Method for preparing high-yield icodextrin by combining ultrasonic field with single enzyme system

Abstract

The invention provides a preparation method of high-yield icodextrin by combining an ultrasonic field with a single enzyme system. The preparation method comprises the following steps: S1, enzymolysis; s2, performing primary ultrafiltration; s3, ultrasonic hydrolysis; s4, carrying out secondary ultrafiltration; and S5, spray drying. According to the method, firstly, the proportion of dextrin with the molecular weight smaller than 1638 Da in the enzymatic hydrolysate is controlled to be not larger than 7%, the working procedures of recycling macromolecular dextrin and conducting ultrasonic hydrolysis are added, and the yield can reach 80-88%. New amylase does not need to be additionally added in ultrasonic hydrolysis, the hydrolysis time is greatly shortened, the pertinence is high, ultrasonic hydrolysis after enzyme deactivation belongs to physical modification, the reaction degree is conveniently controlled, the generation proportion of dextrin with the molecular weight smaller than 1638 Da is accurately controlled, the production cost is low, the preparation efficiency is high, the consistency of a hydrolysis product and an original product is good, and the method is suitable for industrial production. The generation of 5-hydroxymethylfurfural is completely eradicated, so that the peritoneal dialysis can be continuously carried out.

Description

A method for preparing high-yield icodextrin using an ultrasonic field combined with a single enzyme system Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a method for preparing high-yield icodextrin using an ultrasonic field combined with a single enzyme system. Background Technology

[0002] Icodextrin PD solution is widely used clinically due to its suitability for long-term ultrafiltration, improvement of metabolic disorders, and high biocompatibility. As a core component of peritoneal dialysis fluid, icodextrin achieves ultrafiltration through colloid osmotic pressure, replacing traditional glucose dialysis fluid and reducing metabolic complications (such as hyperglycemia and peritoneal fibrosis). Icodextrin is composed of glucose linked by α-1,4 glycosidic bonds and less than 10% α-1,6 glycosidic bonds, with a weight-average molecular weight of 13,000-19,000 Da and a number-average molecular weight of 5,000-6,500 Da. Its molecular weight distribution and branching degree (α-1,6 glycosidic bonds <10%) are key parameters affecting efficacy and safety.

[0003] Chinese patent CN103467608A discloses icodextrin and its preparation method. The method includes the following steps: acid hydrolysis of starch slurry by adding acid and heating; monitoring the viscosity of the hydrolysate using an Ubbelohde viscometer; ultrafiltration; and spray drying. First, starch and acid solution are stirred and hydrolyzed at 70–93°C. The reaction process is monitored. When the outflow time of the reaction solution, measured by an Ubbelohde viscometer with a capillary inner diameter of 0.9–1.0 mm, is 2–4 min, the reaction solution is neutralized to pH 7 with an alkaline solution to terminate the hydrolysis reaction, yielding the product. The reaction time is 0.5–4 h. This acid-hydrolyzed starch preparation method directly adds acid and heats for hydrolysis without gelatinization. The conditions are harsh, the hydrolysis time is long, and the resulting molecular weight distribution is wide. Significant losses occur during molecular weight screening via ultrafiltration, leading to a reduced yield. Under prolonged high-temperature reaction conditions, strong acid may promote the formation of small amounts of aldoses or anhydrides from the starch, which is detrimental to impurity control.

[0004] Chinese patent CN106755199A discloses a method for preparing icodextrin, which includes the following steps: ultrasonic washing, adding lysozyme, centrifugation washing, and drying; debranching enzyme hydrolysis, followed by α-amylase hydrolysis, ultrasonic treatment with modified activated carbon, and ultrafiltration. First, starch is adjusted to a concentration of 5-40 wt% with water at 20-40℃, ultrasonically washed for 10-20 minutes, then lysozyme is added, and the mixture is stirred for 1-6 hours, followed by centrifugation, washing, and drying. Then, the starch is added to water at 40-80℃ for debranching enzyme treatment, stirred for 1-5 hours, then heated to 80-100℃, a certain amount of calcium chloride and α-amylase are added, and the mixture is stirred for 1-3 hours. Acid is added to inactivate the enzyme, and a certain amount of sodium hydroxide solution is added to adjust the pH to 5.0-7.0. Ultrafiltration is performed, the filtrate is treated with activated carbon and ultrasonically, the carbon is removed by filtration, and the filtrate is spray-dried. This method employs ultrafiltration for washing and impurity removal at low temperatures. Ultrasound, however, fails to hydrolyze starch at low temperatures. Lysozyme is also used to disrupt microbial cell structure and accelerate the release of endotoxins and peptidoglycans. However, this method results in an infinitely high concentration of endotoxins and peptidoglycans in the hydrolysate, making it difficult to reduce their levels to acceptable levels through filtration and adsorption. Instead, it adds washing and drying steps, increasing water, energy, and time consumption. A combined debranching enzyme and α-amylase hydrolysis scheme is used, but the reaction process is difficult to precisely control the molecular weight distribution of the hydrolysate, and the operation is complex, making it unsuitable for industrial-scale production.

[0005] Chinese patent CN119824056A discloses a method for high-yield preparation of icodextrin raw material using a single enzyme system. The method includes the following steps: using an α-high-temperature amylase, performing two enzymatic hydrolysis processes, combined with ultrafiltration and spray drying to obtain icodextrin. First, starch is added to water and stirred, then heated to 85-100℃ for gelatinization. α-high-temperature amylase is added for enzymatic hydrolysis for 40-70 minutes. The resulting hydrolysate is filtered through diatomaceous earth. The filtrate is then ultrafiltered to intercept large-molecule dextrin and α-amylase. The intercepted solution is further heated for hydrolysis. The hydrolysate is combined with the ultrafiltration permeate and then ultrafiltered again to obtain icodextrin with a suitable molecular weight. This method involves first heating and gelatinizing the starch, then enzymatically hydrolyzing it. In industrial production, corn starch milk begins to gelatinize at 75°C, resulting in significant sticking to the walls, making stirring difficult, hindering heat transfer through the tank walls, and causing uneven gelatinization. The starch paste sticking to the walls tends to gel as the temperature rises and it sticks for a long time, leading to incomplete hydrolysis. Ultimately, more than 10% of the limiting dextrin cannot be hydrolyzed and forms flocculents suspended in the hydrolysate, causing filtration difficulties, increasing filter material wear, increasing time and energy consumption, and reducing yield. This method is not suitable for industrial production. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a method for preparing high-yield icodextrin using an ultrasonic field combined with a single enzyme system. This method improves starch utilization and icodextrin yield, has high preparation efficiency, is simple to operate, and is suitable for industrial production.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing high-yield icodextrin using an ultrasonic field combined with a single enzyme system, comprising the following steps:

[0009] S1. Enzymatic hydrolysis: A starch milk solution with a mass concentration of 15-35% and α-amylase are placed in a starch hydrolysis tank, stirred and heated to 70-100℃ and maintained at the temperature for 20-120 minutes. At the same time, the proportion of dextrin with a molecular weight of less than 1638 Da in the hydrolysate is controlled at 4-7% (detected by GPC molecular exclusion method). Then the enzyme is inactivated, the pH is adjusted to neutral, and the enzymatic hydrolysate is obtained. The enzymatic hydrolysate is adsorbed and filtered to obtain the first filtrate.

[0010] S2. First ultrafiltration: The first filtrate is subjected to ultrafiltration and cross-flow filtration to separate macromolecular dextrin and α-amylase with a molecular weight of over 45,000 Da, which is the first retentate. The permeate is the second filtrate.

[0011] S3. Ultrasonic hydrolysis: Return the first retentate to the starch hydrolysis tank, add purified water until the solid content of the liquid is 10-15%, heat to 75-100℃, turn on the ultrasonic waves to hydrolyze for 30-60 minutes to obtain hydrolysate, and perform ultrafiltration on the hydrolysate. Separate the large molecular weight dextrin and α-amylase with a molecular weight of more than 45000 Da by cross-flow filtration. Combine the permeate with the second filtrate to obtain the third filtrate.

[0012] S4. Secondary ultrafiltration: The third filtrate is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 500 Da to obtain a second filtrate with a weight average molecular weight of 13000-19000 Da, a number average molecular weight of 5000-6500 Da, and a molar ratio of α-1,6 glycosidic bonds of less than 6%.

[0013] S5. Drying: Dry the second retentate to obtain icodextrin.

[0014] Furthermore, in step S1, the amount of α-amylase used is 12-20 U per gram of dry starch.

[0015] Furthermore, in step S1, the starch used in the starch emulsion solution is one of corn starch, glutinous rice starch, sweet potato starch, and tapioca starch.

[0016] Furthermore, in step S1, the starch milk solution is prepared with purified water at 15-25°C. The enzyme inactivation method is to add hydrochloric acid to adjust the pH to 2.0-3.0 and stir for 20 minutes. The enzyme hydrolysate is adsorbed with activated carbon for 2 hours and then filtered.

[0017] Furthermore, in steps S2 and S3, the ultrafiltration membrane used during ultrafiltration has a molecular weight cutoff range of 30–100 kDa.

[0018] Furthermore, in step S3, the ultrasonic frequency is set to 30–40 kHz and the power is 4000–6000 W.

[0019] Furthermore, in step S4, the ultrafiltration pressure is 0.2–0.7 MPa, and the ultrafiltration membrane used during ultrafiltration has a molecular weight cutoff of 500 Da.

[0020] Furthermore, step S5 employs spray drying, with a feed temperature of 80–90°C, an inlet air temperature of 160–260°C, an outlet air temperature of 80–110°C, a feed pump frequency of 20–40 Hz, and an atomizing high-speed centrifuge speed of 15,000–25,000 rpm.

[0021] Compared with the prior art, the present invention has the following beneficial technical effects:

[0022] (1) In the enzymatic hydrolysis process of the present invention, α-amylase is added to hydrolyze starch. When the temperature is raised to 75-85℃, which is still a relatively low gelatinization temperature, the starch begins to gelatinize while being sheared by α-amylase from the α-1,4 glycosidic bonds in the amylose. At the same time, the hydrogen bonds and van der Waals forces between starch chain molecules are destroyed due to the temperature rise, the double helix structure is dissociated, and the starch granules are continuously disintegrated, so that the liquid always maintains good fluidity and effectively avoids starch sticking to the wall and gelling at high temperature.

[0023] (2) In this invention, after a short enzymatic hydrolysis time, the proportion of small dextrin molecules with a molecular weight less than 1638 Da in the hydrolysate is detected by GPC molecular exclusion method. When the proportion reaches 4-7%, the enzyme is inactivated and the enzymatic hydrolysis is stopped. Then, high molecular weight dextrin is separated by ultrafiltration. Subsequently, the separated high molecular weight dextrin is subjected to secondary hydrolysis by ultrasound. The advantage of ultrasonic hydrolysis is that it can effectively cleave the α-1,6 glycosidic bonds of amylopectin, break the amylopectin side chains, reduce the intermolecular hydrogen bond binding sites, reduce the tendency of dextrin retrogradation after hydrolysis, and eliminate the residue of limit dextrin. The hydrolysate is clear and free of flocculents. After enzyme inactivation, the ultrasonic hydrolysis method can be used to stop the machine at any time to detect the molecular weight distribution of the hydrolysate, accurately control the degree of hydrolysis, and minimize the proportion of small dextrin molecules with a molecular weight less than 1638 Da generated during the hydrolysis process. This greatly improves the utilization rate of starch and the product yield, which can be as high as 80-88%, while the yield of traditional acid hydrolysis or enzymatic hydrolysis of icodextrin is only 45-65%.

[0024] (3) The present invention uses less enzyme, has a shorter ultrasonic hydrolysis time, more flexible central control detection, a smaller increase in production cost, and higher preparation efficiency, making it suitable for large-scale industrial production. Detailed Implementation

[0025] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways than those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0026] Example 1

[0027] S1. Enzymatic hydrolysis: At room temperature, corn starch was prepared into a starch slurry solution with a mass percentage concentration of 20% using 2000L of purified water. After stirring evenly, the solution was transferred to a 3000L starch hydrolysis tank and 50g of α-amylase was added. The steam pressure was controlled at 0.08-0.2MPa, and the temperature was raised to 75℃ at a uniform rate. After incubation for 40 minutes, hydrochloric acid was added to adjust the pH to 2.4, and the enzyme was inactivated by stirring for 20 minutes. Hydroxide was then added to adjust the pH to 6.8 to obtain the enzymatic hydrolysate. While still hot, the enzymatic hydrolysate was adsorbed onto activated carbon for 2 hours, and the first filtrate was obtained by filtration. A sample was taken to detect the molecular weight distribution of the enzymatic hydrolysate.

[0028] Table 1. Results of enzyme hydrolysate detection

[0029]

[0030] S2. First ultrafiltration: The first filtrate is transferred to ultrafiltration machine No. 1 for ultrafiltration. The large molecular weight dextrin and α-amylase with a pore size of 80KD are separated by cross-flow filtration using an 80KD ultrafiltration membrane. This is the first retentate, and the permeate is the second filtrate.

[0031] S3. Ultrasonic Hydrolysis: The first retentate is returned to the starch hydrolysis tank, purified water is added until the solid content of the liquid is 10%, the mixture is stirred and heated to 80°C, the ultrasonic waves are turned on, the ultrasonic frequency is set to 30kHz, the power is 4500W, and the ultrasonic treatment is carried out for 70 minutes to obtain the hydrolysate. The hydrolysate is transferred to ultrafiltration unit 1 and treated with an 80KD large-pore ultrafiltration membrane to obtain the permeate. The permeate is combined with the second filtrate to obtain the third filtrate. The molecular weight distribution of the third filtrate is analyzed by sampling in the central control room.

[0032] Table 2 Results of Third Filtrate Test

[0033]

[0034] S4. Secondary Ultrafiltration: The third filtrate is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 500 Da to obtain a second retentate with a weight-average molecular weight of 13000-19000 Da, a number-average molecular weight of 5000-6500 Da, and a molar proportion of α-1,6 glycosidic bonds of less than 5%. A sample is taken to analyze the molecular weight distribution of the second retentate.

[0035] Table 3. Results of the second retentate test

[0036]

[0037] S5. Spray drying: The second retentate was spray dried with the feed temperature set at 80℃, the feed pump frequency at 30Hz, the inlet air temperature at 220±5℃, the outlet air temperature at 105±5℃, and the speed of the atomizing high-speed centrifuge at 18000rpm to obtain icodextrin for peritoneal dialysis fluid with a yield of 83.2%.

[0038] Example 2

[0039] S1. Enzymatic hydrolysis: At room temperature, corn starch was prepared into a starch slurry solution with a mass percentage concentration of 25% using 2000L of purified water. After stirring evenly, the solution was transferred to a 3000L starch hydrolysis tank and 55g of α-amylase was added. The steam pressure was controlled at 0.08-0.2MPa, and the temperature was raised to 80℃ at a uniform rate. After incubation for 95 minutes, hydrochloric acid was added to adjust the pH to 2.6, and the enzyme was inactivated by stirring for 20 minutes. Sodium hydroxide was then added to adjust the pH to 7.0 to obtain the enzymatic hydrolysate. While still hot, the enzymatic hydrolysate was adsorbed onto activated carbon for 2 hours and filtered to obtain the first filtrate. A sample was taken to detect the molecular weight distribution of the enzymatic hydrolysate.

[0040] Table 4. Results of Enzyme Hydrolysate Detection

[0041]

[0042] S2. First ultrafiltration: The first filtrate is transferred to ultrafiltration machine No. 1 for ultrafiltration. The large molecular weight dextrin and α-amylase with a pore size of 80KD are separated by cross-flow filtration using an 80KD ultrafiltration membrane. This is the first retentate, and the permeate is the second filtrate.

[0043] S3. Ultrasonic Hydrolysis: The first retentate is returned to the starch hydrolysis tank, and purified water is added until the solid content of the liquid is 15%. The mixture is stirred and heated to 85°C, and ultrasonic treatment is initiated at a frequency of 30kHz and a power of 4500W for 55 minutes to obtain the hydrolysate. The hydrolysate is then transferred to ultrafiltration unit 1 and treated with an 80KD large-pore ultrafiltration membrane to obtain the permeate. The permeate is combined with the second filtrate to obtain the third filtrate. Sampling and analysis of the molecular weight distribution of the third filtrate are performed in the central control area.

[0044] Table 5. Results of the third filtrate test

[0045]

[0046] S4. Secondary Ultrafiltration: The third filtrate is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 500 Da to obtain a second retentate with a weight-average molecular weight of 13000-19000 Da, a number-average molecular weight of 5000-6500 Da, and a molar proportion of α-1,6 glycosidic bonds of less than 5%. A sample is taken to analyze the molecular weight distribution of the second retentate.

[0047] Table 6 Results of Second Retention Fluid Test

[0048]

[0049] S5. Spray drying: The second retentate was spray dried with the feed temperature set at 80℃, the feed pump frequency at 30Hz, the inlet air temperature at 220±5℃, the outlet air temperature at 105±5℃, and the speed of the atomizing high-speed centrifuge at 19000rpm to obtain icodextrin for peritoneal dialysis fluid with a yield of 84.3%.

[0050] Example 3

[0051] S1. Enzymatic hydrolysis: At room temperature, corn starch was prepared into a 30% starch slurry solution using 2000L of purified water. After stirring evenly, the solution was transferred to a 3000L starch hydrolysis tank and 55g of α-amylase was added. The steam pressure was controlled at 0.08-0.2MPa, and the temperature was raised to 95℃ at a uniform rate. After incubation for 60 minutes, hydrochloric acid was added to adjust the pH to 2.3, and the enzyme was inactivated by stirring for 20 minutes. Sodium hydroxide was then added to adjust the pH to 6.6 to obtain the enzymatic hydrolysate. While still hot, the enzymatic hydrolysate was adsorbed onto activated carbon for 2 hours and filtered to obtain the first filtrate. A sample was taken to detect the molecular weight distribution of the enzymatic hydrolysate.

[0052] Table 7. Results of Enzyme Hydrolysate Detection

[0053]

[0054] S2. First ultrafiltration: The first filtrate is transferred to ultrafiltration machine No. 1 for ultrafiltration. The large molecular weight dextrin and α-amylase with a pore size of 80KD are separated by cross-flow filtration using an 80KD ultrafiltration membrane. This is the first retentate, and the permeate is the second filtrate.

[0055] S3. Ultrasonic Hydrolysis: The first retentate is returned to the starch hydrolysis tank, and purified water is added until the solid content of the liquid is 12%. The mixture is stirred and heated to 75°C, and ultrasonic treatment is initiated at a frequency of 30kHz and a power of 6000W for 40 minutes to obtain the hydrolysate. The hydrolysate is then transferred to ultrafiltration unit 1 and treated with an 80KD large-pore ultrafiltration membrane to obtain the permeate. The permeate is combined with the second filtrate to obtain the third filtrate. Sampling and analysis of the molecular weight distribution of the third filtrate are performed in the central control area.

[0056] Table 8 Results of Third Filtrate Detection

[0057]

[0058] S4. Secondary Ultrafiltration: The third filtrate is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 500 Da to obtain a second retentate with a weight-average molecular weight of 13000-19000 Da, a number-average molecular weight of 5000-6500 Da, and a molar proportion of α-1,6 glycosidic bonds of less than 5%. A sample is taken to analyze the molecular weight distribution of the second retentate.

[0059] Table 9. Results of Second Retention Fluid Test

[0060]

[0061] S5. Spray drying: The second retentate was spray dried with the feed temperature set at 90℃, the feed pump frequency at 40Hz, the inlet air temperature at 220±5℃, the outlet air temperature at 100±5℃, and the speed of the atomizing high-speed centrifuge at 19000rpm to obtain icodextrin for peritoneal dialysis fluid with a yield of 85.2%.

[0062] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. However, any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing high-yield icodextrin using an ultrasonic field combined with a single enzyme system, characterized in that, Includes the following steps: S1. Enzymatic hydrolysis: A starch emulsion solution with a mass concentration of 15-35% and α-amylase are placed in a starch hydrolysis tank, stirred, and heated to 70-100℃ and maintained at the temperature for 20-120 minutes. Simultaneously, the proportion of dextrin with a molecular weight less than 1638 Da in the hydrolysate is controlled at 4-7%. Then, the enzyme is inactivated, and the pH is adjusted to neutral to obtain the enzymatic hydrolysate. The hydrolysate is adsorbed and filtered to obtain the first filtrate; S2. Single ultrafiltration: The first filtrate is ultrafiltered, and cross-flow filtration is used to separate high molecular weight dextrins (over 45000 Da) and α-amylase, i.e., the first retentate. The permeate is the second filtrate; S3. Ultrasonic hydrolysis: The first retentate is returned to the starch hydrolysis tank, purified water is added until the solid content of the liquid is 10-15%, the temperature is raised to 75-100℃, and ultrasonic hydrolysis is performed for 30-60 minutes to obtain hydrolysate. The hydrolysate is then subjected to ultrafiltration and cross-flow filtration to separate macromolecular dextrin and α-amylase with a molecular weight of over 45000 Da. The permeate is combined with the second filtrate to obtain the third filtrate. S4. Secondary ultrafiltration: The third filtrate is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 500 Da to obtain a second retentate with a weight average molecular weight of 13000-19000 Da, a number average molecular weight of 5000-6500 Da, and a molar proportion of α-1,6 glycosidic bonds of less than 6%; S5. Drying: The second retentate is dried to obtain icodextrin.

2. The method for preparing high-yield icodextrin using an ultrasonic field combined with a single enzyme system according to claim 1, characterized in that: In step S1, the amount of α-amylase used is 12-20 U per gram of dry starch.

3. The method for preparing high-yield icodextrin using an ultrasonic field combined with a single enzyme system according to claim 2, characterized in that: In step S1, the starch used in the starch emulsion solution is one of corn starch, glutinous rice starch, sweet potato starch, and tapioca starch.

4. The method for preparing high-yield icodextrin using an ultrasonic field combined with a single enzyme system according to claim 3, characterized in that: In step S1, the starch milk solution is prepared with purified water at 15-25℃. The enzyme inactivation method is to add hydrochloric acid to adjust the pH value to 2.0-3.0 and stir for 20 minutes. The enzyme hydrolysate is adsorbed with activated carbon for 2 hours and then filtered.

5. The method for preparing high-yield icodextrin using an ultrasonic field combined with a single enzyme system according to any one of claims 1-4, characterized in that: In steps S2 and S3, the ultrafiltration membrane used during ultrafiltration has a molecular weight cutoff range of 30–100 kDa.

6. The method for preparing high-yield icodextrin using an ultrasonic field combined with a single enzyme system according to claim 5, characterized in that: In step S3, the ultrasonic frequency is set to 30-40kHz and the power is 4000-6000W.

7. The method for preparing high-yield icodextrin using an ultrasonic field combined with a single enzyme system according to claim 6, characterized in that: In step S4, the ultrafiltration pressure is 0.2 to 0.7 MPa, and the ultrafiltration membrane used during ultrafiltration has a molecular weight cutoff of 500 Da.

8. The method for preparing high-yield icodextrin using an ultrasonic field combined with a single enzyme system according to claim 7, characterized in that: Step S5 employs spray drying, with a feed temperature of 80–90°C, an inlet air temperature of 160–260°C, an outlet air temperature of 80–110°C, a feed pump frequency of 20–40 Hz, and an atomizing high-speed centrifuge speed of 15,000–25,000 rpm.

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