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CN-122011055-A - Preparation method of D-allose

CN122011055ACN 122011055 ACN122011055 ACN 122011055ACN-122011055-A

Abstract

The invention discloses a preparation method of D-allose, which relates to the technical field of D-allose production, and D-allose conversion liquid is subjected to centrifugation, decolorization, desalination, concentration, chromatographic separation and re-concentration, ethanol is added in batches for segmented cooling crystallization, suction filtration is carried out, white crystals are obtained after ethanol washing, and D-allose is obtained after drying. The production cost is reduced, the crystal morphology of the obtained product is good, and the quality of the product is improved.

Inventors

  • ZHU LIPING
  • XU LIANGPING
  • Huai Jianlu
  • SONG WENZHU
  • Qiu Chongshun

Assignees

  • 诸城市浩天药业有限公司

Dates

Publication Date
20260512
Application Date
20260126

Claims (10)

  1. 1. The preparation method of the D-allose is characterized by comprising the following steps of: D-allose conversion liquid is centrifugated and decolored, and then decolored liquid is collected; Desalting the decolorized solution by a cation resin column and an anion resin column respectively to obtain desalted solution; concentrating the desalted liquid to solid content of 50-60% w/w to obtain concentrated liquid I; separating the concentrated solution I by using a chromatographic column filled with calcium type cationic resin, and concentrating the separated solution to obtain concentrated solution II; E, adding ethanol with the volume of 0.5 times of the feed liquid into the concentrated solution II, stirring and fully mixing, adding seed crystals, cooling to 40 ℃ at the speed of 2-3 ℃ per hour, controlling the stirring rotation speed to 30-80rpm, adding ethanol with the volume of 1.0 times of the concentrated solution II, preserving heat for 0.5-1h, cooling to 10-15 ℃ at the speed of 3-5 ℃ per hour, stopping crystallization, carrying out suction filtration, washing with ethanol to obtain white crystals, and drying to obtain the D-allose.
  2. 2. The method of claim 1, wherein the D-psicose conversion solution in the step A is prepared by adding whole cell wet thalli containing recombinant ribose-5-phosphate isomerase, mnCl 2 and phosphate buffer into psicose solution, and converting at 60-80 ℃.
  3. 3. The method for producing D-psicose according to claim 2, wherein the psicose solution has a concentration of 600-700g/L, the recombinant ribose-5-phosphate isomerase has a total cell wet cell mass of 50-100g/L, mnCl 2 has a mass of 0.5-1mM, the phosphate buffer has a mass of 20-50mM, and the phosphate buffer has a pH of 7-8.
  4. 4. A process for preparing D-allose as claimed in claim 1, wherein the centrifugation speed in step A is 5000-6000rpm, and the collected cells after centrifugation are re-suspended and washed with 50mM phosphate buffer solution and can be reused for the conversion of D-allose.
  5. 5. A process for preparing D-allose as claimed in claim 1, wherein the supernatant liquid obtained in step A is added with 3-5% v/v of activated carbon and 1-2% v/v of diatomite by volume of liquid, decolorized at 60-80 ℃ for 30-50min, filtered and collected to obtain decolorized liquid.
  6. 6. The process for preparing D-allose according to claim 1, wherein the flow rate of the cation resin column and the anion resin column in the step B is 1-3BV, and the electric conductivity of the obtained desalted liquid is <50us/cm; Before the cationic resin column is used, the cationic resin column is soaked in 4-5% wt of hydrochloric acid for 1-2 hours, then is washed with deionized water until the cationic resin column is neutral for standby, and before the anionic resin column is used, the cationic resin column is soaked in 4-5% wt of sodium hydroxide for 1-2 hours, and then is washed with deionized water until the cationic resin column is neutral for standby.
  7. 7. The process for preparing D-allose as claimed in claim 1, wherein the concentration conditions of the desalting solution in the step C and the separating solution in the step D are 50-70 ℃ and the vacuum degree is < -0.09Mpa.
  8. 8. The process of preparing D-allose as claimed in claim 1, wherein the chromatographic column in the step D has a height-diameter ratio of >10, the resin is filled with 50 ℃ water, the resin temperature is maintained by introducing the resin into the jacket of the chromatographic column, the concentrated solution I with the resin volume of 10% v/v is taken into the column, the flow rate is 1-1.5BV/h, 50 ℃ deionized water is used for eluting after the feeding is completed, the effluent liquid at the discharge port starts to be collected when the effluent liquid has solid content, the collection is stopped when the effluent liquid has solid content of 3.8-4.0% w/w, the separation liquid is separated, the chromatographic column is then subjected to top washing with deionized water until the solid content is zero, and the concentrated solution is again fed for separation.
  9. 9. A process for preparing D-allose as claimed in claim 1, wherein the seed crystals in step E are added in an amount of 0.5 to 1.5% w/w based on the solids in the concentrate II.
  10. 10. A process for the preparation of D-allose according to claim 1, characterized in that: And E, adding seed crystals into the feed liquid in the step, performing ultrasonic treatment at 20-40kHz for 30min, and then performing cooling crystallization.

Description

Preparation method of D-allose Technical Field The invention relates to the technical field of D-allose production, in particular to a preparation method of D-allose. Background Allose, also known as D-allose, is a aldohexose, a rare monosaccharide, and is the C-3 epimer of glucose. Is soluble in water but hardly soluble in methanol. It is currently known to be very small in nature, mainly isolated from leaves of african shrubs. Recent researches show that the rare saccharide D-allose has important application in the fields of food, health care, medicine and the like, and becomes a research hotspot in the rare saccharide due to the wide physiological functions. D-allose itself has an effect of inhibiting proliferation of cancer cells, which means that there will be a hope of developing a new drug without side effects. Chinese patent CN108473991a discloses a strain for producing allose from fructose and a method for producing allose using the strain, in which the conversion rate of allose is about 12-15%, and the components contain allose and allose, which increases the separation cost. Chinese patent CN112521429B discloses a method for extracting D-allose from nut shells by a microwave radiation method, which has complex process and needs to use microwave radiation, has certain potential safety hazard, uses diethyl ether as eluent during separation, and is harmful to human bodies. Chinese patent CN102839184a discloses a recombinant ribose-5-phosphate isomerase and its application, the recombinant enzyme catalyzes the ketoaldehyde isomerization reaction between D-psicose and D-psicose to produce D-psicose, the conversion rate after the reaction reaches equilibrium can reach 31%, but only stays in the upstream stage, and there is no mention of subsequent extraction, separation and crystallization. Therefore, it is important to find a safe, low-cost method for preparing high-purity D-allose. Disclosure of Invention Aiming at the defects existing in the prior art, the invention provides the preparation method of the D-allose, which has the advantages of high safety, low cost and high purity of the prepared D-allose. In order to solve the technical problems, the technical scheme of the invention is as follows: a method for preparing D-allose, comprising the following steps: D-allose conversion liquid is centrifugated and decolored, and then decolored liquid is collected; Desalting the decolorized solution by a cation resin column and an anion resin column respectively to obtain desalted solution; concentrating the desalted liquid to solid content of 50-60% w/w to obtain concentrated liquid I; separating the concentrated solution I by using a chromatographic column filled with calcium type cationic resin, and concentrating the separated solution to obtain concentrated solution II; E, adding ethanol with the volume of 0.5 times of the feed liquid into the concentrated solution II, stirring and fully mixing, adding seed crystals, cooling to 40 ℃ at the speed of 2-3 ℃ per hour, controlling the stirring rotation speed to be 30-80rpm, adding ethanol with the volume of 1.0 times of the concentrated solution II, preserving heat for 0.5-1h, cooling to 10-15 ℃ at the speed of 3-5 ℃ per hour, stopping crystallization, carrying out suction filtration, washing with ethanol to obtain white crystals, and drying to obtain D-allose with the purity of more than 99%. Preferably, the D-allose conversion solution in the step A is prepared by adding whole-cell wet thalli containing recombinant ribose-5-phosphate isomerase, mnCl 2 and phosphate buffer solution into an allose solution and converting at 60-80 ℃, wherein the whole-cell wet thalli of the recombinant ribose-5-phosphate isomerase is selected from the recombinant ribose-5-phosphate isomerase with the amino acid sequence of SEQ ID NO:2 in Chinese patent CN 102839184A. Preferably, the concentration of the psicose solution is 600-700g/L, the total cell wet bacterial body addition amount of the recombinant ribose-5-phosphate isomerase is 50-100g/L, the addition amount of MnCl 2 is 0.5-1mM, the addition amount of the phosphate buffer solution is 20-50mM, and the pH value of the phosphate buffer solution is 7-8. Preferably, the centrifugal rotation speed in the step A is 5000-6000rpm, and the thalli collected by centrifugation can be reused for the conversion of D-allose after being resuspended and washed by 50mM phosphate buffer solution, so that the conversion of multiple batches is realized. And (C) adding 3-5% of activated carbon and 1-2% of diatomite by volume of the liquid in the step (A), decoloring for 30-50min at 60-80 ℃, filtering, collecting decolored liquid, and removing impurities such as pigment, protein, macromolecular organic matters and the like in the liquid through decoloring. In the step B, the flow rate of the cation resin column and the anion resin column passing through the column is 1-3BV, and the conductivity of the obtained desalted liquid is less than 50