阅读说明：本技术 一种高结晶度水不溶性右旋糖酐及其制备和应用 (High-crystallinity water-insoluble dextran, and preparation and application thereof ) 是由 马江锋 姜岷 李进 吴昊 王寅竹 方艳 于 2020-12-22 设计创作，主要内容包括：本发明公开了一种高结晶度水不溶性右旋糖酐及其制备方法和应用。制备方法如下：使用右旋糖酐蔗糖酶合成5000-10000分子量的右旋糖酐再进行膜分离,收集截留液,对截留液进行干燥处理。将干燥得到的右旋糖酐溶解在去离子水中,控制pH至7.0-8.0,加入极少量果糖和蔗糖,通过旋转蒸发的方法浓缩体积至40-50mL,将浓缩液置于4oC下放置30小时,取出后离心,获得的沉淀烘干即为高结晶度水不溶性右旋糖酐。本方法操作简单,产品纯度较高,原料成本低,整个过程不涉及有害物质,制备所得右旋糖酐以α(1,6)键为主链,高结晶度且不溶于水,在医学和生物材料化学方面都巨大的应用潜力。(The invention discloses a high-crystallinity water-insoluble dextran, a preparation method and application thereof. The preparation method comprises the following steps: dextran with 5000-10000 molecular weight is synthesized by dextran sucrase, membrane separation is carried out, trapped fluid is collected, and drying treatment is carried out on the trapped fluid. Dissolving dextran obtained by drying in deionized water, controlling the pH value to 7.0-8.0, adding a very small amount of fructose and sucrose, concentrating the volume to 40-50mL by a rotary evaporation method, placing the concentrated solution at 4 ℃ for 30 hours, taking out the concentrated solution, centrifuging, and drying the obtained precipitate to obtain the high-crystallinity water-insoluble dextran. The method has the advantages of simple operation, high product purity, low raw material cost, no harmful substances involved in the whole process, high crystallinity and water insolubility of the prepared dextran by taking alpha (1, 6) bonds as main chains, and huge application potential in the aspects of medicine and biomaterial chemistry.)

1. The high-crystallinity water-insoluble dextran is characterized in that the dextran takes alpha (1, 6) bonds as a main chain, has high crystallinity and is insoluble in water.

2. The method for preparing high-crystallinity water-insoluble dextran of claim 1, comprising the steps of:

1) synthesizing dextran with molecular weight of 5000-10000 by using dextran sucrase to catalyze sucrose;

2) membrane separation: dissolving dextran, performing membrane separation, and collecting trapped fluid to obtain water-soluble 5000-10000-molecular-weight dextran;

3) drying the obtained trapped fluid;

4) preparing a dextran solution: dissolving dextran obtained by drying in water solution with pH of 7.0-8.0, and adding fructose and sucrose;

5) heating and concentrating treatment: heating the dextran solution obtained in the step 1) by using a rotary evaporator and concentrating the solution volume to 40-50mL by reduced pressure evaporation;

6) cooling and crystallizing: and (3) cooling the concentrated solution obtained in the step 2) for 30 hours at a low temperature for crystallization, centrifugally collecting the precipitate, and drying the precipitate at a high temperature to obtain the high-crystallinity water-insoluble dextran.

3. The method of claim 2, wherein: in the step 1), the pH value of the dextran sucrase is 5-6; the temperature is 20-40 ℃, and the preferred temperature is 25-35 ℃; the acting concentration of the dextran sucrase is 1-3U/mL, the acting time is 3-18h, and the preferable time is 4-6 h.

4. The method of claim 2, wherein: in the step 2), the membrane separation molecular weight cut-off is 3000-10000.

5. The method of claim 2, wherein: in the step 3), the drying treatment adopts freeze drying or spray drying.

6. The method of claim 2, wherein: in the step 4), the mass volume fraction of the dextran in the aqueous solution is 15%, the mass volume fraction of the fructose is 0.1%, and the mass volume fraction of the sucrose is 0.05%.

7. The method of claim 2, wherein: in the step 5), the evaporation temperature of the rotary evaporator is 60 ℃, and the pressure is 0.09-0.1 MPa.

8. The method of claim 2, wherein: in the step 6), the temperature for placing the dextran concentrated solution is 4 ℃.

9. The method of claim 2, wherein: in the step 6), the centrifugal rotation speed is 8000rpm/min, the centrifugal temperature is 10 ℃, the centrifugal time is 10 minutes, the drying temperature is 90 ℃, and the drying time is 2 hours.

10. Use of a high crystallinity water-insoluble dextran according to claim 1.

Technical Field

The invention relates to the field of medical technology and daily chemical products, in particular to high-crystallinity water-insoluble dextran and a preparation method and application thereof.

Background

The glucan is a general name of polymers taking glucose as a monomer, and is mainly high-molecular viscous polysaccharide generated in the sugar making process. Wherein dextran is a dextran having a main chain consisting essentially of D-glucopyranose residues linked by α - (1, 6) glycosidic linkages. Dextran may also have alpha- (1, 4), alpha- (1, 3) or alpha- (1, 2) branching bonds in the side chains, and typically the water solubility of dextran increases with increasing alpha- (1, 6) glycosidic linkages and decreases with increasing branching.

Dextran and its derivatives have wide application in pharmaceutical industry, 10⁴<Mw<10⁵Dextran, a molecular weight fragment of Da, is most suitable for clinical use as a plasma substitute; in the food industry, dextran is commonly used as a humectant, excipient, stabilizer, thickener, and the like, high molecular weight dextran (Mw = 1 × 10)⁶～2×10⁶Da) can be added to bread as a food additive to increase the softness and volume of bread and improve the texture of bread; it can also be used in fructose syrup and candy to prevent sucrose crystallization; in alkaline solution, high molecular weight dextran (Mw)> 10⁶Da) reacts with epichlorohydrin to generate cross-linked dextran, which is used for molecular sieves and as a filler of chromatographic columns to separate and purify biomacromolecules.

The insoluble dextran mostly contains more branched chains mainly comprising alpha (1, 3) bonds, and the water-soluble dextran is improved through chemical treatment in medicine, so that the dextran has the effects of resisting cytotoxicity and regulating immunity. The partially insoluble dextran can also be used as a mutagen of enzyme and a heavy metal adsorbent, and has wide application in the aspects of biomaterial chemistry.

Disclosure of Invention

The invention aims to provide high-crystallinity insoluble dextran taking alpha- (1, 6) bonds as a main chain and a preparation method thereof. The method is simple to operate, and alpha- (1, 6) insoluble dextran with higher purity can be obtained. The method comprises the following steps:

1) synthesizing dextran with molecular weight of 5000-10000 by using dextran sucrase to catalyze sucrose;

2) membrane separation: dissolving dextran, performing membrane separation, collecting trapped fluid to obtain water-soluble 5000-10000-molecular-weight dextran, and removing residual zymoprotein and fructose in the solution through membrane separation;

3) drying the trapped fluid obtained by membrane separation, wherein the dried powder state is beneficial to long-term preservation of dextran;

4) preparing a dextran solution: dissolving dextran in water solution of pH7.0-8.0, adding fructose and sucrose to prepare dextran solution with neutral pH value for crystallization;

5) heating and concentrating treatment: heating the dextran solution obtained in the step 4) by using a rotary evaporator, and concentrating the solution volume to 40-50mL by reduced pressure evaporation, wherein the lower the volume, the faster the crystallization speed is, but the lower the crystallization speed is not preferably lower than 40 mL;

6) cooling and crystallizing: and (3) cooling and crystallizing the concentrated solution obtained in the step 5) at a low temperature for 30 hours, centrifuging, collecting precipitate, and drying the precipitate at a high temperature to obtain the dextran with high crystallinity.

A small amount of the obtained dextran solid is taken, 50 times of water is added, and the solid is difficult to dissolve after shaking, so that the dextran prepared is high-crystallinity water-insoluble dextran.

In the step 1), the dextran sucrase has a pH value of 5-6, a temperature of 20-40 ℃, preferably a temperature of 25-35 ℃, an action concentration of 1-3U/mL, an action time of 3-18h, and preferably a time of 4-6 h.

In the step 2), the membrane separation molecular weight cut-off is 3000-10000.

In the step 3), the drying treatment adopts freeze drying or spray drying.

In the step 4), the mass volume fraction of the dextran in the aqueous solution is 15%, the mass volume fraction of the fructose is 0.1%, and the mass volume fraction of the sucrose is 0.05%.

In the step 5), the evaporation temperature of the rotary evaporator is 60 ℃, and the pressure of the rotary evaporator is 0.09-0.1 MPa.

In the step 6), the temperature for placing the dextran concentrated solution is 4 ℃, the centrifugal rotation speed is 8000rpm/min, the centrifugal temperature is 10 ℃, the centrifugal time is 10 minutes, the precipitation drying temperature is 90 ℃, and the drying time is 2 hours.

The beneficial expenditures of the invention are: the preparation method has the advantages of simple operation, high product purity, low equipment investment and low raw material cost, does not relate to harmful substances in the whole process, and can avoid the damage to human bodies and equipment in the production process. The method comprises the steps of producing low molecular weight dextran through dextransucrase catalysis, adding extremely low amount of fructose and sucrose into low molecular weight dextran solution, concentrating to high concentration, cooling and crystallizing, and has high purity and yield. The prepared dextran takes alpha (1, 6) bond as a main chain, can be used for preparing anti-cytotoxic drugs and immunoregulation drugs, can also be used as biological materials such as mutagens of enzymes and heavy metal adsorbents, and has wide application prospect.

Drawings

FIG. 1: infrared absorption spectrum of high crystallinity dextran.

FIG. 2: infrared absorption spectrum of dextran obtained by spray drying.

FIG. 3: x-ray diffraction pattern of high crystallinity dextran.

FIG. 4: x-ray diffraction pattern obtained by spray drying.

Detailed Description

The technique of the present invention is further illustrated by the following examples.

Example 1

200g of sucrose was dissolved in 0.8L of deionized water, and 200mL of 1U/mL dextran sucrase solution was added. And (3) adjusting the pH value of the conversion reaction system to 5.3, setting the temperature to be 30 ℃ and reacting for 4 h. After the reaction is finished, the conversion solution is purified by using membranes with molecular weights of 1000 and 3000, the obtained trapped fluid is collected, dextran with molecular weight of 5000-. 3300-3400 cm on dextran infrared spectrogram obtained by spray drying^-1And 2800-2900 cm^-1The peak at (A) demonstrates that the sample is dextran, 916cm^-1And 857cm^-1The peaks indicate that the glycosidic linkages are predominantly α (1, 6) linkages. The spectrum of XRD shows that the dextran obtained by spray drying has no strong absorption peak and is mainly in amorphous form. 15g of water-soluble dextran was weighed to dissolve in 100mL of deionized water, the pH of the solution was measured to be 7.2, 0.1g of fructose and 0.05g of sucrose were weighed, added to the dextran solution and stirred until all dissolved. The dextran solution is heated by a rotary evaporator and the volume of the solution is concentrated to 45mL by reduced pressure evaporation, the evaporation temperature of the rotary evaporator is set to 60 ℃, and the pressure is 0.09 MPa.

And (3) placing the solution subjected to evaporation concentration in a refrigerator with the temperature of 4 ℃, observing crystal generation after 3 hours, taking the solution out of the refrigerator with a large crystal generation amount after 30 hours, centrifuging the solution at 10 ℃ and 8000rpm/min for 10 minutes, pouring out supernate, collecting precipitate, and placing the precipitate in an oven with the temperature of 90 ℃ for drying for 2 hours to obtain dextran solid.

The obtained dextran solid was weighed at 2g and put into 100mL of pure water, and the solid was found to be difficult to dissolve by shaking. The sample is subjected to infrared and X-ray diffraction analysis, and the spectra are shown in figures 1 and 3. The result shows that the infrared spectrum is similar to the standard alpha (1, 6) dextran, the characteristic peak of the finished product shows that the glycosidic bond is alpha (1, 6) configuration, and the product is mainly alpha (1, 6) dextran. A plurality of high-intensity crystallization peaks appear in an X-ray diffraction pattern, and the generated dextran solid has high crystallinity. The higher concentration and temperature conditions during the formation of this highly crystalline dextran demonstrate that the phenomenon of self-assembly that occurs with hydrogen bonds as the main driving force is the main reason for its formation.

Example 2

200g of sucrose was dissolved in 0.8L of deionized water, and 200mL of 1.5U/mL dextran sucrase solution was added. And (3) adjusting the pH value of the conversion reaction system to 5.5, setting the temperature to be 30 ℃ and reacting for 4 h. And after the reaction is finished, 10000 and 3000 molecular weight membranes are used for purifying the conversion solution, the obtained trapped fluid is collected, and the dextran with the molecular weight of 5000-10000 is obtained by spray drying. 15g of water-soluble dextran was weighed to dissolve in 100mL of deionized water, the pH of the solution was measured to be 7.5, 0.1g of fructose and 0.05g of sucrose were weighed, added to the dextran solution and stirred until all dissolved. The dextran solution is heated by a rotary evaporator and the volume of the solution is concentrated to 50mL by reduced pressure evaporation, the evaporation temperature of the rotary evaporator is set to 60 ℃, and the pressure is 0.09 MPa.

And (3) placing the solution subjected to evaporation concentration in a refrigerator at 4 ℃, observing crystal generation after 3.5 hours, obtaining more crystal generation after 30 hours, taking out the solution from the refrigerator, centrifuging the solution at 10 ℃ and 8000rpm/min for 10 minutes, pouring out a supernatant, collecting a precipitate, and placing the precipitate in an oven at 90 ℃ to dry for 2 hours to obtain a dextran solid.

The obtained dextran solid was weighed at 2g and put into 100mL of pure water, and the solid was found to be difficult to dissolve by shaking. And (4) carrying out infrared spectrum and X-ray diffraction spectrum analysis on the sample. The result shows that the infrared spectrum is similar to the standard alpha (1, 6) dextran, the characteristic peak of the finished product shows that the glycosidic bond is alpha (1, 6) configuration, and the product is mainly alpha (1, 6) dextran. A plurality of high-intensity crystallization peaks appear in an X-ray diffraction pattern, and the generated dextran solid has high crystallinity.

Example 3

200g of sucrose was dissolved in 0.8L of deionized water, and 200mL of 3U/mL dextran sucrase solution was added. And (3) adjusting the pH value of the conversion reaction system to 6, setting the temperature to be 35 ℃ and reacting for 6 h. And after the reaction is finished, 10000 and 3000 molecular weight membranes are used for purifying the conversion solution, the obtained trapped fluid is collected, and the dextran with the molecular weight of 5000-10000 is obtained by spray drying. 30g of water-soluble dextran is weighed and dissolved in 200mL of deionized water, the pH of the solution is measured to be 7.5, 0.2g of fructose and 0.1g of sucrose are weighed and added into the dextran solution and stirred until all the dextran is dissolved. The dextran solution is heated by a rotary evaporator, the volume of the solution is concentrated to 50mL by reduced pressure evaporation, the evaporation temperature of the rotary evaporator is set to 60 ℃, and the pressure is 0.1 MPa.

And (3) placing the solution subjected to evaporation concentration in a refrigerator at 4 ℃, observing crystal generation after 3.5 hours, obtaining more crystal generation after 30 hours, taking out the solution from the refrigerator, centrifuging the solution at 10 ℃ and 8000rpm/min for 10 minutes, pouring out a supernatant, collecting a precipitate, and placing the precipitate in an oven at 90 ℃ to dry for 2 hours to obtain a dextran solid.

The obtained dextran solid was weighed into pure water and shaken to find that the solid was difficult to dissolve. And (4) carrying out infrared spectrum and X-ray diffraction spectrum analysis on the sample. The result shows that the infrared spectrum is similar to the standard alpha (1, 6) dextran, the characteristic peak of the finished product shows that the glycosidic bond is alpha (1, 6) configuration, and the product is mainly alpha (1, 6) dextran. A plurality of high-intensity crystallization peaks appear in an X-ray diffraction pattern, and the generated dextran solid has high crystallinity.