Method for high-yield preparation of icodextrin bulk drug by single enzyme system

Abstract

The invention relates to a method for high-yield preparation of an icodextrin bulk drug by a single enzyme system, which comprises the following steps: gelatinizing starch at high temperature, adding alpha-thermoamylase for hydrolysis, carrying out ultrafiltration separation on primary enzymatic hydrolysate to obtain high-molecular-weight dextrin and alpha-thermoamylase, returning the high-molecular-weight dextrin and the alpha-thermoamylase to a gelatinization tank for secondary enzymolysis, and combining ultrafiltration permeates of the secondary enzymatic hydrolysate and the primary enzymatic hydrolysate, and performing molecular weight screening by ultrafiltration to obtain icodextrin of which the weight-average molecular weight and the number-average molecular weight simultaneously meet the requirements of alpha-1, 6 glucosidic bonds. On the basis of a traditional icodextrin manufacturing process, the working procedures of recovering macromolecular dextrin and performing secondary enzymolysis are added, and the yield can reach 85-91%. Particularly, new amylase does not need to be additionally added in the secondary enzymolysis, the original amylase is continuously used, new equipment is not added, the production cost increase is small, and the preparation efficiency is high.

Description

Technical Field

[0001] The invention belongs to the technical field of drug preparation, and particularly relates to a method for preparing icodextrin raw material medicine in high yield using a single enzyme system. Background Art

[0002] Peritoneal dialysis (PD) is an important renal replacement therapy, mainly suitable for patients with acute renal injury, chronic renal failure, etc., and is also used for acute drug and poison poisoning. Icodextrin, as a commonly used new type of peritoneal dialysis fluid osmotic agent, uses colloidal osmotic pressure difference for ultrafiltration, and has significant advantages in improving ultrafiltration effect, protecting the structure and function of the peritoneum, and reducing related complications. Icodextrin is glucose connected by α-1,4 glycosidic bonds and less than 10% α-1,6 glycosidic bonds. The weight average molecular weight is 13000-19000Da, and the number average molecular weight is 5000-6500Da. Compared with glucose peritoneal dialysis fluid, it can reduce the risk of congestive heart failure in patients and reduce the impact on blood sugar. Chinese patent CN 112979831 B discloses a method for efficiently preparing the raw material drug icodextrin, which includes starch gelatinization, acid hydrolysis, decolorization and ultrafiltration steps in sequence. First, starch, insoluble organic particles and water are stirred and gelatinized at 90-100°C, then the temperature is controlled at 85-100°C, and a pre-prepared acid solution is slowly added to react. After acid hydrolysis, activated carbon and chitosan are used for decolorization 1-2 times at room temperature between pH 3 and 4, and finally, ultrafiltration membranes of different molecular weights are used for molecular weight screening. When acid hydrolysis of starch is used to prepare icodextrin, the conditions are severe, and the molecular weight distribution range obtained by hydrolysis is large. When molecular weight screening is performed by ultrafiltration, the loss is large, resulting in a reduced yield.

[0003] Chinese patent CN 106397616A discloses a method for preparing icodextrin for starch-based peritoneal dialysis fluid, which includes starch gelatinization, enzymolysis, debranching, and ultrafiltration purification steps in sequence. First, starch is prepared into a starch solution with a phosphate buffer solution, and gelatinized at high temperature, and α-amylase is added to hydrolyze the starch at a temperature of 70 to 95°C, and then the enzymolysis solution is debranched by a debranching enzyme at a pH of 5.5 to 6.5 and 40 to 60°C. After the enzyme is inactivated at high temperature, the enzymolysis solution is subjected to alcohol precipitation, centrifugal separation, washing and drying steps, and finally ultrafiltration and gel chromatography columns are used for separation and purification to obtain icodextrin that meets the requirements. The enzymatic method for preparing icodextrin uses α-amylase, debranching enzyme, etc. for reaction, and the reaction process is difficult to control and the operation is complicated, which is not suitable for industrial scale-up.

[0004] Chinese patent CN 117384305 A discloses a preparation process of icodextrin raw material, which combines acid hydrolysis and enzymatic debranching. The method includes starch gelatinization, acid hydrolysis, debranching, ultrafiltration, decolorization, concentration and drying steps in sequence. First, the starch raw material is gelatinized at high temperature, then acid is added to the gelatinized liquid for hydrolysis, then the debranching enzyme is used to cut the branches, the enzyme is inactivated and filtered and concentrated, then sodium hypochlorite decolorization, activated carbon adsorption and decolorization are performed, and finally the icodextrin decolorized filtrate is removed by impurities and molecular weight screening using an ultrafiltration membrane combination, and the icodextrin finished product is obtained by reduced pressure concentration and drying.

[0005] In fact, the α-1,6 glycosidic bond content of most corn starches is within the range specified by icodextrin, and there is no need to use a debranching enzyme to debranch. Therefore, the use of a debranching enzyme in the above two invention processes increases production costs and has no technical significance. Chinese patent CN 114605563 B discloses a single enzyme system preparation method for icodextrin raw materials for peritoneal dialysis fluid, which includes enzymatic hydrolysis, ultrafiltration, and spray drying steps. First, the starch solution is gelatinized at high temperature, α-high temperature amylase is added for enzymatic hydrolysis, and then the pH is adjusted with acid to inactivate the enzyme under high temperature conditions. After the obtained enzymatic hydrolyzate is filtered through diatomaceous earth, the molecular weight is screened using ultrafiltration membranes of different pore sizes, and finally the ultrafiltration liquid is sprayed dry to obtain icodextrin for peritoneal dialysis fluid. Icodextrin is prepared by a single enzyme system through a single enzymatic hydrolysis process, and some starch is not completely hydrolyzed, resulting in losses during molecular weight screening in the ultrafiltration process, resulting in a reduction in product yield.

[0006] It can be seen from the above processes that in the existing processes, the separated macromolecular dextrins are all discarded and not recycled, which causes great waste and significantly reduces the dextrin yield. Although these macromolecular dextrins exceed the molecular weight range of icodextrin, they cannot be used directly in icodextrin. How to reduce waste, can they be recycled and reused? Therefore, this team tried to recycle these macromolecular dextrins and then enzymolyze them again. The results showed that most dextrins can be reused. However, a new problem has emerged. The enzyme added for re-enzymolysis increases the production cost and partially offsets the advantage of recycling dextrin. How to solve the new problem? This team continued to research and develop, and finally thought that the original enzyme could be directly used for secondary enzymolysis. Moreover, no new process equipment was added, and the existing equipment was directly used. Finally, the present invention proposes an innovative process for the problems of large waste of raw materials and low dextrin yield in the existing dextrin production process, using the original α-amylase in the enzymolysis reaction to enzymolyze the separated macromolecular dextrin again so that its molecular weight meets the requirements of the molecular weight range of icodextrin, thereby greatly improving the icodextrin yield. Summary of the invention

[0007] The object of the present invention is to provide a method for preparing icodextrin raw materials for peritoneal dialysis fluid in a high yield with a single enzyme system with high yield and simple process, using an α-high temperature amylase, undergoing two enzymolysis steps, combined with ultrafiltration and spray drying processes to obtain icodextrin, thereby improving starch utilization and icodextrin yield, having high preparation efficiency, simple operation, and being suitable for industrial scale-up. The present invention uses gel permeation chromatography (GPC) to detect the weight average molecular weight and number average molecular weight of the prepared icodextrin product; using 1 H-NMR was used to determine the content of α-1,6 glycosidic bonds in the product. The weight average molecular weight, number average molecular weight, and the ratio of α-1,4 glycosidic bonds to α-1,6 glycosidic bonds of the icodextrin product obtained by the preparation method of the present invention all met the requirements of the quality standard of icodextrin raw materials.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A method for preparing icodextrin raw material in high yield using a single enzyme system comprises the following steps:

[0010] (1) primary enzymolysis: in a starch gelatinization tank, starch is prepared with 15-35° C. purified water to form a starch slurry solution with a mass concentration of 5%-30%, calcium chloride is added, and an aqueous solution of alkali is added to adjust the pH value to 5.4-7.4, the mixture is heated to 85-100° C. and maintained at the temperature for 30-60 minutes to obtain a gelatinized solution, α-high temperature amylase is added for enzymolysis for 40-70 minutes to obtain an enzymolysis solution 1, and the enzymolysis solution 1 is filtered through diatomaceous earth while hot to obtain a filtrate 1;

[0011] (2) Secondary enzymolysis: The filtrate 1 obtained in step (1) is separated into macromolecular dextrin and α-high temperature amylase using a large-pore ultrafiltration membrane, i.e., the retentate 1, and the permeate is the filtrate 2. The retentate 1 is returned to the starch gelatinization tank, heated to 80-95° C., and continued to react for 20-30 minutes to obtain the enzymolysis solution 2, which is combined with the filtrate 2, and the pH value is adjusted to 2.0-4.0 using acid, and the high temperature is maintained for 10-20 minutes to inactivate the enzyme, and then treated with a large-pore ultrafiltration membrane to obtain the permeate.

[0012] (3) ultrafiltration: the permeate obtained in step (2), i.e., filtrate 3, is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 1000Da-2000Da to obtain a retentate 2 having a weight average molecular weight of 13000Da-19000Da, a number average molecular weight of 5000Da-6500Da, and a molar ratio of α-1,6 glycosidic bonds of less than 10%;

[0013] (4) Spray drying: spray drying the retentate 2 obtained by ultrafiltration to obtain icodextrin for peritoneal dialysis fluid, with a yield of 85-91%;

[0014] In order to better achieve the purpose of the present invention, wherein:

[0015] Preferably, in step (1), the starch is one of corn starch, wheat starch, sweet potato starch and potato starch.

[0016] Preferably, in step (1), the starch slurry solution has a mass concentration of 5%-10%.

[0017] Preferably, in step (1), the amount of calcium chloride added is 10-80 ppm per gram of starch slurry.

[0018] Preferably, in step (1), the dosage of the α-high temperature amylase is 4-12 U per gram of dry starch.

[0019] Preferably, in step (2), the large-pore ultrafiltration membrane has a molecular weight cut-off range of 40KD-60KD.

[0020] Preferably, in step (3), the ultrafiltration pressure is 0.2-0.6 MPa.

[0021] Preferably, in step (3), the weight average molecular weight and number average molecular weight are determined using gel permeation chromatography (GPC), and the proportion of α-1,6 glycosidic bonds is determined by 1 H-NMR determination.

[0022] Preferably, in step (4), the spray drying operation is performed at: a feed temperature of 90-100°C, an air inlet temperature of 190-210°C, and an air outlet temperature of 110-130°C.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] 1. After the first enzymolysis, the present invention separates high molecular weight dextrin and α-high temperature amylase, and then uses the separated α-high temperature amylase to perform secondary enzymolysis on the separated macromolecular dextrin, which greatly improves the utilization rate of starch and the product yield, and the yield can be as high as 85-91%. However, the icodextrin production process without the secondary enzymolysis of the present invention has an icodextrin yield of only 50-65%. In particular, under the process cleverly designed by the present invention, the secondary enzymolysis continues to use the original enzyme, the production cost increases less, and the preparation efficiency is high.

[0025] 2. The present invention adopts a bio-enzyme method to hydrolyze the starch raw material. Compared with the traditional acid method, the reaction conditions are milder, the hydrolysis has a certain selectivity for the glycosidic bonds in the starch, and the degree of hydrolysis of the reaction is easier to control, thereby improving the preparation efficiency of icodextrin.

[0026] 3. The present invention adopts a single enzyme system to prepare icodextrin. The starch raw material is hydrolyzed only by α-high-temperature amylase. The degree of enzymatic hydrolysis of α-1,4 glycosidic bonds and α-1,6 glycosidic bonds is controlled by combining parameters such as pH, temperature, enzyme dosage, and enzymatic hydrolysis time to obtain the target product. The preparation efficiency is high, the process is simple, and it is suitable for industrial scale-up. DETAILED DESCRIPTION

[0027] In order to better understand the present invention, the present invention is further described below in conjunction with the examples. The present invention has many successful embodiments, and specific embodiments are listed below, but the scope of protection claimed by the present invention is not limited to the scope described in the embodiments. The molecular weight distribution range of the obtained icodextrin product was determined by gel permeation chromatography (GPC)-multi-angle laser light scattering detector (MALS)-differential refractive index detector (RI). The specific measurement parameters are: the chromatographic column is a hydrophilic gel chromatographic column with an exclusion limit of 2000Da and an exclusion limit of 50000Da connected in series, the mobile phase is ultrapure water (standby after 20 minutes of ultrasound), the column temperature is 35°C; the flow rate is 0.8mL / min, and the detector temperature is 35°C.

[0028] use 1 The weight average molecular weight and number average molecular weight of the obtained icodextrin product were determined by H-NMR. The specific measurement parameters were: deuterated H2O as solvent and the measurement temperature was 25°C. The chemical shift corresponding to the α-1,4 glycosidic bond was 5.14ppm, and the chemical shift corresponding to the α-1,6 glycosidic bond was 4.78ppm. DETAILED DESCRIPTION

[0030] Example 1

[0031] The specific operation steps of the present invention are as follows:

[0032] (1) Primary enzymatic hydrolysis. At room temperature, in a starch gelatinization tank, corn starch was prepared into a starch slurry with 500 mL of purified water at a mass percentage concentration of 10%. After stirring evenly, 13.75 mg of anhydrous calcium chloride was added, and 0.2 M NaOH solution was added dropwise to adjust the pH to 6.8. After the temperature was maintained at 93°C and the gelatinization was stirred for 40 minutes, 19.7 mg of α-high temperature amylase (Novozymes) was added, and after enzymatic hydrolysis for 50 minutes, an enzymatic hydrolyzate 1 was obtained. The enzymatic hydrolyzate 1 was filtered through diatomaceous earth while hot to obtain 480 mL of filtrate 1.

[0033] (2) Secondary enzymatic hydrolysis. The filtrate 1 obtained in step (1) is first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 50KD at an ultrafiltration pressure of 0.3MPa to separate the macromolecular dextrin and enzyme, namely the retentate 1, and the permeate is the filtrate 2. The retentate 1 is returned to the starch gelatinization tank and the reaction is continued at 85°C for 30 minutes to obtain the enzymatic hydrolyzate 2 of the secondary reaction, which is combined with the filtrate 2, and a 0.5M hydrochloric acid solution is added dropwise to adjust the pH value to 3.0. The mixture is heated to boiling and maintained at this temperature for 20 minutes to inactivate the enzyme, and then ultrafiltered using an ultrafiltration membrane with a pressure of 0.3MPa to remove the macromolecules, and the mixture is washed with 300mL of purified water for 3 times, and the permeates are combined;

[0034] (3) Separation of small molecule dextrin. The permeate obtained in step (2), i.e., filtrate 3, is used to separate small molecules using an ultrafiltration membrane with a pressure of 0.3 MPa and a molecular weight cutoff of 1500 Da. After the volume of the feed liquid is concentrated to 200 mL, 200 mL of purified water is added for filtration twice. When the volume is concentrated to 200 mL again, ultrafiltration is stopped to obtain retentate 2.

[0035] (4) Spray drying. The parameters were set as follows: feed temperature of 95°C, air inlet temperature of 200°C, and air outlet temperature of 120°C. The obtained retentate 2 was spray dried to obtain the finished product icodextrin. The calculated yield after drying was 85.4%.

[0036] The product obtained in Example 1 was tested and found that the proportion of α-1,6 glycosidic bonds was 3.8%, the proportion of α-1,4 glycosidic bonds was 96.2%, and the weight-average molecular weight distribution was as follows: less than 1638 was 4.3%, 1638-5000 was 14.6%, 5000-20000 was 54.4%, 20000-45000 was 21.3%, and 1638-45000 was 90.3%, which met the quality standards of icodextrin.

[0037] Example 2

[0038] (1) Primary enzymatic hydrolysis. At room temperature, in a starch gelatinization tank, corn starch was prepared with 5 L of purified water into a starch slurry with a mass percentage concentration of 8%. After stirring evenly, 134.4 mg of anhydrous calcium chloride was added, and 0.2 M NaOH solution was added dropwise to adjust the pH to 7.0. After the temperature was maintained at 90°C and the gelatinization was stirred for 50 minutes, 173.6 mg of α-high temperature amylase (Novozymes) was added. After enzymatic hydrolysis for 50 minutes, an enzymatic hydrolyzate 1 was obtained. The enzymatic hydrolyzate 1 was filtered through diatomaceous earth while hot to obtain 4850 mL of filtrate 1.

[0039] (2) Secondary enzymatic hydrolysis. The filtrate 1 obtained in step (1) is first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 50KD at an ultrafiltration pressure of 0.3MPa to separate the macromolecular dextrin and α-high-temperature amylase, namely the retentate 1, and the permeate is the filtrate 2. The retentate 1 is returned to the starch gelatinization tank and the reaction is continued at 92°C for 25 minutes to obtain the enzymatic hydrolyzate 2 of the secondary reaction, which is combined with the filtrate 2, and a 1M hydrochloric acid solution is added dropwise to adjust the pH value to 3.0. The mixture is heated to boiling and maintained at this temperature for 20 minutes to inactivate the enzyme, and then ultrafiltered using an ultrafiltration membrane with a pressure of 0.3MPa and a 50KD ultrafiltration membrane to remove the macromolecules. The mixture is washed with 3000mL of purified water for 3 times, and the permeates are combined;

[0040] (3) Separation of small molecule dextrin. The permeate obtained in step (2), i.e., filtrate 3, is ultrafiltered to separate small molecules using an ultrafiltration membrane with a pressure of 0.3 MPa and a molecular weight cutoff of 1500 Da. After the volume of the feed liquid is concentrated to 500 mL, 500 mL of purified water is added for two washes. When the volume is concentrated to 500 mL again, ultrafiltration is stopped to obtain retentate 2.

[0041] (4) Spray drying. The parameters were set as follows: feed temperature of 95°C, air inlet temperature of 200°C, and air outlet temperature of 120°C. The obtained retentate 2 was spray dried to obtain the finished product icodextrin. The calculated yield after drying was 91%.

[0042] The product obtained in Example 2 was tested and found that the proportion of α-1,6 glycosidic bonds was 4.2%, the proportion of α-1,4 glycosidic bonds was 95.8%, and the weight-average molecular weight distribution was as follows: less than 1638 was 5.9%, 1638-5000 was 15.0%, 5000-20000 was 56.6%, 20000-45000 was 20.3%, and 1638-45000 was 91.9%, which met the quality standards of icodextrin.

[0043] Example 3

[0044] (1) Primary enzymatic hydrolysis. At room temperature, in a starch gelatinization tank, corn starch was prepared into a starch slurry with 500 mL of purified water at a mass percentage concentration of 15%. After stirring evenly, 14.67 mg of anhydrous calcium chloride was added, and 0.2 M NaOH solution was added dropwise to adjust the pH to 6.8. After the temperature was maintained at 93°C and the gelatinization was stirred for 40 minutes, 31.2 mg of α-high temperature amylase (Novozymes) was added, and after enzymatic hydrolysis for 50 minutes, an enzymatic hydrolyzate 1 was obtained. The enzymatic hydrolyzate was filtered through diatomaceous earth while hot to obtain 475 mL of filtrate 1.

[0045] (2) Secondary enzymatic hydrolysis. The filtrate 1 obtained in step (1) is first ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 50KD at an ultrafiltration pressure of 0.3MPa to separate the macromolecular dextrin and α-high-temperature amylase, namely the retentate 1, and the permeate is the filtrate 2. The retentate 1 is returned to the starch gelatinization tank and the reaction is continued at 95°C for 20 minutes to obtain the enzymatic hydrolyzate 2 of the secondary reaction, which is combined with the filtrate 2, and a 0.5M hydrochloric acid solution is added dropwise to adjust the pH value to 3.0. The mixture is heated to boiling and maintained at this temperature for 20 minutes to inactivate the enzyme, and then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 50KD at an ultrafiltration pressure of 0.3MPa to remove the macromolecules, and the mixture is washed with 300mL of purified water for 3 times, and the permeates are combined;

[0046] (3) Separation of small molecule dextrin. The permeate obtained in step (2), i.e., filtrate 3, is ultrafiltered to separate small molecules using an ultrafiltration membrane with a pressure of 0.3 MPa and a molecular weight cutoff of 1500 Da. After the volume of the feed liquid is concentrated to 200 mL, 200 mL of purified water is added for 2 washes. When the volume is concentrated to 200 mL again, ultrafiltration is stopped to obtain retentate 2.

[0047] (4) Spray drying. The parameters were set as follows: feed temperature of 95°C, air inlet temperature of 200°C, and air outlet temperature of 120°C. The obtained retentate 2 was spray dried to obtain the finished product icodextrin. The calculated yield after drying was 88.6%.

[0048] The product obtained in Example 3 was tested and found that the proportion of α-1,6 glycosidic bonds was 4.7%, the proportion of α-1,4 glycosidic bonds was 95.3%, and the weight-average molecular weight distribution was as follows: less than 1638 was 6.1%, 1638-5000 was 14.8%, 5000-20000 was 57.1%, 20000-45000 was 18.9%, and 1638-45000 was 90.8%, which met the quality standards of icodextrin.

[0049] Comparative Example 1

[0050] Except for the secondary enzymolysis, all other reaction conditions and operation steps are the same as those in Example 1. That is, in Comparative Example 1, the macromolecular dextrin is no longer recovered and subjected to secondary enzymolysis. The molecular weight distribution and α-1,6 glycosidic bond content of the obtained icodextrin meet the quality standards of icodextrin. The yield is 51%. Since the macromolecular dextrin is not recovered and re-enzymolyzed, this part of dextrin is wasted, so the yield of icodextrin is greatly reduced.

[0051] Comparative Example 2

[0052] Except for the secondary enzymolysis, all other reaction conditions and operation steps are the same as those in Example 2. That is, in Comparative Example 2, the macromolecular dextrin is no longer recovered and subjected to secondary enzymolysis. The molecular weight distribution and α-1,6 glycosidic bond content of the obtained icodextrin meet the quality standards of icodextrin. The yield is 64%. Since the macromolecular dextrin is not recovered and re-enzymolyzed, this part of dextrin is wasted, so the yield of icodextrin is greatly reduced.

Claims

1. A method for preparing icodextrin raw materials in high yield using a single enzyme system, characterized in that: The following steps are involved: (1) Enzymatic hydrolysis: In a starch gelatinization tank, starch is prepared into a starch slurry solution with a mass concentration of 5%-30% by using room temperature purified water, calcium chloride is added, and an aqueous solution of alkali is added to adjust the pH value to 5.4-7.4, the mixture is heated to 85-100° C. and maintained at the temperature for gelatinization for 30-60 minutes with stirring, α-high temperature amylase is added for enzymatic hydrolysis for 40-70 minutes to obtain an enzymatic hydrolyzate 1, and the enzymatic hydrolyzate 1 is filtered through diatomaceous earth while hot to obtain a filtrate 1; (2) Secondary enzymolysis: The filtrate 1 obtained in step (1) is ultrafiltered using an ultrafiltration membrane to separate the macromolecular dextrin and α-high-temperature amylase, i.e., the retentate 1, and the permeate is the filtrate 2; the retentate 1 is returned to the starch gelatinization tank, heated to 80-95° C., and continued to react for 20-30 minutes to obtain the enzymolysis solution 2, which is combined with the filtrate 2, and the pH value is adjusted to 2.0-4.0 using acid, and the high temperature is maintained for 10-20 minutes to inactivate the enzyme, and then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 50KD; (3) ultrafiltration: the permeate obtained in step (2), i.e., filtrate 3, is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 1000Da-2000Da to obtain a retentate 2 having a weight average molecular weight of 13000Da-19000Da, a number average molecular weight of 5000Da-6500Da, and a molar ratio of α-1,6 glycosidic bonds of less than 10%; (4) Spray drying: The retentate 2 obtained by ultrafiltration is spray dried to obtain icodextrin for peritoneal dialysis solution.