ratio >10. After the resin was packed, 50°C water was passed through the column jacket to maintain the resin temperature. 10% v / v of concentrated solution I was injected into the column at a flow rate of 1.5 BV / h. After injection, the column was eluted with 50°C deionized water. Collection began when the effluent contained solids, and samples were taken for monitoring during collection. Collection was stopped when the solids content in the effluent reached 3.9%. The column was then eluted with deionized water until the solids content was zero, and concentrated solution I was reinjected for separation.
[0052] 7. The collected liquid was concentrated to a solid content of 55% w / w under the conditions of 60℃ and vacuum ≤ -0.09MPa to obtain concentrated liquid II.
[0053] 8. When 0.8 L of concentrated solution II is heated to 55 °C, 0.5 times the volume of the solution is added to ethanol. After stirring and mixing thoroughly, 1.0% w / w of the concentrated solution solids is added as seed crystals. After adding the seed crystals, low-frequency ultrasound (30 kHz) is applied for 30 min to promote uniform crystal nucleus formation. Then, the temperature is lowered to 40 °C at a rate of 2.5 °C / h, and the stirring speed is controlled at 50 rpm. Then, 1.0 times the volume of concentrated solution II is added to ethanol, and the temperature is maintained for 0.8 h. Then, the temperature is lowered to 12 °C at a rate of 4 °C / h to stop crystallization. After filtration and washing with ethanol, white crystals are obtained. After drying, 329 g of D-allose is obtained, with a yield of 72.31% and a purity of 99.4%. Example 3
[0054] 1. Take 2L of allulose solution with a concentration of 700g / L, add whole-cell wet bacterial cells containing recombinant ribose-5-phosphate isomerase at a concentration of 100g / L, add 1mM MnCl2, and incubate at 80℃ for 4h in 50mM phosphate buffer (pH 8) to obtain a conversion solution containing D-allose with a conversion rate of 35%.
[0055] 2. The conversion solution is centrifuged, and 2L of the supernatant and bacterial cells are collected separately. The centrifugation speed is 6000rpm. After collection, the bacterial cells are resuspended and washed with 50mM phosphate buffer and can be reused in step 1 for transformation again to achieve multiple batches of transformation.
[0056] 3. Add 5% v / v activated carbon and 2% v / v diatomaceous earth to the supernatant after centrifugation, decolorize at 80℃ for 30 min, filter, and collect the decolorized liquid.
[0057] 4. The decolorizing solution was first passed through a cation exchange resin column, and then through an anion exchange resin column for desalting, yielding 4.2 L of desalted solution. The column flow rate was 3 BV, and the conductivity of the desalted solution was <50 μS / cm. The cation exchange resin was model SQ-605, and the anion exchange resin was model SQD-936. Before use, the cation exchange resin was soaked in 5% wt hydrochloric acid for 1 hour, and then washed with deionized water until neutral. Before use, the anion exchange resin was soaked in 5% wt sodium hydroxide for 1 hour, and then washed with deionized water until neutral.
[0058] 5. Concentrate the desalting solution to a solid content of 60% w / w to obtain concentrated solution I. Concentration conditions: temperature 70℃, vacuum degree < -0.09MPa, and keep concentrated solution I at 50-60℃ for later use.
[0059] 6. Concentrate I was separated using a chromatographic column packed with calcium-type cation exchange resin. The high-content fraction of D-aloose was collected for later use. The resin type was LS-4850.
[0060] The chromatographic column was selected with a height-to-diameter ratio >10. After the resin was packed, 50°C water was passed through the column jacket to maintain the resin temperature. 10% v / v of concentrated solution I was injected into the column at a flow rate of 1.5 BV / h. After injection, the column was eluted with 50°C deionized water. Collection began when the effluent contained solids, and samples were taken for monitoring during collection. Collection was stopped when the solids content in the effluent reached 4.0%. The column was then eluted with deionized water until the solids content was zero, and concentrated solution I was reinjected for separation.
[0061] 7. The collected liquid was concentrated to a solid content of 60% w / w under the conditions of 70℃ and vacuum ≤ -0.09MPa to obtain concentrated liquid II.
[0062] 8. When 0.76 L of concentrate II was heated to 60 °C, 0.5 times the volume of the concentrate was added to ethanol. After stirring and mixing thoroughly, 1.5% w / w of the concentrate solids was added as seed crystals. After adding the seed crystals, low-frequency ultrasound (40 kHz) was applied for 30 min to promote uniform crystal nucleus formation. Then, the temperature was lowered to 40 °C at a rate of 3 °C / h, and the stirring speed was controlled at 80 rpm. Then, 1.0 times the volume of concentrate II was added to ethanol, and the temperature was maintained for 1 h. Then, the temperature was lowered to 10 °C at a rate of 5 °C / h to stop crystallization. After filtration and washing with ethanol, white crystals were obtained. After drying, 360 g of D-allose was obtained, with a yield of 73.47% and a purity of 99.5%.
[0063] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.
Claims
1. A method for preparing D-allose, characterized in that... Includes the following steps: A: The D-allose conversion solution was centrifuged and decolorized, and the decolorized solution was collected. B: The decolorizing solution is desalted by passing it through a cation exchange resin column and an anion exchange resin column, respectively, to obtain a desalted solution; C: Concentrate the desalting solution to a solid content of 50-60% w / w to obtain concentrated solution I; D: Concentrate I was separated using a chromatographic column packed with calcium-type cation exchange resin. After the separated solution was concentrated, concentrate II was obtained. E: Add 0.5 times the volume of ethanol to concentrated solution II, stir to mix thoroughly, add seed crystals, then cool to 40°C at a rate of 2-3°C / h, stirring at 30-80 rpm, then add 1.0 times the volume of ethanol to concentrated solution II, keep warm for 0.5-1h, then cool to 10-15°C at a rate of 3-5°C / h, stop crystallization, filter, wash with ethanol to obtain white crystals, and dry to obtain D-allose.
2. The method for preparing D-allose as described in claim 1, characterized in that: The D-allose conversion solution in step A is prepared by adding whole-cell wet bacterial cells containing recombinant ribose-5-phosphate isomerase, MnCl2, and phosphate buffer to allulose solution and converting it at 60-80℃.
3. The method for preparing D-allose as described in claim 2, characterized in that: The concentration of allulose solution is 600-700 g / L, the amount of whole-cell wet bacterial cells of recombinant ribose-5-phosphate isomerase is 50-100 g / L, the amount of MnCl2 is 0.5-1 mM, the amount of phosphate buffer is 20-50 mM, and the pH of phosphate buffer is 7-8.
4. The method for preparing D-allose as described in claim 1, characterized in that: In step A, the centrifugation speed is 5000-6000 rpm. The bacterial cells collected by centrifugation can be resuspended and washed with 50 mM phosphate buffer and then reused for the conversion of D-allose.
5. The method for preparing D-allose as described in claim 1, characterized in that: In step A, add 3-5% v / v activated carbon and 1-2% v / v diatomaceous earth to the supernatant after centrifugation, decolorize at 60-80℃ for 30-50 min, filter, and collect the decolorized liquid.
6. The method for preparing D-allose as described in claim 1, characterized in that: In step B, the flow rate through the cation exchange resin column and the anion exchange resin column is 1-3 BV, and the conductivity of the resulting desalting solution is <50 μS / cm. Before use, cation exchange resin columns should be soaked in 4-5% wt hydrochloric acid for 1-2 hours, then washed with deionized water until neutral. Before use, anion exchange resin columns should be soaked in 4-5% wt sodium hydroxide for 1-2 hours, then washed with deionized water until neutral.
7. The method for preparing D-allose as described in claim 1, characterized in that: The concentration conditions for the desalting solution in step C and the separation solution in step D are both a temperature of 50-70℃ and a vacuum degree of <-0.09Mpa.
8. The method for preparing D-allose as described in claim 1, characterized in that: In step D, the height-to-diameter ratio of the chromatographic column is >10. After the resin is packed, 50°C water is passed through the column jacket to maintain the resin temperature. 10% v / v of the concentrated solution I is injected into the column at a flow rate of 1-1.5 BV / h. After the injection is complete, the column is eluted with 50°C deionized water. Collection begins when the effluent contains solids and stops when the solids content in the effluent reaches 3.8-4.0% w / w, yielding the separated solution. The column is then eluted with deionized water until the solids content is zero, and the concentrated solution is reinjected for separation.
9. The method for preparing D-allose as described in claim 1, characterized in that: In step E, the amount of seed crystals added accounts for 0.5-1.5% w / w of the solids in concentrate II.
10. The method for preparing D-allose as described in claim 1, characterized in that: In step E, after adding seed crystals to the liquid, the mixture is sonicated at 20-40 kHz for 30 minutes, and then cooled for crystallization.