Specific molecular weight oligomeric dextran as well as continuous production method, device and application thereof

Abstract

The invention relates to oligomeric dextran with specific molecular weight as well as a continuous production method, device and application thereof, the continuous production method comprises the following steps: mixing a dextran substrate with a dextranase solution for pre-reaction to obtain pre-reaction feed liquid; the pre-reaction feed liquid is subjected to enzymolysis through an enzyme membrane reactor, an enzymolysis product is synchronously and continuously separated out, meanwhile, a dextran substrate is continuously supplemented into the pre-reaction feed liquid, and the enzymolysis product is oligomeric dextran feed liquid with the specific molecular weight; in the long-term operation process, the molecular weight and composition of the product are kept stable depending on a molecular weight self-adjusting mechanism, the continuous high and stable yield of oligomeric dextran with specific molecular weight and narrow molecular weight distribution is achieved, and dextranase is fully used; the continuous production method is simple and safe to operate, efficient and energy-saving, and the continuous production device is high in integration level, low in investment cost and easy for industrial and large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme membrane reaction and dextran production, in particular to an oligodextran with a specific molecular weight and a continuous production method, device and application thereof, in particular to an oligodextran with an average molecular weight of 3-6 kDa and a continuous production method, device and application thereof. Background Art

[0002] Dextran is a high molecular weight glucose polymer synthesized by fermentation of sucrose by Leuconostoc mesenteroides, and is hydrolyzed into products of different molecular weight ranges. Oligodextran has important application value in the biomedical and pharmaceutical fields due to its biological activities such as anticoagulation, antithrombosis, antioxidant and anti-inflammatory. The traditional production method relies on acid hydrolysis and organic solvent precipitation and classification, which not only consumes a large amount of organic solvent, but also reduces the yield of the target product, making it difficult to effectively control the molecular weight and distribution of dextran, and may cause allergies and side effects in clinical applications. The method of dextranase-catalyzed hydrolysis has the characteristics of strong specificity, is suitable for the preparation of low molecular weight oligodextran, and can improve its molecular weight distribution. However, the problems of uncontrollable enzymatic hydrolysis process, difficulty in recycling and reusing enzymes, many by-products and low product yield have become bottlenecks restricting the industrial application of enzymatic production of oligodextran.

[0003] CN116606894A discloses a technology for preparing molecular weight controllable dextran by precursor fermentation, wherein a precursor having a weight average molecular weight less than that of the target dextran and a distribution coefficient within 1.6 is added for fermentation to control the molecular weight and synthesize a dextran product, and then an ultrafiltration membrane is used to separate and directly extract dextran products of different molecular weight specifications. The method controls the molecular weight of the product depending on the pore size of the ultrafiltration membrane. Currently, the pore size distribution range of commercial ultrafiltration membranes is relatively wide, resulting in a relatively wide range of final molecular weights.

[0004] CN102942638A discloses a method for producing dextran with a weight average molecular weight of 3000-6000Da. The method uses dextranase to directly degrade dextran with a weight average molecular weight of 7000-80000Da into ultra-micromolecule dextran with a weight average molecular weight of 3000-6000Da. In the method, the dextranase is inactivated and cannot be reused, resulting in a high production cost.

[0005] CN107988284A discloses a method for producing small molecule dextran, its product and use. The method utilizes dextran raw material to perform simultaneous enzymatic hydrolysis and separation in an integrated enzyme membrane reactor to obtain a small molecule dextran product; the integrated enzyme membrane reactor comprises an ultrafiltration membrane system adsorbed with dextranase; wherein the immobilization of the enzyme leads to a decrease in its catalytic activity, and after the enzyme is immobilized, the pore size distribution of the ultrafiltration membrane is reduced, and the efficiency of membrane separation is also limited, thereby affecting the overall production efficiency of the enzyme membrane reactor, and it is difficult to meet the needs of large-scale production.

[0006] The prior art has the following defects: the molecular weight of oligodextran depends on the pore size of the ultrafiltration membrane; the molecular weight range of the oligodextran product is relatively wide; the dextranase is inactivated, which makes it impossible to reuse it and difficult to carry out continuous production; the enzyme reactor has low production efficiency, etc. Therefore, how to provide a continuous production method and device for oligodextran of a specific molecular weight to efficiently and stably produce oligodextran of a specific molecular weight has become a problem to be solved. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a specific molecular weight oligodextran and a continuous production method, device and application thereof, which realizes the molecular weight self-regulation mechanism of the oligodextran, achieves the effect of continuous high and stable production, and solves the problems of low production efficiency of the current enzyme membrane reactor, wide molecular weight distribution of the dextran product, low yield and inability to produce continuously, and the technical problems of high production cost caused by the inability to reuse dextranase.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for continuous production of oligodextran of a specific molecular weight, the continuous production method comprising the following steps:

[0010] (1) mixing a dextran substrate and a dextranase solution for pre-reaction to obtain a pre-reaction liquid;

[0011] (2) The pre-reaction liquid in step (1) is hydrolyzed by an enzyme membrane reactor and the hydrolysis product is separated synchronously and continuously, and the dextran substrate is continuously added to the pre-reaction liquid. The hydrolysis product is an oligodextran liquid with a specific molecular weight.

[0012] The continuous production method of the present invention utilizes the characteristic that dextranase preferentially reacts with dextran substrates with larger molecular weights, and avoids the situation that insufficient substrate degradation leads to excessive molecular weight of target products, and excessive degradation of target products leads to excessive molecular weight of target products, by combining pre-reaction with continuous substrate supplementation, thereby achieving regulation of molecular weight of target product oligodextran, without relying on the membrane pore size of the membrane assembly in the enzyme membrane reactor to achieve control of product molecular weight, successfully preparing oligodextran with narrow molecular weight distribution and high yield, and dextranase can be reused, thereby reducing production costs.

[0013] Preferably, the flow rate of the continuously replenished dextran substrate in step (2) is the same as the flow rate of the enzymatic hydrolysis product.

[0014] Among them, the flow rate of the enzymolysis product is the speed at which the enzymolysis product vertically penetrates the membrane. The present invention further preferably makes the flow rate of the continuously supplemented dextran substrate the same as the flow rate of the enzymolysis product, and cooperates with the mass ratio of the dextran raw material to the dextranase and the residence time of the pre-reaction liquid in the enzyme membrane reactor to achieve continuous production of oligomeric dextran with a specific molecular weight and a narrow molecular weight distribution; if the flow rate of the continuously supplemented dextran substrate is too fast or too slow, the molecular weight distribution of the product will become wider.

[0015] Preferably, the weight average molecular weight of the dextran raw material in the dextran substrate in step (1) is 40-1000 kDa, for example, 40 kDa, 70 kDa, 100 kDa, 150 kDa, 200 kDa, 400 kDa, 600 kDa, 800 kDa or 1000 kDa, etc.

[0016] Preferably, the dextran raw material in the dextran substrate includes a dextran raw material with a customized molecular weight and a dextran industrial raw material produced by fermentation.

[0017] Preferably, the concentration of the dextran raw material in the dextran substrate in step (1) is 10-200 g / L, for example, 10 g / L, 30 g / L, 50 g / L, 100 g / L, 150 g / L or 200 g / L, etc.

[0018] Preferably, the concentration of dextranase in the dextranase solution of step (1) is 10-200 g / L, for example, 10 g / L, 30 g / L, 50 g / L, 100 g / L, 150 g / L or 200 g / L.

[0019] Preferably, the mass ratio of the dextran raw material to the dextranase is (50-1000):1, for example, it can be 50:1, 100:1, 200:1, 400:1, 600:1, 800:1 or 1000:1, etc., preferably (150-600):1.

[0020] Preferably, the total volume of the dextran substrate and the dextranase solution in step (1) is 0.2-50 L, for example, 0.2 L, 0.5 L, 1 L, 5 L, 10 L, 20 L, 30 L, 40 L or 50 L, etc., preferably 0.2-20 L.

[0021] Preferably, the pre-reaction time in step (1) is 5-30 min, for example, it can be 5 min, 10 min, 15 min, 20 min, 25 min or 30 min.

[0022] Preferably, the membrane pressure of the enzymatic hydrolysis reactor in step (2) is 0.1-0.4 MPa, for example, it can be 0.1 MPa, 0.15 MPa, 0.2 MPa, 0.25 MPa, 0.3 MPa, 0.35 MPa or 0.4 MPa, etc.

[0023] In the membrane separation process of the present invention, there is no need to screen and separate the oligodextran products after enzymatic hydrolysis according to their molecular weight. It is only necessary to use a membrane to separate all the enzymatic hydrolysis products, i.e., oligodextran with a specific molecular weight. Since the molecular weight difference between the enzymatic hydrolysis products and the enzyme is large, a smaller membrane pressure can be used for separation.

[0024] Preferably, the temperature of the enzymatic hydrolysis in step (2) is 25-45°C, for example, 25°C, 35°C or 45°C.

[0025] Preferably, the residence time of the pre-reaction liquid in step (2) in the enzyme membrane reactor is 0.5-4 h, for example, 0.5 h, 1 h, 2 h, 3 h or 4 h, etc., preferably 1-2 h.

[0026] The residence time of the pre-reaction liquid, i.e., the reaction time of the dextran substrate, is defined as the ratio of the volume of the pre-reaction liquid to the volume flow rate of the oligomeric dextran liquid.

[0027] It is worth noting that, in the present invention, since the mass of the dextran raw material and the dextranase is relatively large, and the concentrations of the two are preferably equal during the experiment, the volume of the dextranase solution is not counted in the calculation process, that is, the volume of the pre-reaction liquid is only the volume of the dextran substrate in the calculation process.

[0028] The present invention further preferably has a mass ratio of the dextran raw material to the dextran enzyme of (150-600): 1, and further preferably has a residence time of the pre-reaction liquid in the enzyme membrane reactor of 1-2h, and the two cooperate with each other to achieve the regulation of the molecular weight of the oligomeric dextran. Specifically, in the enzyme membrane reactor, after the product reaches a certain molecular weight, it passes through the membrane material of the membrane assembly, and the dextran substrate and the dextran enzyme that have not reacted to the target molecular weight continue to react with the intercepted liquid reflux; as the mass ratio of dextran to dextran enzyme increases, the dextran enzyme needs more time to hydrolyze the dextran substrate to the product of the target molecular weight, that is, the pre-reaction liquid needs a longer residence time; if the residence time is too short, it cannot be hydrolyzed to the target molecular weight.

[0029] That is, in the continuous production method of the present invention, when the mass ratio of the dextran raw material to the dextranase is kept the same, if the residence time of the pre-reaction liquid is too short, the molecular weight of the product is too large; if the residence time of the pre-reaction liquid is too long, the molecular weight of the product is too small; when the residence time of the pre-reaction liquid is kept the same, if the mass ratio of the dextran raw material to the dextranase is too small, the molecular weight of the product is too small; if the mass ratio of the dextran raw material to the dextranase is too large, the molecular weight of the product is too large.

[0030] Preferably, the cross-flow rate of the enzymatic hydrolysis product in step (2) is 0.6-1 L / min, for example, it can be 0.6 L / min, 0.8 L / min or 1 L / min.

[0031] The present invention further preferably has a cross-flow velocity of the enzymatic hydrolysis product of 0.6-1 L / min, which reduces the accumulation of substances on the membrane surface and avoids membrane contamination affecting the membrane flux, thereby affecting the residence time of the pre-reaction liquid and causing changes in the molecular weight distribution of the product, wherein the cross-flow velocity refers to the flow velocity of the enzymatic hydrolysis product parallel to the membrane surface.

[0032] Preferably, the retentate obtained by separation in step (2) is circulated to the pre-reaction feed liquid.

[0033] Preferably, the retentate in step (2) comprises dextranase.

[0034] The invention selects the cut-off molecular weight of the membrane component so that the membrane component can completely intercept the dextranase, thereby realizing the reuse of the dextranase and reducing the production cost.

[0035] Preferably, the membrane component in the enzyme membrane reactor in step (2) comprises a porous ultrafiltration membrane component.

[0036] It is worth noting that the enzyme membrane reactor described in the present invention preferably adopts a cross-flow enzyme membrane reactor, that is, in the cross-flow mode, the reaction liquid flows parallel to the membrane surface, avoiding the rapid accumulation of solutes on the membrane surface in traditional dead-end filtration, reducing concentration polarization and membrane pollution; thereby allowing products of suitable molecular weight to permeate through the membrane, while the enzyme and macromolecular substrate are retained by the membrane and circulated back to the pre-reaction tank.

[0037] Preferably, the porous ultrafiltration membrane assembly comprises any one of a rolled ultrafiltration membrane assembly, a tubular ultrafiltration membrane assembly, a flat ultrafiltration membrane assembly or a hollow fiber ultrafiltration membrane assembly, or a combination of at least two of them, wherein typical but non-limiting combinations include a combination of a rolled ultrafiltration membrane assembly and a tubular ultrafiltration membrane assembly, a combination of a flat ultrafiltration membrane assembly and a hollow fiber ultrafiltration membrane assembly, a combination of a tubular ultrafiltration membrane assembly and a flat ultrafiltration membrane assembly, etc.

[0038] Preferably, the membrane material in the porous ultrafiltration membrane assembly includes any one of polysulfone, polyethersulfone, sulfonated polyethersulfone, polyacrylonitrile, polyvinylidene fluoride, polyvinyl alcohol, polyester, regenerated cellulose or inorganic ceramics, or a combination of at least two of them, wherein typical but non-limiting combinations include a combination of polysulfone and polyethersulfone, a combination of sulfonated polyethersulfone and polyacrylonitrile, a combination of polypropylene and polyvinylidene fluoride, a combination of polyethersulfone and polyvinylidene fluoride, a combination of polyester and polyethersulfone, a combination of polysulfone, polyacrylonitrile and polypropylene, and the like.

[0039] Preferably, the molecular weight cutoff of the membrane in the porous ultrafiltration membrane assembly is from 20 kDa to the molecular weight of dextranase, for example, it can be 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa or 67 kDa, etc., preferably 20-50 kDa.

[0040] Since the molecular weight of the oligomeric dextran described in the present invention depends on the mass ratio of the dextran raw material to the dextranase and the time of the pre-reaction feed solution, the porous ultrafiltration membrane component described in the present invention selects a high-throughput ultrafiltration membrane that can only completely intercept the dextranase (the molecular weight of the dextranase is ≈67 kDa) without controlling the molecular weight of the product, thereby achieving rapid separation of oligomeric dextran products with a specific molecular weight and reuse of the dextranase.

[0041] It is worth noting that the present invention further prefers that the molecular weight cutoff of the membrane in the porous ultrafiltration membrane assembly is 20-50kDa. This is because the pore size of commercially available membrane materials is uneven and may cause slight leakage of the enzyme. This does not contradict the fact that the molecular weight of the product proposed by the present invention is independent of the membrane pore size.

[0042] Preferably, the water flux of the membrane in the porous ultrafiltration membrane assembly is 60-150LMH bar -1 , for example, it can be 60LMHbar -1 、90LMH bar-1 、100LMH bar -1 、120LMH bar -1 or 150LMH bar -1 wait.

[0043] The present invention further preferably has a water flux of 60-150 LMH bar in the porous ultrafiltration membrane assembly. -1 , further improving the yield of dextran products with specific molecular weight. If the water flux of the membrane is low, the yield will decrease. If the membrane has a higher water flux while being able to intercept the enzyme, the yield can be increased in conjunction with a faster enzymatic reaction.

[0044] The water flux of the membrane described in the present invention refers to the pure water flux, and the pure water refers to deionized water used in the laboratory.

[0045] As a further preferred technical solution of the present invention, the continuous production method comprises the following steps:

[0046] (1) mixing a dextran substrate having a concentration of 10-200 g / L and a weight average molecular weight of 40-1000 kDa with a dextran enzyme solution having a concentration of 10-200 g / L at a mass ratio of dextran raw material to dextran enzyme of (50-1000):1 and performing a pre-reaction for 5-30 min to obtain a pre-reaction feed solution;

[0047] The volume of the pre-reaction liquid is 0.2-50L;

[0048] (2) at 25-45° C., the pre-reaction liquid in step (1) is subjected to enzymatic hydrolysis in an enzyme membrane reactor, and the enzymatic hydrolysis product is synchronously and continuously separated at a membrane pressure of 0.1-0.4 MPa, and the dextran substrate is continuously added to the pre-reaction liquid, wherein the enzymatic hydrolysis product is an oligodextran liquid with a specific molecular weight;

[0049] The residence time of the pre-reaction liquid in the enzyme membrane reactor is 0.5-4h; the cross-flow rate of the enzymatic hydrolysis product is 0.6-1L / min; the separated retentate is circulated to the pre-reaction liquid; the retentate includes dextranase;

[0050] The membrane assembly in the enzyme membrane reactor includes a porous ultrafiltration membrane assembly; the membrane in the porous ultrafiltration membrane assembly has a molecular weight cutoff of 20 kDa to the molecular weight of dextranase, and a water flux of 60-150 LMH bar. -1 .

[0051] Optionally, the continuous production method further comprises purifying the oligodextran of specific molecular weight to further narrow the molecular weight distribution size.

[0052] Optionally, the molecular weight cutoff of the membrane component used for the purification is 1-8 kDa, for example, 1 kDa, 3 kDa, 5 kDa or 8 kDa.

[0053] In a second aspect, the present invention provides a specific molecular weight oligomeric dextran, wherein the specific molecular weight oligomeric dextran is prepared by the continuous production method of the specific molecular weight oligomeric dextran described in the first aspect.

[0054] The specific molecular weight oligodextran of the present invention has a narrow molecular weight distribution and high biocompatibility.

[0055] Preferably, the average molecular weight of the specific molecular weight oligosaccharide anhydride is 3-6 kDa.

[0056] In a third aspect, the present invention provides an application of the specific molecular weight oligomeric dextran described in the second aspect, wherein the specific molecular weight oligomeric dextran is used as a raw material for veterinary iron supplement injections, plasma substitutes or blood volume expanders.

[0057] The specific molecular weight oligodextran of the present invention is an excellent raw material for veterinary iron supplement injection, plasma substitute or blood volume expander, and has important application value in the fields of biomedicine and pharmacy.

[0058] In a fourth aspect, the present invention provides a continuous production device for oligodextran of a specific molecular weight, wherein the continuous production method described in the first aspect is carried out using the continuous production device;

[0059] The continuous production device comprises a substrate storage component, an enzyme membrane reactor and a product collection component which are connected in sequence;

[0060] Along the flow direction of the feed liquid, the enzyme membrane reactor comprises a pre-reaction component, a feed liquid conveying pipeline, a membrane assembly and a reflux pipeline which are cyclically connected in sequence;

[0061] The substrate storage component is connected to the pre-reaction component; the product collection component is arranged on the lower side of the membrane assembly;

[0062] The pre-reaction component is provided with a liquid level monitoring component; and the product collecting component is placed on the flow monitoring component.

[0063] The continuous production device of specific molecular weight oligo dextran described in the present invention is provided with a liquid level monitoring component and a flow monitoring component on the basis of an enzyme membrane reactor, wherein the liquid level monitoring component is used to monitor the volume of the pre-reaction liquid in the pre-reaction component and replenish the dextran substrate in time, and the flow monitoring component is used to monitor the flow of the product specific molecular weight oligo dextran, and the two work synergistically to achieve continuous production. During the entire operation process, the molecular weight of the product is always kept stable with the self-regulation of the speed of replenishing the dextran substrate; and the activity loss of the dextranase and the decrease of the membrane flux reach a steady-state balance in the system, that is, the self-regulation of the molecular weight of the specific molecular weight oligo dextran is achieved.

[0064] Preferably, a first pressurizing component is provided on the pipeline between the substrate storage component and the pre-reaction component.

[0065] Preferably, a second pressurizing component is provided on the liquid conveying pipeline in the enzyme membrane reactor.

[0066] Preferably, the substrate storage part and the pre-reaction part are placed in a water bath thermostatic part.

[0067] Compared with the prior art, the present invention has at least the following beneficial effects:

[0068] (1) The method for continuous production of oligodextran of specific molecular weight provided by the present invention utilizes the characteristic that dextranase preferably reacts with large molecular weight dextran substrate, and realizes the regulation and continuous production of the molecular weight of the target product oligodextran by means of pre-reaction and continuous supplementation of substrate. Furthermore, by regulating the mass ratio of the dextran raw material and the dextranase, the residence time of the pre-reaction liquid, and the speed of continuous supplementation of the dextran substrate, the molecular weight of the oligodextran of the target product is further precisely regulated (for example, the average molecular weight is precisely regulated to 3-6 kDa), and the molecular weight distribution is narrow, the PDI value is in the range of 1.3-1.4, and the molecular weight distribution is narrow; the regulation of the molecular weight is no longer dependent on the pore size of the membrane, which broadens the selection range of the membrane, and at the same time realizes the full use of the dextranase, thereby improving the yield of the target product.

[0069] (2) The specific molecular weight oligomeric dextran provided by the present invention has a narrow molecular weight distribution, exhibits high stability during storage and use, and has high biocompatibility.

[0070] (3) The application of the oligodextran with a specific molecular weight provided by the present invention has important application value in the fields of biomedicine and pharmaceuticals, for example, it can be used as a raw material for veterinary iron supplement injections, plasma substitutes or blood volume expanders.

[0071] (4) The continuous production device of oligodextran of specific molecular weight provided by the present invention is provided with a liquid level monitoring component and a flow rate monitoring component on the basis of a traditional enzyme membrane reactor. The two components work synergistically to enable the enzyme membrane reactor to operate continuously and self-regulate the molecular weight of the product. The device has high integration, simple operation, safety and energy saving, and is easy to industrialize and mass-produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 It is a schematic diagram of the structure and connection relationship of the continuous production device of oligomeric dextran of a specific molecular weight provided in Example 1 of the present invention;

[0073] In the figure: 1. Substrate storage tank; 2. Pre-reaction tank; 3. Feed liquid delivery pipeline; 4. Membrane assembly; 5. Reflux pipeline; 6. Product collection tank; 7. First pressure pump; 8. Second pressure pump; 9. Liquid level monitoring sensor; 10. Electronic balance; 11. Water bath thermostatic tank. DETAILED DESCRIPTION

[0074] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0075] The dextranase used in the following examples and comparative examples was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., model FDY-2207. The present invention only needs to use dextranase with endoglycosidic bond function without special restrictions; and the following examples and comparative examples are exemplified by polyethersulfone spiral ultrafiltration membrane components, but are not intended to limit the present invention.

[0076] 1. Implementation

[0077] Example 1

[0078] This embodiment provides a continuous production device for oligomeric dextran of a specific molecular weight, such as Figure 1 As shown, the continuous production device comprises a substrate storage tank 1, an enzyme membrane reactor and a product collection tank 6 which are connected in sequence;

[0079] Along the flow direction of the feed liquid, the enzyme membrane reactor comprises a pre-reaction tank 2, a feed liquid delivery pipeline 3 (pre-reaction feed liquid), a membrane assembly 4 (polyethersulfone rolled ultrafiltration membrane assembly 4) and a reflux pipeline 5 which are cyclically connected in sequence;

[0080] The substrate storage tank 1 is connected to the pre-reaction tank 2; a first pressure pump 7 is provided on the pipeline between the substrate storage tank 1 and the pre-reaction tank 2; a second pressure pump 8 is provided on the feed liquid delivery pipeline 3 in the enzyme membrane reactor;

[0081] A liquid level monitoring sensor 9 is provided in the pre-reaction tank 2; the product collection tank 6 is arranged at the lower side of the membrane assembly 4; the product collection tank 6 is placed on a flow monitoring component, and the flow monitoring component is an electronic balance 10; the substrate storage tank 1 and the pre-reaction tank 2 are placed in a water bath thermostatic tank.

[0082] This embodiment provides a method for continuous production of oligodextran with a specific molecular weight, which is carried out using the above-mentioned continuous production device. The continuous production method comprises the following steps:

[0083] (1) 300 mL of 10 g / L dextran substrate (the weight average molecular weight of the dextran raw material is 70 kDa) and 10 g / L dextran enzyme solution are mixed at a mass ratio of 200:1 and pre-reacted for 5 min to obtain a pre-reaction solution;

[0084] (2) at 25° C., the pre-reaction liquid in step (1) is subjected to enzymatic hydrolysis in an enzyme membrane reactor, and the enzymatic hydrolysis product is synchronously and continuously separated at a membrane pressure of 0.25 MPa, and the dextran substrate is continuously added to the pre-reaction liquid at a flow rate equal to the flow rate of the enzymatic hydrolysis product;

[0085] The membrane module in the enzyme membrane reactor is a rolled ultrafiltration membrane module, the membrane material is polyethersulfone, the molecular weight cutoff is 30kDa, and the pure water flux is 97LMH bar. -1 ;

[0086] The cross-flow rate of the enzymatic hydrolysis product is 0.7 L / min; the residence time of the pre-reaction liquid in the enzyme membrane reactor is 1 h; experimental measurements show that the average molecular weight of the oligodextran liquid obtained within an operation time of 1-3 h is in the range of 3-6 kDa.

[0087] Example 2

[0088] This embodiment provides a method for continuous production of oligomeric dextran of a specific molecular weight, which is carried out using the same continuous production device as in Example 1, and comprises the following steps:

[0089] (1) mixing 300 mL of 50 g / L dextran substrate (the weight average molecular weight of the dextran raw material is 850 kDa) with 50 g / L dextranase solution at a mass ratio of 150:1 and performing a pre-reaction for 15 min to obtain a pre-reaction solution;

[0090] (2) at 30° C., the pre-reaction liquid in step (1) is subjected to enzymatic hydrolysis in an enzyme membrane reactor, and the enzymatic hydrolysis product is synchronously and continuously separated at a membrane pressure of 0.4 MPa, and the dextran substrate is continuously added to the pre-reaction liquid at a flow rate equal to the flow rate of the enzymatic hydrolysis product;

[0091] The membrane module in the enzyme membrane reactor is a rolled ultrafiltration membrane module, the membrane material is polyethersulfone, the molecular weight cutoff is 50kDa, and the pure water flux is 120LMH bar -1 ;

[0092] The cross-flow rate of the enzymatic hydrolysis product is 0.6 L / min; the residence time of the pre-reaction liquid in the enzyme membrane reactor is 0.8 h; experimental measurements show that the average molecular weight of the oligodextran liquid obtained within an operation time of 1-3 h is in the range of 3-6 kDa.

[0093] Example 3

[0094] This embodiment provides a method for continuous production of oligomeric dextran of a specific molecular weight, which is carried out using the same continuous production device as in Example 1, and comprises the following steps:

[0095] (1) mixing 300 mL of a 100 g / L dextran substrate (the weight average molecular weight of the dextran raw material is 1000 kDa) with a 100 g / L dextranase solution at a mass ratio of 600:1 and performing a pre-reaction for 30 min to obtain a pre-reaction solution;

[0096] (2) at 45° C., the pre-reaction liquid in step (1) is subjected to enzymatic hydrolysis in an enzyme membrane reactor, and the enzymatic hydrolysis product is synchronously and continuously separated at a membrane pressure of 0.1 MPa, and the dextran substrate is continuously added to the pre-reaction liquid at a flow rate equal to the flow rate of the enzymatic hydrolysis product;

[0097] The membrane module in the enzyme membrane reactor is a rolled ultrafiltration membrane module, the membrane material is polyethersulfone, and its molecular weight cutoff is 20kDa and the pure water flux is 50LMH bar. -1 ;

[0098] The cross-flow rate of the enzymatic hydrolysis product is 1 L / min; the residence time of the pre-reaction liquid in the enzyme membrane reactor is 2 hours; experimental measurements show that the average molecular weight of the oligodextran liquid obtained within an operation time of 1-3 hours is in the range of 3-6 kDa.

[0099] Example 4

[0100] This embodiment provides a method for continuous production of oligomeric dextran of a specific molecular weight. The method is the same as that of Embodiment 1 except that the mass ratio of the dextran raw material to the dextranase is changed to 600:1 and the residence time of the pre-reaction liquid is changed to 2 hours.

[0101] In this embodiment, the average molecular weight measured in the experiment when the operation time is 1-3 hours is in the range of 3-6 kDa.

[0102] Example 5

[0103] This embodiment provides a method for continuous production of oligomeric dextran of a specific molecular weight. The method is the same as that of Embodiment 1 except that the mass ratio of the dextran raw material to the dextranase is changed to 300:1 and the residence time of the pre-reaction liquid is changed to 1.5 h.

[0104] In this embodiment, the average molecular weight measured in the experiment when the operation time is 1-3 hours is in the range of 3-6 kDa.

[0105] Example 6

[0106] This embodiment provides a method for continuous production of oligomeric dextran of a specific molecular weight. The method is the same as that of Example 1 except that the volume of the dextran substrate is changed to 500 mL, so that the residence time of the pre-reaction liquid in the enzyme membrane reactor is increased to 4.6 h.

[0107] Compared with Example 1, since the mass ratio of the dextran raw material to the dextranase in this embodiment remains unchanged, and the residence time of the pre-reaction liquid increases, resulting in excessive enzymatic hydrolysis reaction, the experimentally measured average molecular weight of the operation time of 1-3 hours shows a downward trend, and the average molecular weight measured at 3 hours has dropped to 1.4 kDa, which does not meet the requirements of the product molecular weight.

[0108] Example 7

[0109] The present embodiment provides a method for continuous production of oligomeric dextran of a specific molecular weight, wherein the continuous production method only changes the mass ratio of the dextran raw material to the dextranase to 100:1, and changes the volume of the pre-reaction liquid to 500 mL so that the residence time of the pre-reaction liquid is maintained at 1 h, and the rest is the same as in Example 1.

[0110] Compared with Example 1, since the residence time of the pre-reaction liquid in this embodiment remains unchanged, and the mass ratio of the dextran raw material to the dextranase is reduced, resulting in an excessively fast enzymatic hydrolysis reaction rate, the experimentally measured average molecular weight for an operation time of 1-3 hours is in the range of 1.8-2 kDa, and the average molecular weight of the product is lower than the product molecular weight requirement.

[0111] Example 8

[0112] This embodiment provides a method for continuous production of oligomeric dextran of a specific molecular weight, wherein the continuous production method only changes the mass ratio of the dextran raw material to the dextranase to 300:1, and by changing the volume of the pre-reaction liquid to 250 mL, the residence time of the pre-reaction liquid is maintained at 1 h, and the rest is the same as in Example 1.

[0113] Compared with Example 1, since the residence time of the pre-reaction liquid in this embodiment remains unchanged, and the mass ratio of the dextran raw material to the dextranase increases, resulting in a slow enzymatic hydrolysis reaction rate, the experimentally measured average molecular weight for an operation time of 1-3 hours is in the range of 5-8 kDa, and the average molecular weight of the product is higher than the product molecular weight requirement.

[0114] Example 9

[0115] This embodiment provides a method for continuous production of oligomeric dextran with a specific molecular weight, wherein the method only changes the pure water flux of the polyethersulfone membrane material of the spiral ultrafiltration membrane assembly to 63LMH bar. -1 , the rest are the same as in Example 1.

[0116] Compared with Example 1, the average molecular weight of the experimentally measured operation time in this example is still in the range of 3-6 kDa at 1-3 hours, but due to the low pure water flux of the membrane material, the yield of the obtained product is reduced by about 24% compared with the product yield of Example 1.

[0117] Example 10

[0118] This embodiment provides a method for continuous production of oligomeric dextran with a specific molecular weight, wherein the method only changes the pure water flux of the polyethersulfone membrane material of the spiral ultrafiltration membrane assembly to 80LMH bar. -1 , the rest are the same as in Example 1.

[0119] Compared with Example 1, the average molecular weight of the experimentally measured operation time in this example is still in the range of 3-6 kDa at 1-3 hours, but due to the low water flux of the membrane material, the yield of the obtained product is reduced by about 12% compared with the product yield of Example 1.

[0120] Embodiment 11

[0121] This embodiment provides a method for continuous production of oligomeric dextran with a specific molecular weight, wherein the method only changes the pure water flux of the polyethersulfone membrane material of the spiral ultrafiltration membrane assembly to 130LMH bar. -1 , the rest are the same as in Example 1.

[0122] Compared with Example 1, the average molecular weight of the experimentally measured operation time in this example is still in the range of 3-6 kDa when the operation time is 1-3 hours. However, due to the higher pure water flux of the membrane material, the yield of the obtained product is increased by about 18% compared with the product yield of Example 1.

[0123] Example 12

[0124] This embodiment provides a method for continuous production of oligodextran with a specific molecular weight. The continuous production method only changes the molecular weight cutoff of the polyethersulfone membrane material of the spiral ultrafiltration membrane assembly to 20 kDa, and the rest is the same as that of Example 1.

[0125] Compared with Example 1, since the selected membrane cutoff molecular weight is still greater than the product molecular weight and has a better retention effect on dextranase, the average molecular weight measured by the experiment when the operation time is 1-3 hours is still in the range of 3-6 kDa.

[0126] Embodiment 13

[0127] This embodiment provides a method for continuous production of oligodextran of specific molecular weight. The continuous production method only changes the molecular weight cutoff of the polyethersulfone membrane material of the spiral ultrafiltration membrane assembly to 50 kDa, and the rest is the same as that of embodiment 1.

[0128] Compared with Example 1, since the selected membrane cutoff molecular weight is much larger than the product molecular weight, it still has a good retention effect on dextranase. The average molecular weight measured by the experiment when the operation time is 1-3 hours is still in the range of 3-6 kDa.

[0129] Embodiment 14

[0130] This embodiment provides a method for continuous production of oligodextran with a specific molecular weight. The continuous production method only changes the molecular weight cutoff of the polyethersulfone membrane material of the spiral ultrafiltration membrane assembly to 70 kDa, and the rest is the same as that of Example 1.

[0131] Compared with Example 1, since the selected membrane cutoff molecular weight is much larger than the product molecular weight, but the retention effect on dextranase is poor, dextranase leakage occurs, resulting in the continued participation of the permeate side product in the reaction. The experimentally measured average molecular weight for an operation time of 1-3 hours is less than 1 kDa, which does not meet the product requirements.

[0132] Embodiment 15

[0133] This embodiment provides a method for continuous production of oligodextran with a specific molecular weight. The method is the same as that of Embodiment 1 except that the pre-reaction time in step (1) is 2 minutes.

[0134] Compared with Example 1, the pre-reaction time described in this embodiment is shortened, which only makes the initial molecular weight at the beginning of the reactor operation larger. The enzymatic reaction rate is not enough to match the membrane separation rate. The molecular weight of the output product in the initial stage is relatively large. As the time for the reactor to stably produce products of suitable molecular weight increases, the operation of the reactor tends to be stable after 5-15 minutes. The average molecular weight measured in the experiment for an operation time of 1-3 hours is 3-6 kDa, but its molecular weight distribution range becomes wider.

[0135] Example 16

[0136] This embodiment provides a method for continuous production of oligodextran with a specific molecular weight. The method is the same as that of Embodiment 1 except that the actual operation time is extended to 28 hours.

[0137] Compared with Example 1, the average molecular weight measured in the experiment of this example is still in the range of 3-6 kDa when the operation time is 1-28 hours, and the yield of dextran increases with time.

[0138] Embodiment 17

[0139] This embodiment provides a method for continuous production of oligomeric dextran of a specific molecular weight. The method is the same as that of Embodiment 1 except that the flow rate of continuously replenishing the dextran substrate in step (2) is lower than the flow rate of the enzymatic hydrolysis product.

[0140] Compared with Example 1, in this embodiment, the flow rate of supplementing the dextran substrate is too small to provide the substrate required for the dextranase reaction. The average molecular weight of the operation time of 1-3 hours is experimentally measured to be 1.4-2.2 kDa, and as the reaction time increases, the circulatory reaction liquid in the reactor continues to decrease, and the steady-state operation of the reactor cannot be supported.

[0141] Embodiment 18

[0142] This embodiment provides a method for continuous production of oligodextran of specific molecular weight. The method is the same as that of embodiment 1 except that the flow rate of continuously replenishing the dextran substrate in step (2) is higher than the flow rate of the enzymatic hydrolysis product.

[0143] Compared with Example 1, in this embodiment, due to the large flow rate of supplementing the dextran substrate, the dextranase cannot convert the macromolecular substrate to the target molecular weight in time. The average molecular weight of the operation time of 1-3 hours is 6-15 kDa, and as the reaction time increases, the macromolecular reaction liquid circulating in the reactor continues to accumulate, and the average molecular weight of the product continues to increase.

[0144] 2. Comparison

[0145] Comparative Example 1

[0146] This comparative example provides a method for the continuous production of oligodextran, which is the same as Example 1 except that the pre-reaction in step (1) is not performed.

[0147] Compared with Example 1, this comparative example reduces the time of the pre-reaction. In the initial stage of the reactor operation, the enzymatic reaction rate is not enough to match the membrane separation rate. The molecular weight of the output product in the initial stage is relatively large. Although the time for the reactor to stably produce products of suitable molecular weight increases, the operation of the reactor tends to be stable after 5-15 minutes. The experimental measurement shows that the average molecular weight of the operation time of 1-3 hours is 3-6 kDa, but its molecular weight distribution range becomes wider.

[0148] Comparative Example 2

[0149] This comparative example provides a method for producing oligodextran, which is the same as Example 1 except that dextran substrate is not supplemented in step (2).

[0150] Compared with Example 1, this comparative example lacks the supplement of dextran substrate. As the product is separated, the dextran substrate in the reactor continues to decrease, the dextranase remains unchanged, and is insufficient to provide the substrate required for the dextranase reaction. The experimental measurement shows that the average molecular weight of the product shows a continuous downward trend when the operation time is 1-3 hours, and as the reaction time increases, the circulatable reaction liquid in the reactor continues to decrease, and the steady-state operation of the reactor cannot be supported.

[0151] 3. Test and its results

[0152] The average molecular weight of the oligodextran with an operation time of 1-3h in the continuous production method described in the above embodiments and comparative examples was tested, and its polydispersity index (PDI value) and the yield of the oligodextran product with a specific molecular weight at 3h were calculated. Since the average molecular weight and PDI values ​​of the samples tested at different time periods fluctuated, range data were presented. The results are shown in Table 1;

[0153] Table 1

[0154]

[0155]

[0156] From the data in Table 4, we can see that:

[0157] (1) It can be seen from Examples 1 to 5 that the method for continuous production of oligodextran of a specific molecular weight provided by the present invention, through pre-reaction and continuous supplementation of substrate, and by further regulating the mass ratio of the dextran raw material to the dextranase, the residence time of the pre-reaction liquid, and controlling the flow rate of the continuously supplemented dextran substrate to be the same as the flow rate of the enzymatic hydrolysis product, achieves the continuous production of oligodextran with an average molecular weight of 3-6 kDa (operation time of 1-3 h), and the molecular weight distribution is narrow, and the PDI value is in the range of 1.3-1.4.

[0158] (2) Combining Example 1 with Examples 6 to 8, it can be seen that: compared with Example 1, Example 6 maintains the mass ratio of the dextran raw material to the dextranase unchanged, while increasing the residence time of the pre-reaction liquid, resulting in the average molecular weight decreasing to less than 1.4 kDa at 3 h; Example 7 maintains the residence time of the pre-reaction liquid unchanged, reduces the mass ratio of the dextran raw material to the dextranase, resulting in the average molecular weight within the range of 1.8-2 kDa during the operation time of 1-3 h; Example 8 maintains the residence time of the pre-reaction liquid unchanged, increases the mass ratio of the dextran raw material to the dextranase, resulting in the average molecular weight within the range of 5-8 kDa during the operation time of 1-3 h; thus, the present invention further achieves continuous production of oligodextran with an average molecular weight of 3-6 kDa (operation time of 1-3 h) by further regulating the mass ratio of the dextran raw material to the dextranase to (150-600):1 and the residence time with the pre-reaction liquid to (1-2) h.

[0159] (3) Combining Example 1 with Examples 9 to 14, it can be seen that: compared with Example 1, the pure water flux of the polyethersulfone membrane material described in Examples 9 to 10 is reduced, the average molecular weight of the product remains unchanged, and the yield is reduced by 24% and 12% respectively compared with the yield of the product in Example 1; the pure water flux of the polyethersulfone membrane material described in Example 11 is increased, the average molecular weight of the product remains unchanged, and the yield is increased by 18% compared with the yield of the product in Example 1; in Examples 12 and 13, only the molecular weight cutoff of the polyethersulfone membrane material is changed (to ensure that the membrane can completely pass through the target molecular weight product and completely intercept the enzyme), but the average molecular weight of the product remains unchanged; the molecular weight cutoff of the polyethersulfone membrane material described in Example 14 is too high to effectively intercept the enzyme molecules, resulting in enzyme leakage and continued reaction on the product side, causing changes in the molecular weight of the product; this shows that the average molecular weight of the oligodextran product produced by the continuous production method of the present invention does not depend on the pure water flux of the membrane and its molecular weight cutoff, and by further optimizing the water flux of the membrane in the porous ultrafiltration membrane assembly to be 60-150LMH bar -1The molecular weight cutoff of the membrane in the porous ultrafiltration membrane assembly is 20-50 kDa, and a membrane that can effectively intercept the enzyme and has a larger flux is screened out, further increasing the yield of the product.

[0160] (4) Combining Example 1 with Examples 15 to 16, it can be seen that: compared with Example 1, the pre-reaction time in Example 15 is shorter, resulting in a longer time for the reactor to stably produce a product of suitable molecular weight, and the molecular weight of the product produced in the initial stage is relatively large, which shows that the present invention further prefers that the reaction time is 5 to 30 minutes, which is conducive to keeping the product molecular weight in the range of 3-6 kDa; in Example 16, the actual operation time is extended to 28 hours, and the average molecular weight of the product is still in the range of 3-6 kDa (within 1-28 hours), which shows that the continuous production method of the present invention has long-term stability and can achieve long-term stable continuous production.

[0161] (5) Combining Example 1 with Examples 17 to 18, it can be seen that compared with Example 1, the flow rate of continuously replenishing the dextran substrate in Example 17 is relatively low, resulting in a low average molecular weight of the product, which is 1.4-2.2 kDa, which does not meet the production requirements of oligodextran; the flow rate of continuously replenishing the dextran substrate in Example 18 is relatively high, resulting in a high average molecular weight of the product, which is 6-15 kDa, and the molecular weight distribution is relatively wide, with a PDI value of 1.5-3; this shows that the present invention further preferably has the flow rate of continuously replenishing the dextran substrate be the same as the flow rate of the enzymatic hydrolysis product, which can further make the molecular weight distribution of the obtained product narrower.

[0162] (6) It can be seen from the combination of Example 1 and Comparative Examples 1 to 2 that: since the method described in Comparative Example 1 does not carry out continuous pre-reaction, the time for the reactor to stably produce a product of suitable molecular weight is prolonged within the actual operation time of 1-3 hours, and the molecular weight distribution of the product becomes wider; since the method described in Comparative Example 2 does not carry out substrate supplementation, the average molecular weight of the product continues to decrease within the actual operation time of 1-3 hours, the molecular weight distribution of the product is relatively wide (the maximum value of PDI is 3), and the dextranase cannot be fully used, resulting in reduced economic benefits.

[0163] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for continuous production of oligodextran of specific molecular weight, characterized in that: The continuous production method comprises the following steps: (1) mixing a dextran substrate and a dextranase solution for pre-reaction to obtain a pre-reaction liquid; (2) The pre-reaction liquid in step (1) is hydrolyzed by an enzyme membrane reactor and the hydrolysis product is separated synchronously and continuously, and the dextran substrate is continuously added to the pre-reaction liquid. The hydrolysis product is an oligodextran liquid with a specific molecular weight.

2. The continuous production method according to claim 1, characterized in that: The weight average molecular weight of the dextran raw material in the dextran substrate of step (1) is 40-1000 kDa; Preferably, the concentration of the dextran raw material in the dextran substrate in step (1) is 10-200 g / L; Preferably, the concentration of dextranase in the dextranase solution in step (1) is 10-200 g / L; Preferably, the mass ratio of the dextran raw material to the dextranase is (50-1000):1, preferably (150-600):1; Preferably, the volume of the pre-reaction liquid in step (1) is 0.2-50 L, preferably 0.2-20 L; Preferably, the pre-reaction time in step (1) is 5-30 min.

3. The continuous production method according to claim 1 or 2, characterized in that: The membrane pressure of the enzyme membrane reactor in step (2) is 0.1-0.4 MPa; Preferably, the temperature of the enzymatic hydrolysis in step (2) is 25-45°C; Preferably, the residence time of the pre-reaction liquid in step (2) in the enzyme membrane reactor is 0.5-4h, preferably 1-2h; Preferably, the cross-flow rate of the enzymatic hydrolysis product in step (2) is 0.6-1 L / min; Preferably, the retentate obtained by separation in step (2) is circulated to the pre-reaction liquid; Preferably, the retentate in step (2) comprises dextranase.

4. The continuous production method according to any one of claims 1 to 3, characterized in that: The membrane assembly in the enzyme membrane reactor in step (2) comprises a porous ultrafiltration membrane assembly; Preferably, the porous ultrafiltration membrane assembly comprises any one of a rolled ultrafiltration membrane assembly, a tubular ultrafiltration membrane assembly, a flat ultrafiltration membrane assembly or a hollow fiber ultrafiltration membrane assembly, or a combination of at least two thereof; Preferably, the molecular weight cutoff of the membrane in the porous ultrafiltration membrane assembly is from 20 kDa to the molecular weight of dextranase, preferably 20-50 kDa; Preferably, the water flux of the membrane in the porous ultrafiltration membrane assembly is 60-150LMH bar -1 .

5. The continuous production method according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: (1) mixing a dextran substrate having a concentration of 10-200 g / L and a weight average molecular weight of 40-1000 kDa with a dextran enzyme solution having a concentration of 10-200 g / L at a mass ratio of dextran raw material to dextran enzyme of (50-1000):1 and performing a pre-reaction for 5-30 min to obtain a pre-reaction feed solution; The volume of the pre-reaction liquid is 0.2-50L; (2) at 25-45° C., the pre-reaction liquid in step (1) is subjected to enzymatic hydrolysis in an enzyme membrane reactor, and the enzymatic hydrolysis product is synchronously and continuously separated at a membrane pressure of 0.1-0.4 MPa, and the dextran substrate is continuously added to the pre-reaction liquid, wherein the enzymatic hydrolysis product is an oligodextran liquid with a specific molecular weight; The residence time of the pre-reaction liquid in the enzyme membrane reactor is 0.5-4h; the cross-flow rate of the enzymatic hydrolysis product is 0.6-1L / min; the separated retentate is circulated to the pre-reaction liquid; the retentate includes dextranase; The membrane assembly in the enzyme membrane reactor includes a porous ultrafiltration membrane assembly; the membrane in the porous ultrafiltration membrane assembly has a molecular weight cutoff of 20 kDa to the molecular weight of dextranase, and a water flux of 60-150 LMH bar. -1 .

6. A specific molecular weight oligodextran, characterized in that: The specific molecular weight oligodextran is prepared by the continuous production method of the specific molecular weight oligodextran according to any one of claims 1 to 5.

7. The specific molecular weight oligodextran according to claim 6, characterized in that: The average molecular weight of the specific molecular weight oligosaccharide anhydride is 3-6 kDa.

8. A use of the specific molecular weight oligodextran as claimed in claim 6 or 7, characterized in that: The specific molecular weight oligodextran is used as a raw material for veterinary iron supplement injection, plasma substitute or blood volume expansion medicine.

9. A continuous production device for oligodextran of specific molecular weight, characterized in that: The continuous production method according to any one of claims 1 to 5 is carried out using the continuous production device; The continuous production device comprises a substrate storage component, an enzyme membrane reactor and a product collection component which are connected in sequence; Along the flow direction of the feed liquid, the enzyme membrane reactor comprises a pre-reaction component, a feed liquid conveying pipeline, a membrane assembly and a reflux pipeline which are cyclically connected in sequence; The substrate storage component is connected to the pre-reaction component; the product collection component is arranged on the lower side of the membrane assembly; The pre-reaction component is provided with a liquid level monitoring component; and the product collecting component is placed on the flow monitoring component.

10. The continuous production device according to claim 9, characterized in that: A first pressurizing component is provided on the pipeline between the substrate storage component and the pre-reaction component; Preferably, a second pressurizing component is provided on the liquid delivery pipeline in the enzyme membrane reactor; Preferably, the substrate storage part and the pre-reaction part are placed in a water bath thermostatic part.

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