High activity dextranase production system and production method

Abstract

The application discloses a high-activity dextranase preparation system and a preparation method. The high-activity dextranase preparation system comprises a fermentation device and a purification device. The fermentation device comprises a fermentation tank, a monitoring mechanism, a feeding mechanism, a continuous flow circulation mechanism, a microfluid impedance spectrum analysis mechanism and a control mechanism. The monitoring mechanism is connected to the fermentation tank. The feeding mechanism is connected to the fermentation tank to feed liquid into the fermentation tank. The continuous flow circulation mechanism is connected between the bottom and the top of the fermentation tank to cyclically process the fermentation liquid in the fermentation tank. The microfluid impedance spectrum analysis mechanism is connected to the continuous flow circulation mechanism to collect metabolic flow characteristic spectrum. The control mechanism can execute an adaptive pulse feeding algorithm according to the metabolic flow characteristic spectrum and control the feeding mechanism to feed. The purification device purifies the fermentation product from the fermentation tank through a nine-fold filtration purification process. The application improves the purity, the enzyme activity, the production efficiency and reduces the cost.

Description

Technical Field

[0001] This application relates to the fields of bioenzyme engineering and biomanufacturing technology, and in particular to a high-activity dextranase preparation system and preparation method. Background Technology

[0002] Dextranase, a key industrial enzyme, is widely used in food processing (such as sucrose refining) to remove dextran impurities, in the pharmaceutical field (such as preventing and treating dextran-induced thrombosis), in the bio-fermentation industry (such as reducing the interference of dextran in fermentation systems on product extraction), and in oral care. For example, the application of dextranase in oral care products stems primarily from its highly efficient and specific ability to degrade dextran. The main matrix component of dental plaque biofilm is dextran (especially α-1,6-glycosidic linked dextran), which is synthesized by cariogenic bacteria (such as Streptococcus mutans) using sucrose and promotes bacterial adhesion and plaque aggregation. Dextranase can hydrolyze the α-1,6-glycosidic bonds of dextran, disrupting the plaque matrix structure and preventing its formation and accumulation. It is also environmentally friendly. This technology is often combined with fluoride, other enzymes (such as lysozyme), or low-concentration antibacterial agents to achieve sustained release and synergistic effects through toothpaste, mouthwash, and other dosage forms.

[0003] Traditional dextranase production mainly relies on microbial fermentation, with commonly used strains including molds and bacteria. However, existing strains suffer from drawbacks such as low enzyme activity expression and limited product conversion efficiency. Traditional fermentation processes often employ a batch-feed model, where the supply of nutrients (carbon and nitrogen sources) is not synchronized with the metabolic demands of microbial growth and enzyme synthesis. This leads to disordered energy metabolism, long fermentation cycles, and low product yields, thus limiting enzyme synthesis efficiency. Summary of the Invention

[0004] Therefore, it is necessary to provide a high-activity dextranase preparation system that can improve purity, enzyme activity, production efficiency, and reduce costs.

[0005] One embodiment of this application provides a high-activity dextranase preparation system.

[0006] A high-activity dextranase preparation system according to an embodiment of this application includes a fermentation device and a purification device. The fermentation device includes a fermenter, a monitoring mechanism, a feeding mechanism, a continuous flow circulation mechanism, a microfluidic impedance spectroscopy analysis mechanism, and a control mechanism. The monitoring mechanism is connected to the fermenter to monitor at least one parameter value of the fermentation broth in the fermenter. The feeding mechanism is connected to the fermenter to feed the broth into the fermenter. The continuous flow circulation mechanism is connected to the bottom and top of the fermenter to circulate the fermentation broth in the fermenter. The microfluidic impedance spectroscopy analysis mechanism is connected to the continuous flow circulation mechanism to collect metabolic flow characteristic spectra. The control mechanism is electrically connected to the monitoring mechanism, the feeding mechanism, the continuous flow circulation mechanism, and the microfluidic impedance spectroscopy analysis mechanism. The control mechanism can execute an adaptive pulse feeding algorithm based on the parameter values ​​and the metabolic flow characteristic spectra and control the feeding mechanism to feed the broth.

[0007] A purification device that purifies fermentation products from a fermenter using a nine-stage filtration and purification process.

[0008] In some embodiments, the fermenter is equipped with a stirring mechanism that can control the rotation speed from 100 rpm to 500 rpm.

[0009] In some embodiments, the monitoring mechanism includes at least one of a dissolved oxygen sensor, a pH sensor, a temperature sensor, and a metabolic heat sensor, and the control mechanism is electrically connected to the dissolved oxygen sensor, the pH sensor, the temperature sensor, and the metabolic heat sensor. The control mechanism is used to acquire parameter values ​​including dissolved oxygen, pH value, temperature, and metabolic heat.

[0010] In some embodiments, the feeding mechanism includes a multi-channel peristaltic pump, each channel of which is connected to a carbon source, a nitrogen source, and a metal ion source, respectively.

[0011] In some embodiments, the continuous flow circulation mechanism is equipped with a dual-screw peristaltic pump.

[0012] In some embodiments, the inlet end of the dual-screw peristaltic pump is connected to the bottom of the fermenter, and the outlet end of the dual-screw peristaltic pump is connected to the top of the fermenter.

[0013] In some embodiments, the inlet diameter of the dual-screw peristaltic pump is 70mm to 90mm.

[0014] In some embodiments, the outlet diameter of the dual-screw peristaltic pump is 110mm to 130mm.

[0015] In some embodiments, the purification device includes at least one of a plate and frame filter, a polypropylene pleated filter element, a polyethersulfone pleated microfiltration membrane module, a hydrophilic modified polysulfone ultrafiltration membrane module, a polyethersulfone ultrafiltration membrane module, a polyamide composite nanofiltration membrane module, a hydrophilic polyvinylidene fluoride membrane module, an aromatic polyamide reverse osmosis membrane module, and a polytetrafluoroethylene sterile pleated filter element.

[0016] In some embodiments, the filter media surface of the plate and frame filter has a diatomaceous earth filter aid layer with a thickness of 1 mm to 2 mm.

[0017] In some embodiments, the pore size of the polypropylene pleated filter element is 5μm to 6μm.

[0018] In some embodiments, the polyethersulfone pleated microfiltration membrane module meets the following conditions: it can withstand operating pressures ranging from 0.1 MPa to 0.4 MPa, and its continuous filtration flux attenuation rate is ≤5% / 10h.

[0019] In some embodiments, the hydrophilic modified polysulfone ultrafiltration membrane module has a molecular weight cutoff of 800KD to 820KD.

[0020] In some embodiments, the polyethersulfone ultrafiltration membrane module has a molecular weight cutoff of 5KD to 6KD.

[0021] In some embodiments, the polyamide composite nanofiltration membrane module has a molecular weight cutoff of 1KD to 2KD.

[0022] In some embodiments, the hydrophilic polyvinylidene fluoride membrane module has a pore size of 0.4 μm to 0.5 μm.

[0023] In some embodiments, the aromatic polyamide reverse osmosis membrane module meets the following conditions: it can withstand operating pressures ranging from 1.5 MPa to 2 MPa, and it is effective against monovalent ions (such as Na+) in the feed solution. + Cl - The retention rate is ≥99.2%, and the water permeability is ≥1.2m. 3 / (m 2 •d).

[0024] In some embodiments, the PTFE sterile pleated filter element has a pore size of 0.22 μm to 0.25 μm.

[0025] In some embodiments, the high-activity dextranase preparation system further includes a drying device connected to the purification device, the drying device being used to perform low-temperature drying of the target enzyme obtained from the purification device.

[0026] In some embodiments, the drying apparatus includes a freeze dryer.

[0027] In some embodiments, the drying apparatus is capable of gradient drying, and the gradient drying process includes:

[0028] Pre-freezing: Drying at -45℃ to -37℃ under 15Pa to 25Pa pressure for 1.5h to 2.5h;

[0029] Main drying: -35℃~-25℃, 8Pa~12Pa pressure for 8h~12h;

[0030] Drying: Dry at -20℃ to -15℃ and 4Pa to 6Pa pressure for 12 to 15 hours.

[0031] An embodiment of this application also provides a method for preparing a highly active dextranase.

[0032] A method for preparing a highly active dextranase according to an embodiment of this application includes the following steps:

[0033] Activate the bacterial strain to obtain seed liquid;

[0034] The seed liquid is fermented to obtain fermentation products. During the fermentation process, the fermentation liquid in the fermenter is circulated, at least one parameter value of the fermentation liquid in the fermenter is monitored, and the metabolic flow characteristic spectrum is collected. An adaptive pulse feeding algorithm is executed and feeding is performed based on the metabolic flow characteristic spectrum.

[0035] In addition, the fermentation products are purified.

[0036] In some embodiments, the bacterial species includes *Chaetoceros spp.*

[0037] In some embodiments, the seed culture is inoculated at a rate of 0.8% to 1.2% (v / v) relative to the fermentation medium in the fermenter.

[0038] In some implementations, the fermentation process meets the following conditions:

[0039] Fermentation temperature: 25℃~30℃;

[0040] Stirring speed: 100 rpm to 500 rpm;

[0041] Ventilation rate is 0.8 vvm to 1.2 vvm;

[0042] The total fermentation time is 40-42 hours.

[0043] In some embodiments, the following conditions are met during the circulation process of the fermentation broth in the fermenter:

[0044] The circulation flow rate is 1.8 L / h to 2.2 L / h;

[0045] The dissolved oxygen concentration in the fermentation broth is 20%~40%.

[0046] In some implementations, the following conditions are met during the execution of the adaptive pulse feeding algorithm and the feeding process:

[0047] (1) Carbon and nitrogen source replenishment: When the 1.2kHz component of the microfluidic impedance spectrum decays by more than 15%, a 0.5-second nitrogen source pulse injection is triggered to simultaneously replenish the carbon source and maintain the carbon-nitrogen ratio dynamically stable at (10~15):1; when the 3.8kHz component of the microfluidic impedance spectrum rises by more than 20%, the circulation flow rate is reduced by 15%~25% to control the fluctuation of the microbial ATP synthesis rate to <5%;

[0048] (2) Metal ion source feeding: After the preset fermentation time, add a metal ion mixture.

[0049] The preset time is ~24h: a magnesium source is added to control the Mg content in the fermentation broth. 2+ The concentration increased linearly to 5 mmol / L~10 mmol / L;

[0050] 24h~36h: Add manganese source to control Mn in fermentation broth 2+ The concentration increased linearly to 1 mmol / L~3 mmol / L;

[0051] 36h ~ Fermentation endpoint: Add zinc source to increase Zn concentration in the fermentation broth. 2+ The concentration increased linearly to 0.5 mmol / L~1.5 mmol / L.

[0052] In some embodiments, the carbon source comprises a glucose solution with a concentration of 200 g / L to 220 g / L.

[0053] In some embodiments, the nitrogen source comprises soybean meal hydrolysate with a concentration of 50 g / L to 60 g / L.

[0054] In some implementations, the preset time is any time point between 12h and 15h.

[0055] In some embodiments, the magnesium source comprises a MgSO4•7H2O solution.

[0056] In some embodiments, the manganese source comprises a MnSO4•H2O solution.

[0057] In some embodiments, the zinc source comprises a ZnSO4•7H2O solution.

[0058] In some embodiments, the filtration and purification process of the fermentation product is performed in accordance with the following nine-stage filtration and purification process conditions: the fermentation product is subjected to at least one of the following in sequence: first stage plate and frame pretreatment, second stage prefiltration, third stage microfiltration, fourth stage selective ultrafiltration, fifth stage fine ultrafiltration, sixth stage nanofiltration, seventh stage fine filtration and polishing, eighth stage reverse osmosis, and ninth stage aseptic terminal filtration.

[0059] In some embodiments, the purification process of the fermentation product is performed under the following nine-stage filtration purification process conditions: the fermentation product is subjected to the following stages in sequence: first stage plate and frame pretreatment, second stage prefiltration, third stage microfiltration, fourth stage selective ultrafiltration, fifth stage fine ultrafiltration, sixth stage nanofiltration, seventh stage fine filtration and polishing, eighth stage reverse osmosis, and ninth stage aseptic terminal filtration.

[0060] In some embodiments, the plate and frame pretreatment meets the following conditions: the fermentation product is introduced into a plate and frame filter press, the filter medium of which has a diatomaceous earth filter aid layer with a thickness of 1 mm to 2 mm, and the filter is filtered at 0.3 MPa to 0.4 MPa and 25°C to 28°C to obtain crude enzyme solution.

[0061] In some embodiments, the pre-filtration process meets the following conditions: 0.1% to 0.3% (w / v) of flocculant is added to the crude enzyme solution, stirred at 180 rpm to 220 rpm for 15 min to 20 min, and then filtered through a 5 μm to 6 μm polypropylene pleated filter cartridge to obtain the filtrate.

[0062] In some embodiments, the flocculant includes a chitosan-magnesium silicate composite flocculant.

[0063] In some embodiments, the microfiltration process satisfies the following conditions: the filtrate is introduced into a polyethersulfone pleated microfiltration membrane module and cross-flow filtration is performed at 0.15MPa~0.2MPa, 18℃~20℃, and a cross-flow velocity of 1.5m / s~2m / s to obtain the permeate.

[0064] In some embodiments, backwashing is performed for 1 to 2 minutes after every 30 to 40 minutes of filtration to control the turbidity of the permeate to ≤0.1 NTU.

[0065] In some implementations, an online air-water backflushing system is used for backflushing.

[0066] In some embodiments, the selective ultrafiltration process satisfies the following conditions: the permeate is introduced into a hydrophilic modified polysulfone ultrafiltration membrane module with a molecular weight cutoff of 800KD~820KD, and ultrafiltration is performed at 0.2MPa~0.3MPa, 18℃~20℃, and cross-flow velocity of 1.5m / s~2m / s to obtain a first ultrafiltrate.

[0067] In some embodiments, the fine ultrafiltration process satisfies the following conditions: the first ultrafiltrate is introduced into a polyethersulfone ultrafiltration membrane module with a molecular weight cutoff of 5KD~6KD, and concentration and dialysis are performed alternately at 0.3MPa~0.4MPa and 18℃~20℃.

[0068] Concentrate the first ultrafiltrate to 1 / 10 to 1 / 8 of its original volume, add an equal volume of phosphate buffer with a pH of 5.5 to 6.5, and dialyze for 20 to 30 minutes. Repeat this process 2 to 3 times to obtain the second ultrafiltrate. Control the conductivity of the second ultrafiltrate to ≤5 mS / cm.

[0069] In some embodiments, the nanofiltration process satisfies the following conditions: the second ultrafiltrate is introduced into a polyamide composite nanofiltration membrane module with a molecular weight cutoff of 1KD to 2KD, and the pH value is adjusted to 5 to 6. The solution is then concentrated to 1 / 20 to 1 / 18 of its original volume at 0.7MPa to 0.8MPa and 18°C ​​to 20°C to obtain the nanofiltrate.

[0070] In some embodiments, the fine filtration and polishing process satisfies the following conditions: the nanofiltration solution is introduced into a hydrophilic polyvinylidene fluoride membrane module with a pore size of 0.4μm~0.5μm, and filtered at 0.1MPa~0.15MPa and 18℃~20℃ to obtain a fine filtrate, and the fine filtrate is controlled to meet the following conditions: the number of particles greater than or equal to 0.5μm is ≤10 particles / mL.

[0071] In some embodiments, the reverse osmosis treatment satisfies the following conditions: the fine filtrate is introduced into an aromatic polyamide reverse osmosis membrane module and subjected to reverse osmosis treatment at 1.1 MPa~1.2 MPa and 18℃~20℃ to obtain a concentrate.

[0072] In some embodiments, the permeate from the reverse osmosis treatment is returned to the fine ultrafiltration process as dialysis water.

[0073] In some embodiments, the aseptic terminal filtration process meets the following condition: the concentrate is filtered through a polytetrafluoroethylene (PTFE) aseptic pleated filter cartridge with a pore size of 0.22 μm to 0.25 μm, and the aseptic assurance level is controlled to reach 10. -6 .

[0074] In some embodiments, the method for preparing high-activity dextranase further includes the following step: drying the purified target enzyme.

[0075] In some embodiments, the drying process satisfies the following conditions:

[0076] The target enzyme is subjected to gradient drying. The gradient drying procedure includes:

[0077] Pre-freezing: Drying at -45℃ to -37℃ under 15Pa to 25Pa pressure for 1.5h to 2.5h;

[0078] Main drying: -35℃~-25℃, 8Pa~12Pa pressure for 24h~28h;

[0079] Drying: Dry at -20℃ to -15℃ and 4Pa to 6Pa pressure for 12 to 15 hours.

[0080] In some embodiments, the method for preparing high-activity dextranase employs the high-activity dextranase preparation system described in any of the above embodiments.

[0081] One embodiment of this application provides a dextranase.

[0082] According to an embodiment of this application, a dextranase is prepared using at least one of the high-activity dextranase preparation system and the high-activity dextranase preparation method described in any of the above embodiments.

[0083] In some embodiments, the dextranase satisfies the following conditions:

[0084] Enzyme activity expression level: 27000U / mL~28900U / mL;

[0085] Enzyme activity loss rate: 12.9%~15.1%;

[0086] Fermentation time: 40h~42h.

[0087] One embodiment of this application provides an application of dextranase.

[0088] The application of dextranase in the preparation of oral care products includes dextranase prepared using the high-activity dextranase preparation system described in any of the above embodiments, dextranase prepared using the high-activity dextranase preparation method described in any of the above embodiments, and the application of at least one of the dextranases described in any of the above embodiments in the preparation of oral care products.

[0089] In some embodiments, the oral care product includes toothpaste.

[0090] The high-activity dextranase preparation system of this application can improve the purity and activity of enzyme preparations, and the enzyme preparations have high stability and a wide range of applications. It can effectively broaden the application system, improve the conversion capacity, conform to the concept of green manufacturing, and has high production efficiency and low cost.

[0091] This application's method for preparing highly active dextranase addresses the shortcomings of existing dextranase preparation technologies by using a bacterial strain, such as Chaetomium brevicornu, as the starting strain. It combines a refined feeding strategy with metabolic flux characteristic spectrum to execute an adaptive pulse feeding algorithm and perform feeding, achieving precise matching between microbial metabolism and enzyme synthesis. It innovatively introduces specific metal ions, nitrogen sources, and carbon sources to directionally promote metabolism and target the activation of key enzyme gene expression in dextranase synthesis. Attached Figure Description

[0092] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0093] To gain a more complete understanding of this application and its beneficial effects, the following description will be provided in conjunction with the accompanying drawings. In the following description, the same reference numerals denote the same parts.

[0094] Figure 1 This is a schematic diagram of a high-activity dextranase preparation system according to an embodiment of this application;

[0095] Figure 2 This is a schematic diagram of the process for preparing a highly active dextranase according to an embodiment of this application.

[0096] Explanation of reference numerals in the attached figures

[0097] 100. Fermentation device; 101. Fermentation tank; 103. Feeding mechanism; 104. Continuous flow circulation mechanism; 105. Microfluidic impedance spectroscopy analysis mechanism; 107. Stirring mechanism; 108. Multi-channel peristaltic pump; 109. Dual-screw peristaltic pump; 111. Online monitoring module; 112. Data processing unit; 113. Edge computing unit. Detailed Implementation

[0098] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, the specific embodiments of this application are described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0099] In the description of this application, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc., indicating the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application.

[0100] In this application, unless otherwise expressly specified and limited, the terms "installation," "connection," "joining," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise expressly limited. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0101] In this application, unless otherwise expressly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "on top of," and "over" the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0102] In the description of this application, "several" means one or more, "multiple" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the stated number, while "above," "below," and "within" are understood to include the stated number. The use of "first" and "second" in the description is merely for distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the order of the indicated technical features.

[0103] In this document, "optionally," "optionally," and "optional" mean that something is optional, that is, it is selected from either "with" or "without." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent. In this application, descriptions such as "optionally contains" and "optionally includes" indicate "contains or does not contain."

[0104] In this application, unless otherwise stated, the sum of the parts of each component in the composition may be 100 parts by weight. Unless otherwise specified, the percentages (including weight percentages) in this application are based on the total weight of the composition, and "wt%" in this document means mass percentage.

[0105] In this document, unless otherwise stated, the reaction steps may be performed in the order described herein or not. For example, other steps may be included between reaction steps, and the order of reaction steps may be appropriately interchanged. This is something that those skilled in the art can determine based on conventional knowledge and experience. Preferably, the reaction methods described herein are performed sequentially.

[0106] In this application, when numerical intervals (i.e., numerical ranges) are mentioned, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed in this application should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include percentage intervals, ratio intervals, proportion intervals, etc.

[0107] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0108] This application provides a high-activity dextranase preparation system to address at least one of the following technical problems in conventional dextranase preparation techniques: (1) traditional strains exhibit low enzyme activity expression and insufficient dextran conversion efficiency, making it difficult to achieve a synergistic improvement in both enzyme activity and conversion; (2) the traditional feeding mode leads to an imbalance in microbial energy metabolism, resulting in low fermentation efficiency, long cycle, and high production costs. The high-activity dextranase preparation system will be described below with reference to the accompanying drawings.

[0109] It should be noted that the following are explanations of some terms used in this application:

[0110] Dextranase: A hydrolase that specifically hydrolyzes the α-1,6-glycoanhydride bond in dextran (glucan). It can degrade large molecules of dextran into low molecular weight glucan or glucose. EC number (3.2.1.11).

[0111] Chaetomium gracile: A filamentous fungus belonging to the genus Chaetomium in the phylum Ascomycota, possessing the natural potential to synthesize dextranase.

[0112] Refined feeding strategy: Based on the laws of microbial metabolism, this feeding method dynamically adjusts the supply rate of nutrients (carbon source, nitrogen source, metal ions, etc.) by monitoring fermentation system parameters in real time.

[0113] Continuous flow energy metabolism: This mode maintains the stability of dissolved oxygen and metabolic product concentrations in the fermentation system through continuous circulation of the fermentation broth, thereby enabling the continuous and efficient energy metabolism of microorganisms.

[0114] Multistage membrane separation: A hierarchical separation system composed of membrane modules (microfiltration, ultrafiltration, nanofiltration, reverse osmosis) with different pore sizes and functions to achieve the gradual removal of impurities.

[0115] Microfluidic impedance spectroscopy analysis: a non-invasive monitoring technology that reflects the metabolic state of microorganisms in real time by measuring changes in the electrical properties of fermentation broth within a specific frequency range.

[0116] Adaptive pulse feeding algorithm: Based on the microfluidic impedance spectrum analysis results, an intelligent control method for executing millisecond-level precise pulse feeding is used as the trigger condition by characteristic spectrum changes.

[0117] The high-activity dextranase preparation system provided in one embodiment of this application is exemplary; please refer to [link to example]. Figure 1 As shown, Figure 1 This is a schematic diagram of a high-activity dextranase preparation system provided in one embodiment of this application. The high-activity dextranase preparation system of this application can significantly improve the industrial application value and conversion efficiency of enzyme preparations. For example, the dextranase prepared in this application can be used in toothpaste products. By adjusting the pH value through the proportions of humectants, preservatives, surfactants, and moisture content, the enzyme activity can be increased under the same dosage, thereby enhancing the efficacy of removing dental plaque and breaking down dental tartar.

[0118] To more clearly illustrate the structure of the high-activity dextranase preparation system, the following description, in conjunction with the accompanying drawings, will be provided.

[0119] For example, please refer to Figure 1 As shown, a high-activity dextranase preparation system includes a fermentation apparatus 100 and a purification apparatus. The fermentation apparatus 100 includes a fermenter 101, a monitoring mechanism, a feeding mechanism 103, a continuous flow circulation mechanism 104, a microfluidic impedance spectroscopy analysis mechanism 105, and a control mechanism. The monitoring mechanism is connected to the fermenter 101 to monitor at least one parameter value of the fermentation broth within the fermenter 101. The feeding mechanism 103 is connected to the fermenter 101 to replenish the fermentation broth within the fermenter 101. The continuous flow circulation mechanism 104 is connected between the bottom and top of the fermenter 101 to circulate the fermentation broth within the fermenter 101. The microfluidic impedance spectroscopy analysis mechanism 105 is connected to the continuous flow circulation mechanism 104 to acquire metabolic flux characteristic spectra. The control mechanism is electrically connected to the monitoring mechanism, the feeding mechanism 103, the continuous flow circulation mechanism 104, and the microfluidic impedance spectroscopy analysis mechanism 105. The control mechanism can execute an adaptive pulse feeding algorithm based on the parameter values ​​and the metabolic flux characteristic spectra and control the feeding mechanism 103 to perform feeding. The purification unit uses a nine-stage filtration and purification process to purify the fermentation products from fermenter 101.

[0120] The high-activity dextranase preparation system of this application can improve the purity and activity of enzyme preparations, and the enzyme preparations have high stability and a wide range of applications. It can effectively broaden the application system, improve the conversion capacity, conform to the concept of green manufacturing, and has high production efficiency and low cost.

[0121] In some embodiments, a stirring mechanism 107 is provided inside the fermenter 101. The stirring mechanism 107 is capable of controlling a rotation speed of 100 rpm to 500 rpm.

[0122] In some embodiments, the monitoring mechanism includes at least one of a dissolved oxygen sensor, a pH sensor, a temperature sensor, and a metabolic calorific sensor. The control mechanism is electrically connected to the dissolved oxygen sensor, pH sensor, temperature sensor, and metabolic calorific sensor. The parameters monitored by the monitoring mechanism include at least one of dissolved oxygen, pH value, temperature, and metabolic calorific value. The dissolved oxygen sensor, pH sensor, temperature sensor, and metabolic calorific sensor are not shown in the accompanying drawings.

[0123] In some embodiments, the feeding mechanism 103 includes a multi-channel peristaltic pump 108. Each channel of the multi-channel peristaltic pump 108 is connected to a carbon source, a nitrogen source, and a metal ion source, respectively.

[0124] In some embodiments, a dual-screw peristaltic pump 109 is provided on the continuous flow circulation mechanism 104.

[0125] In some embodiments, the inlet end of the dual-screw peristaltic pump 109 is connected to the bottom of the fermenter 101, and the outlet end of the dual-screw peristaltic pump 109 is connected to the top of the fermenter 101.

[0126] In some embodiments, the diameter of the inlet end of the dual-screw peristaltic pump 109 is 70mm to 90mm. For example, the diameter of the inlet end of the dual-screw peristaltic pump 109 may include, but is not limited to, 70mm, 75mm, 80mm, 85mm, 90mm or any range between the two mentioned above.

[0127] In some embodiments, the outlet diameter of the dual-screw peristaltic pump 109 is 110 mm to 130 mm. For example, the inlet diameter of the dual-screw peristaltic pump 109 may be, but is not limited to, 110 mm, 115 mm, 120 mm, 125 mm, 130 mm, or any range between the two mentioned above.

[0128] In some embodiments, the control mechanism includes an online monitoring module 111, a data processing unit 112, and an edge computing unit 113 that are electrically connected to each other. The online monitoring module 111 is electrically connected to the continuous flow circulation mechanism 104. The edge computing unit 113 is electrically connected to the multi-channel peristaltic pump 108 and the dual-screw peristaltic pump 109.

[0129] Furthermore, traditional technologies in the purification and formulation stages often employ methods such as single-stage chromatography, salting out, and spray drying, which suffer from cumbersome purification steps, limited purity improvement, and significant enzyme activity loss. These methods are insufficient to meet the demands of high-end industrial applications for high-purity (≥90%) and high-activity (≥1,000,000 U / g) dextranase. It is evident that traditional purification methods are cumbersome, involve high spray temperatures, result in significant enzyme activity loss, and are costly, failing to efficiently obtain high-activity dextranase. Therefore, this application further refines the purification apparatus to improve purification efficiency.

[0130] In some embodiments, the purification apparatus includes at least one of a plate and frame filter, a polypropylene pleated filter cartridge, a polyethersulfone (PES) pleated microfiltration membrane module, a hydrophilic modified polysulfone ultrafiltration membrane module, a polyethersulfone ultrafiltration membrane module, a polyamide composite nanofiltration membrane module, a hydrophilic polyvinylidene fluoride (PVDF) membrane module, an aromatic polyamide reverse osmosis membrane module, and a polytetrafluoroethylene (PTFE) sterile pleated filter cartridge.

[0131] In some embodiments, the filter media surface of the plate and frame filter press has a diatomaceous earth filter aid layer with a thickness of 1 mm to 2 mm. For example, the thickness of the diatomaceous earth filter aid layer includes, but is not limited to: 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, or any range between the foregoing.

[0132] In some embodiments, the pore size of the polypropylene pleated filter element is 5 μm to 6 μm. For example, the pore size of the polypropylene pleated filter element includes, but is not limited to, 5.1 μm, 5.2 μm, 5.3 μm, 5.4 μm, 5.5 μm, 5.6 μm, 5.7 μm, 5.8 μm, 5.9 μm, 6 μm, or any range between the foregoing.

[0133] In some embodiments, the polyethersulfone (PES) pleated microfiltration membrane module meets the following conditions: it can withstand operating pressures ranging from 0.1 MPa to 0.4 MPa, and the continuous filtration flux attenuation rate is ≤5% / 10h.

[0134] In some embodiments, the hydrophilic modified polysulfone ultrafiltration membrane module has a molecular weight cutoff of 800KD to 820KD.

[0135] In some embodiments, the polyethersulfone ultrafiltration membrane module has a molecular weight cutoff of 5KD to 6KD.

[0136] In some embodiments, the polyamide composite nanofiltration membrane module has a molecular weight cutoff of 1KD to 2KD.

[0137] In some embodiments, the pore size of the hydrophilic polyvinylidene fluoride (PVDF) membrane module is 0.4 μm to 0.5 μm. For example, the pore size of the hydrophilic PVDF membrane module includes, but is not limited to, 0.4 μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm, 0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.5 μm, or any range between the foregoing.

[0138] In some embodiments, the aromatic polyamide reverse osmosis membrane module meets the following conditions: it can withstand operating pressures ranging from 1.5 MPa to 2 MPa, and it is effective against monovalent ions (such as Na+) in the feed solution. + Cl - The retention rate is ≥99.2%, and the water permeability is ≥1.2m. 3 / (m 2 •d).

[0139] In some embodiments, the pore size of the aseptic pleated filter element of polytetrafluoroethylene (PTFE) is 0.22 μm to 0.25 μm. For example, the pore size of the aseptic pleated filter element of polytetrafluoroethylene (PTFE) includes, but is not limited to, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, or any range between the foregoing.

[0140] In some embodiments, the high-activity dextranase preparation system further includes a drying device. The drying device is connected to the purification device. The drying device is used to perform low-temperature drying of the target enzyme obtained from the purification device.

[0141] In some of these embodiments, the drying apparatus includes a freeze dryer.

[0142] In some embodiments, the drying apparatus is capable of gradient drying, and the gradient drying process includes:

[0143] Pre-freezing: Drying at -45℃ to -37℃ under 15Pa to 25Pa pressure for 1.5h to 2.5h;

[0144] Main drying: -35℃~-25℃, 8Pa~12Pa pressure for 8h~12h;

[0145] Drying: Dry at -20℃ to -15℃ and 4Pa to 6Pa pressure for 12 to 15 hours.

[0146] An embodiment of this application also provides a method for preparing a highly active dextranase.

[0147] See Figure 2 As shown, Figure 2This is a schematic flowchart of a method for preparing a highly active dextranase according to an embodiment of this application. The method includes the following steps:

[0148] Activate the bacterial strain to obtain seed liquid;

[0149] The seed liquid is fermented to obtain fermentation products. During the fermentation process, the fermentation liquid in the fermenter 101 is circulated. At least one parameter value of the fermentation liquid in the fermenter 101 is monitored, and the metabolic flow characteristic spectrum is collected. An adaptive pulse feeding algorithm is executed and feeding is performed based on the parameter value and the metabolic flow characteristic spectrum.

[0150] In addition, the fermentation products are purified.

[0151] This application's method for preparing highly active dextranase addresses the shortcomings of existing dextranase preparation technologies by using a bacterial strain, such as Chaetomium brevicornu, as the starting strain. It combines a refined feeding strategy with metabolic flux characteristic spectrum to execute an adaptive pulse feeding algorithm and perform feeding, achieving precise matching between microbial metabolism and enzyme synthesis. It innovatively introduces specific metal ions, nitrogen sources, and carbon sources to directionally promote metabolism and target the activation of key enzyme gene expression in dextranase synthesis.

[0152] Furthermore, this application can also achieve precise matching between microbial metabolism and enzyme synthesis by optimizing strain-directed domestication, combining a refined feeding strategy with metabolic flux characteristic spectrum execution of an adaptive pulse feeding algorithm.

[0153] In some of these embodiments, the microbial species include Chaetomium spp.

[0154] In some embodiments, the inoculum amount of seed culture relative to the fermentation medium in fermenter 101 is 0.8% (v / v) to 1.2% (v / v). For example, the inoculum amount of seed culture relative to the fermentation medium in fermenter 101 may include, but is not limited to, 0.8% (v / v), 0.9% (v / v), 1% (v / v), 1.1% (v / v), 1.2% (v / v), or a range between any two of the foregoing.

[0155] In some implementations, the fermentation process meets the following conditions:

[0156] Fermentation temperature: 25℃~30℃;

[0157] Stirring speed: 100 rpm to 500 rpm;

[0158] Ventilation rate is 0.8 vvm to 1.2 vvm;

[0159] The total fermentation time is 40-42 hours.

[0160] For example, during fermentation, the fermentation temperature may include, but is not limited to, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, or any range thereof. The stirring speed may include, but is not limited to, 100rpm, 200rpm, 300rpm, 400rpm, 500rpm, or any range thereof. The aeration rate may include, but is not limited to, 0.8vvm, 0.9vvm, 1vvm, 1.1vvm, 1.2vvm, or any range thereof. For example, the total fermentation time may include, but is not limited to, 40h, 40.5h, 41h, 41.5h, 42h, or any range thereof.

[0161] In some embodiments, during the circulation process of the fermentation broth in the fermenter 101, the following conditions are met:

[0162] The circulation flow rate is 1.8 L / h to 2.2 L / h;

[0163] The dissolved oxygen concentration in the fermentation broth is 20%~40%.

[0164] For example, during the circulation treatment of the fermentation broth in fermenter 101, the circulation flow rate may include, but is not limited to, 1.8 L / h, 1.9 L / h, 2 L / h, 2.1 L / h, 2.2 L / h, or any range between the two aforementioned. The dissolved oxygen concentration of the fermentation broth may include, but is not limited to, 20%, 25%, 30%, 35%, 40%, or any range between the two aforementioned.

[0165] In some implementations, the following conditions are met during the execution of the adaptive pulse feeding algorithm and the feeding process:

[0166] Carbon and nitrogen source replenishment: When the 1.2kHz component of the microfluidic impedance spectrum decays by more than 15%, a 0.5-second nitrogen source pulse injection is triggered, and carbon source is replenished simultaneously to maintain the carbon-nitrogen ratio dynamically stable at (10~15):1; when the 3.8kHz component of the microfluidic impedance spectrum rises by more than 20%, the circulation flow rate is reduced by 15%~25% to control the fluctuation of microbial ATP synthesis rate to <5%;

[0167] Metal ion source feeding: Add metal ion mixture after the preset fermentation time.

[0168] The preset time is ~24h: a magnesium source is added to control the Mg content in the fermentation broth. 2+ The concentration increased linearly to 5 mmol / L~10 mmol / L;

[0169] 24h~36h: Add manganese source to control Mn in fermentation broth 2+ The concentration increased linearly to 1 mmol / L~3 mmol / L;

[0170] 36h ~ Fermentation endpoint: Add zinc source to increase Zn concentration in the fermentation broth. 2+ The concentration increased linearly from 0.5 mmol / L to 1.5 mmol / L;

[0171] In some implementations, the preset time can be any time point between 10h and 15h.

[0172] In some implementations, the fermentation endpoint can be any time point between 40h and 42h.

[0173] In some embodiments, the magnesium source includes a MgSO4•7H2O solution.

[0174] In some embodiments, the manganese source includes a MnSO4•H2O solution.

[0175] In some embodiments, the zinc source includes a ZnSO4•7H2O solution.

[0176] In some embodiments, the carbon source includes a glucose solution with a concentration of 200 g / L to 220 g / L. For example, the concentration of the glucose solution may include, but is not limited to, 200 g / L, 201 g / L, 202 g / L, 204 g / L, 205 g / L, 206 g / L, 207 g / L, 208 g / L, 209 g / L, 210 g / L, or any range between the foregoing.

[0177] In some embodiments, the nitrogen source includes soybean meal hydrolysate with a concentration of 50 g / L to 60 g / L. For example, the concentration of the soybean meal hydrolysate may include, but is not limited to, 50 g / L, 52 g / L, 54 g / L, 56 g / L, 58 g / L, 60 g / L, or any range between the foregoing.

[0178] In some embodiments, the purification process of the fermentation product meets the following nine-stage filtration purification process conditions: the fermentation product is subjected to at least one of the following sequential stages: first stage plate and frame pretreatment, second stage prefiltration, third stage microfiltration, fourth stage selective ultrafiltration, fifth stage fine ultrafiltration, sixth stage nanofiltration, seventh stage fine filtration and polishing, eighth stage reverse osmosis, and ninth stage aseptic terminal filtration. The fermentation product is the fermentation supernatant.

[0179] In some embodiments, the purification process of the fermentation product is performed under the following conditions: the fermentation product is subjected to the following in sequence: first plate and frame pretreatment, second prefiltration, third microfiltration, fourth selective ultrafiltration, fifth fine ultrafiltration, sixth nanofiltration, seventh fine filtration and polishing, eighth reverse osmosis, and ninth aseptic terminal filtration.

[0180] In some embodiments, the plate and frame pretreatment meets the following conditions: the fermentation product is introduced into a plate and frame filter press, the filter media of the plate and frame filter press has a diatomaceous earth filter aid layer with a thickness of 1 mm to 2 mm, and the filter is filtered at 0.3 MPa to 0.4 MPa and 25°C to 28°C to remove more than 90% of large solids and obtain crude enzyme solution.

[0181] In some embodiments, the pre-filtration process meets the following conditions: 0.1% (w / v) to 0.3% (w / v) of flocculant is added to the crude enzyme solution, stirred at 180 rpm to 220 rpm for 15 min to 20 min, filtered through a 5 μm to 6 μm polypropylene pleated filter cartridge to retain flocculents and obtain filtrate.

[0182] In some embodiments, the flocculant includes a chitosan-magnesium silicate composite flocculant.

[0183] In some embodiments, the microfiltration process satisfies the following conditions: the filtrate is introduced into a polyethersulfone (PES) pleated microfiltration membrane module and cross-flow filtration is performed at 0.15 MPa to 0.2 MPa, 18°C ​​to 20°C, and a cross-flow velocity of 1.5 m / s to 2 m / s to obtain the permeate.

[0184] In some embodiments, backwashing is performed for 1 to 2 minutes after every 30 to 40 minutes of filtration to control the turbidity of the permeate to ≤0.1 NTU.

[0185] In some implementations, an online air-water backflushing system is used for backflushing.

[0186] In some embodiments, the selective ultrafiltration process satisfies the following conditions: the permeate is introduced into a hydrophilic modified polysulfone ultrafiltration membrane module with a molecular weight cutoff of 800KD~820KD, and ultrafiltration is performed at 0.2MPa~0.3MPa, 18℃~20℃, and a cross-flow velocity of 1.5m / s~2m / s to obtain a first ultrafiltrate, which selectively retains macromolecular heteroprotein complexes and polysaccharide polymers.

[0187] In some embodiments, the fine ultrafiltration process meets the following conditions: the first ultrafiltrate is introduced into a polyethersulfone ultrafiltration membrane module with a molecular weight cutoff of 5KD~6KD, and concentration and dialysis are performed alternately at 0.3MPa~0.4MPa and 18℃~20℃. After the first ultrafiltrate is concentrated to 1 / 10~1 / 8 of its original volume, an equal volume of phosphate buffer with a pH of 5.5~6.5 is added and dialyzed for 20min~30min. This process is repeated 2~3 times to obtain the second ultrafiltrate, and the conductivity of the second ultrafiltrate is controlled to be ≤5mS / cm.

[0188] In some embodiments, the nanofiltration process meets the following conditions: the second ultrafiltrate is introduced into a polyamide composite nanofiltration membrane module with a molecular weight cutoff of 1KD to 2KD, the pH of the feed solution is adjusted to 5 to 6, and the solution is concentrated to 1 / 20 to 1 / 18 of its original volume under conditions of 0.7MPa to 0.8MPa and 18°C ​​to 20°C to deeply remove small molecule impurities and obtain nanofiltrate.

[0189] In some embodiments, the fine filtration polishing process meets the following conditions: the nanofiltration solution is introduced into a hydrophilic polyvinylidene fluoride (PVDF) membrane module with a pore size of 0.4μm~0.5μm, and filtered at 0.1MPa~0.15MPa and 18℃~20℃ to obtain a fine filtrate, and the fine filtrate is controlled to meet the following conditions: the number of particles greater than or equal to 0.5μm is ≤10 particles / mL.

[0190] In some embodiments, the number of particles larger than or equal to 0.5 μm in the filtrate is monitored by an online particle counter.

[0191] In some embodiments, the reverse osmosis treatment meets the following conditions: the fine filtrate is introduced into an aromatic polyamide reverse osmosis membrane module and subjected to reverse osmosis treatment at 1.1 MPa~1.2 MPa and 18℃~20℃ to obtain a concentrate.

[0192] In some embodiments, the permeate from the reverse osmosis treatment is returned to the fine ultrafiltration process as dialysis water.

[0193] In some embodiments, the aseptic terminal filtration process meets the following condition: the concentrate is filtered through a 0.22μm~0.25μm PTFE aseptic pleated filter cartridge, and the sterility assurance level (SAL) is controlled to reach 10. -6 .

[0194] In some embodiments, the PTFE aseptic pleated filter cartridge uses a two-stage series configuration, that is, two PTFE aseptic pleated filter cartridges are connected in series. It is easy to understand that in other examples, the PTFE aseptic pleated filter cartridges can also be connected in multiple stages in series.

[0195] In some implementations, the concentrate is filtered in a Class A clean environment.

[0196] In some implementations, the filter cartridge integrity is tested using the bubble point method before and after filtration to obtain the sterility assurance level (SAL).

[0197] In one embodiment, this application constructs a multi-stage membrane separation high-efficiency purification system and purification method to simultaneously remove impurities such as sugars and proteins; combined with freeze-drying technology, it finally obtains customized dextranase with high purity (≥90%), high enzyme activity (≥1,000,000 U / g) and broad-spectrum temperature and pH application scenarios, which significantly improves the industrial application value and conversion efficiency of enzyme preparations.

[0198] In some embodiments, the method for preparing high-activity dextranase further includes the following step: drying the purified target enzyme.

[0199] In some embodiments, the drying process satisfies the following conditions:

[0200] The target enzyme is subjected to gradient drying. The gradient drying procedure includes:

[0201] Pre-freezing: -45℃ to -37℃, drying for 1.5h to 2.5h;

[0202] Main drying: -35℃~-25℃, 8Pa~12Pa pressure drying for 8h~12h;

[0203] Drying: Dry at -20℃ to -15℃ and 4Pa to 6Pa pressure for 12 to 15 hours.

[0204] For example, the pre-freezing temperature may include, but is not limited to: -45℃, -44℃, -43℃, -42℃, -41℃, -40℃, -39℃, -38℃, -37℃ or any two of the above; the pre-freezing drying time may include, but is not limited to: 1.5h, 1.6h, 1.8h, 1.9h, 2h, 2.1h, 2.2h, 2.3h, 2.4h, 2.5h or any two of the above.

[0205] For example, the main drying temperature may include, but is not limited to, -35℃, -34℃, -33℃, -32℃, -31℃, -30℃, -29℃, -28℃, -27℃, -26℃, -25℃ or any two of the above; the main drying pressure may include, but is not limited to, 8Pa, 9Pa, 10Pa, 11Pa, 12Pa or any two of the above; and the main drying time may include, but is not limited to, 8h, 9h, 10h, 11h, 12h or any two of the above.

[0206] For example, the values ​​of the desorption drying temperature include, but are not limited to: -20℃, -19℃, -18℃, -17℃, -16℃, -15℃ or any two of the above; the values ​​of the desorption drying pressure include, but are not limited to: 4Pa, 4.5Pa, 5Pa, 5.5Pa, 6Pa or any two of the above; the values ​​of the desorption drying time include, but are not limited to: 12h, 13h, 14h, 15h or any two of the above.

[0207] In some embodiments, the method for preparing high-activity dextranase employs the high-activity dextranase preparation system described in any of the above embodiments.

[0208] One embodiment of this application provides a dextranase.

[0209] According to an embodiment of this application, a dextranase is prepared using at least one of the high-activity dextranase preparation system and the high-activity dextranase preparation method described in any of the above embodiments.

[0210] In some embodiments, dextranase satisfies the following conditions:

[0211] Enzyme activity expression level: 27000U / mL~28900U / mL;

[0212] Enzyme activity loss rate: 12.9%~15.1%;

[0213] Fermentation time: 40h~42h.

[0214] One embodiment of this application provides an application of dextranase.

[0215] In some embodiments, the dextranase prepared using the high-activity dextranase preparation system of any of the above embodiments, the dextranase prepared using the high-activity dextranase preparation method of any of the above embodiments, and at least one of the dextranases in any of the above embodiments are used in the preparation of oral care products.

[0216] In some embodiments, the oral care product includes toothpaste. The application of dextranase in toothpaste offers the following advantages: 1) Dextranase can be applied to different formulation frameworks while maintaining enzyme activity, resulting in broader formulation compatibility. 2) Enhanced whitening effect; toothpaste with added dextranase exhibits better whitening efficacy indicators such as gloss and PCR value.

[0217] In one embodiment of this application, dextranase is more stable in formulation and can be used in multiple formulation frameworks, reducing the limitations of formulation use. At the same dosage, the enzyme activity is higher. For example, when used in toothpaste preparation, it has a better effect on removing dental plaque and tartar. Active ingredients in the toothpaste formulation, such as whitening ingredients like phosphates, HAP, and sodium fluoride, are more likely to come into contact with the tooth surface, thus improving the efficacy of the toothpaste.

[0218] Example 1

[0219] This embodiment provides a customized high-activity dextranase and its preparation method.

[0220] I. Experimental Materials and Equipment

[0221] (1) Microbial strains

[0222] Chaetomium gracile.

[0223] (2) Culture medium formulation

[0224] Slant culture medium: 200 g / L potato (boiled and filtered), 20 g / L glucose, 20 g / L agar, 3 g / L KH2PO4, 1 g / L MgSO4•7H2O, pH 6, sterilized at 121℃ for 20 min.

[0225] Seed culture medium: sugarcane molasses hydrolysate (30 g / L glucose), yeast extract 5 g / L, KH2PO4 2 g / L, MgSO4•7H2O 0.5 g / L, MnSO4•H2O 0.1 g / L, pH 6, sterilized at 121℃ for 20 min.

[0226] Fermentation medium: a compound carbon source system (20% modified inulin derivative + 80% sugarcane molasses hydrolysate, 40 g / L based on glucose), soybean meal hydrolysate (nitrogen source, total nitrogen content 3 g / L), KH2PO4 1.5 g / L, CaCl2 0.2 g / L, pH 6, sterilized at 115℃ for 30 min.

[0227] (3) Equipment

[0228] The method for preparing high-activity dextranase adopts Figure 1 The system for preparing highly active dextranase is shown.

[0229] II. Preparation method of high-activity dextranase

[0230] (1) Activation of strains and seed culture

[0231] Strain activation: The *Chaetoceros spp.* strain was inoculated onto an agar slant culture medium and incubated at 28°C for 48 hours to obtain activated strains.

[0232] Seed culture: Pick 5 loops of activated bacterial strain from the slant and inoculate into a 3000mL Erlenmeyer flask. Add 500mL of seed culture medium and incubate at 28℃ with shaking at 180rpm for 24 hours to obtain the seed culture. The bacterial cell count (OD) is... 600 =4.

[0233] (2) Fermentation process control

[0234] Inoculation: Inoculate the seed culture into a 500L fermenter 101 at a rate of 1% (v / v). The fermenter 101 contains 300L of fermentation medium.

[0235] Basic fermentation parameters: fermentation temperature 28℃, initial stirring speed 150rpm, initial aeration rate 1vvm.

[0236] Continuous flow circulation control: Start the dual-screw peristaltic pump 109, set the initial circulation flow rate to 2L / h, and maintain the dissolved oxygen concentration of the fermentation broth at 28%±2%.

[0237] Refined feeding control: Activate the online monitoring module 111 and the edge computing unit 113 to execute the adaptive pulse feeding algorithm.

[0238] Carbon source replenishment: Using 200g / L glucose solution, when the attenuation of the 1.2kHz component of the microfluidic impedance spectrum exceeds 15%, a 0.5s (second) nitrogen source (50g / L soybean meal hydrolysate) pulse injection is triggered to simultaneously replenish the carbon source and maintain the carbon-nitrogen ratio dynamically stable at (10~15):1.

[0239] When the 3.8kHz component increases by more than 20%, the circulation rate will be reduced from 2L / h to 1.5L / h.

[0240] Targeted addition of metal ions: Starting 12 hours into fermentation, the metal ion mixture was added in stages using a multi-channel peristaltic pump (108).

[0241] During the 12-24 h period, MgSO4·7H2O solution was added to control the Mg content in the fermentation broth. 2+ The concentration increased linearly to 8 mmol / L;

[0242] 24h~36h: Add MnSO4·H2O solution to control the Mn content in the fermentation broth. 2+ The concentration increased linearly to 2 mmol / L;

[0243] 36h~42h: Add ZnSO4・7H2O solution to increase the Zn concentration in the fermentation broth. 2+ The concentration increased linearly to 1 mmol / L.

[0244] Fermentation endpoint: Fermentation is stopped after 42 hours, and the fermentation broth is collected.

[0245] III. Purification Process

[0246] The supernatant of the fermentation broth was subjected to the following treatments in sequence: first plate and frame pretreatment, second prefiltration, third microfiltration, fourth selective ultrafiltration, fifth fine ultrafiltration, sixth nanofiltration, seventh fine filtration and polishing, eighth reverse osmosis, and ninth aseptic terminal filtration.

[0247] The plate and frame pretreatment meets the following conditions: the supernatant of the fermentation broth is introduced into a plate and frame filter press, a 2mm thick diatomaceous earth filter aid layer is pre-coated on the surface of the filter medium of the plate and frame filter press, and the filter is filtered at 0.3MPa and 25℃ to remove more than 90% of the large solids and obtain crude enzyme solution.

[0248] The pre-filtration treatment meets the following conditions: 0.2% (w / v) of chitosan-magnesium silicate composite flocculant is added to the crude enzyme solution, stirred at 200 rpm for 15 min, filtered through a 5 μm polypropylene pleated filter cartridge to retain flocculents and obtain filtrate.

[0249] The microfiltration process meets the following conditions: the filtrate is introduced into a polyethersulfone (PES) pleated microfiltration membrane module and cross-flow filtration is performed at 0.15 MPa, 18 °C and a cross-flow velocity of 1.5 m / s to obtain the permeate. After every 30 min of filtration, an online air-water backwashing system is used to backwash for 1 min to control the turbidity of the permeate to ≤0.1 NTU.

[0250] Selective ultrafiltration meets the following conditions: the permeate is introduced into a hydrophilic modified polysulfone ultrafiltration membrane module with a molecular weight cutoff of 800KD, and ultrafiltration is carried out at 0.2MPa, 18℃ and cross-flow rate of 1.5m / s to obtain the first ultrafiltrate, which selectively retains macromolecular heteroprotein complexes and polysaccharide polymers.

[0251] The fine ultrafiltration process meets the following conditions: the first ultrafiltrate is introduced into a polyethersulfone ultrafiltration membrane module with a molecular weight cutoff of 5KD, and the concentration and dialysis are alternately performed at 0.3MPa and 18℃. After the first ultrafiltrate is concentrated to 1 / 10 of its original volume, an equal volume of phosphate buffer with a pH of 5.5 is added and dialyzed for 20 min. This process is repeated twice to obtain the second ultrafiltrate. The conductivity of the second ultrafiltrate is controlled to be ≤5mS / cm.

[0252] Nanofiltration treatment meets the following conditions: the second ultrafiltrate is introduced into a polyamide composite nanofiltration membrane module with a molecular weight cutoff of 1KD~2KD, the pH of the feed solution is adjusted to 5.5, and concentrated to 1 / 20 of the original volume under the conditions of 0.7MPa and 18℃ to deeply remove small molecule impurities and obtain nanofiltrate.

[0253] The fine filtration and polishing process meets the following conditions: the nanofiltration solution is introduced into a hydrophilic polyvinylidene fluoride (PVDF) membrane module with a pore size of 0.45 μm, and filtered at 0.1 MPa and 18 °C to obtain the fine filtrate. The fine filtrate is controlled to meet the following conditions by monitoring with an online particle counter: the number of particles greater than or equal to 0.5 μm is ≤10 particles / mL.

[0254] The reverse osmosis treatment meets the following conditions: the fine filtrate is introduced into an aromatic polyamide reverse osmosis membrane module and subjected to reverse osmosis treatment at 1.1 MPa and 18°C ​​to obtain a concentrate.

[0255] The permeate from the reverse osmosis treatment is returned to the fine ultrafiltration process as dialysis water.

[0256] The aseptic terminal filtration process meets the following conditions: the concentrate is filtered in a Class A clean environment through a dual-stage series of aseptic pleated polytetrafluoroethylene (PTFE) filter cartridges with a pore size of 0.22 μm. Filter cartridge integrity is tested using the bubble point method before and after filtration, and the sterility assurance level (SAL) is controlled to reach 10. -6 .

[0257] The purified target enzyme is then dried.

[0258] In some embodiments, the drying process satisfies the following conditions:

[0259] The target enzyme is subjected to gradient drying. The gradient drying procedure includes:

[0260] Pre-freezing: -40℃, 15Pa pressure drying for 2 hours;

[0261] Main drying: -30℃, 10Pa pressure drying for 24 hours;

[0262] Drying: Dry at -20℃ and 5Pa pressure for 12 hours to obtain the finished enzyme preparation.

[0263] (4) Detection method

[0264] The product conforms to GB 1886.174-2024 "National Food Safety Standard for Food Additives and Enzyme Preparations for Food Industry" or T / COCIA 35-2024 "Dextranase for Toothpaste in Oral Hygiene Products".

[0265] IV. Results and Analysis

[0266] Fermentation enzyme activity expression level: 28000 U / mL, a 40% increase compared to the traditional technology's 20000 U / mL;

[0267] Enzyme activity loss rate: 13.2%, a 41% reduction compared to the 22.5% loss rate of traditional purification processes;

[0268] Fermentation cycle: 42 hours, which is 41.7% shorter than the traditional 72 hours.

[0269] Example 2

[0270] This embodiment provides a customized high-activity dextranase and its preparation method.

[0271] The preparation method of the high-activity dextranase in this embodiment is basically the same as that in Example 1, the difference being the directional addition concentration of metal ions:

[0272] 12h~24h: Add MgSO4•7H2O solution to control the Mg content in the fermentation broth. 2+ The concentration increased linearly to 10 mmol / L;

[0273] 24h~36h: Add MnSO4•H2O solution to control the Mn content in the fermentation broth. 2+ The concentration increased linearly to 3 mmol / L;

[0274] 36h~42h: Add ZnSO4•7H2O solution to increase the Zn concentration in the fermentation broth. 2+ The concentration increased linearly to 1.5 mmol / L.

[0275] Results and Analysis

[0276] Enzyme activity expression level: 28900 U / mL, an increase of 3.2% compared to Example 1.

[0277] Enzyme activity loss rate: 15.1%, higher than in Example 1, due to the high concentration of ions slightly increasing the difficulty of membrane separation.

[0278] Applicable scenarios: More suitable for the pharmaceutical field where enzyme stability is extremely important.

[0279] Example 3

[0280] This embodiment provides a customized high-activity dextranase and its preparation method.

[0281] The preparation method of high-activity dextranase in this embodiment is basically the same as that in Example 1, except that the triggering conditions of the adaptive pulse feeding algorithm are adjusted in this embodiment:

[0282] When the attenuation of the 1.2kHz component of the microfluidic impedance spectrum exceeds 12%, a 0.5-second nitrogen source pulse injection is triggered.

[0283] When the 3.8kHz component increases by more than 18%, the circulation flow rate is adjusted downward in a stepwise manner.

[0284] Results and Analysis

[0285] Enzyme activity expression level: 27000 U / mL, slightly lower than in Example 1, but still 35% higher than the traditional technique;

[0286] Fermentation cycle: 40 hours, which is 4.8% shorter than that of Example 1.

[0287] Enzyme activity loss rate: 12.9%, slightly lower than in Example 1.

[0288] Advantages: Shorter fermentation cycle, further reduced production costs, suitable for large-scale industrial mass production scenarios.

[0289] Example 4

[0290] This embodiment provides an application of dextranase in toothpaste.

[0291] The typical values ​​of the added amounts of each component of the toothpaste are shown in Table 1.

[0292] Table 1

[0293]

[0294] The dextran prepared in Example 1 was applied to toothpaste, and the required amount of each component is shown in Table 2.

[0295] Table 2

[0296]

[0297] As shown in Tables 1 and 2, this application broadens formulation compatibility. Enzymes with greater stability exhibit higher tolerance to acidic and alkaline environments, active additives (such as fluoride and plant extracts), fragrances, preservatives, and surfactants in toothpaste formulations, eliminating the need for excessive stabilizers. This simplifies the formulation system, reduces costs, and allows for compounding with various active ingredients to develop multi-functional high-end oral care products. Dextranase is more effective at breaking down plaque and tartar. Active ingredients in the formulation (such as whitening ingredients like phosphates, HAP, and sodium fluoride) are better able to contact the tooth surface. Whitening ingredients like phosphates can break down pigments on the tooth surface and continuously combine with pigment-causing factors to achieve stain removal and prevention. Active ingredients like HAP and sodium fluoride can effectively adhere to the enamel surface, increasing tooth hardness. Under the same enzyme activity conditions, the required dosage is lower, while broadening the compatibility of toothpaste formulations and reducing formulation costs.

[0298] In summary, in at least one embodiment of this application, the high-activity dextranase preparation system has the following beneficial effects:

[0299] (1) By combining refined feeding strategy with continuous flow energy metabolism, the problems of microbial energy metabolism imbalance and low KLa (volume oxygen mass transfer coefficient) mass transfer efficiency are solved. The fermentation cycle is shortened from 72 hours to 42 hours, a reduction of 41.7%, and the enzyme activity expression level is increased to about 27,000 U / mL, which is 35% higher than the traditional 20,000 U / mL.

[0300] (2) The extraction process adopts a multi-stage membrane separation combined with a -30℃ gradient freeze drying process, which makes the enzyme activity loss rate ≤15%, compared with the traditional purification process loss >20%, making it easier to produce a finished powder with high enzyme activity, with an enzyme activity ≥1000000U / g.

[0301] (3) The compatibility of dextranase in toothpaste formulation is broadened and the enzyme activity is maintained for a long time: Under the same amount of enzyme activity, the activity period is extended, and it can stably play a catalytic role during the storage, transportation and use of toothpaste. It avoids enzyme inactivation due to environmental factors (such as temperature and humidity) or formulation ingredients (such as surfactants, fragrances and preservatives), and ensures that the product has consistent efficacy throughout the three-year shelf life from the time it leaves the factory to the time it is used by consumers.

[0302] In the above embodiments, the descriptions of each embodiment have different focuses. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0303] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0304] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of this patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A system for preparing a high-activity dextranase, characterized in that, include: A fermentation apparatus includes a fermenter, a monitoring mechanism, a feeding mechanism, a continuous flow circulation mechanism, a microfluidic impedance spectroscopy analysis mechanism, and a control mechanism. The monitoring mechanism is connected to the fermenter to monitor at least one parameter value of the fermentation broth within the fermenter. The feeding mechanism is connected to the fermenter to replenish the fermentation broth. The continuous flow circulation mechanism is connected to the bottom and top of the fermenter to circulate the fermentation broth. The microfluidic impedance spectroscopy analysis mechanism is connected to the continuous flow circulation mechanism to acquire metabolic flow characteristic spectra. The control mechanism is integrated with the monitoring mechanism, the feeding mechanism, the continuous flow circulation mechanism, and the microfluidic impedance spectroscopy analysis mechanism. The microfluidic impedance spectroscopy analysis mechanism is electrically connected; the control mechanism can execute an adaptive pulse feeding algorithm and control the feeding mechanism to feed according to the parameter values ​​and the metabolic flow characteristic spectrum. When the 1.2kHz component of the microfluidic impedance spectrum decays by more than 15%, a 0.5-second nitrogen source pulse injection is triggered to simultaneously supplement the carbon source and maintain the carbon-nitrogen ratio dynamically stable at (10~15):1; when the 3.8kHz component of the microfluidic impedance spectrum rises by more than 20%, the circulation flow rate is reduced by 15%~25% to control the fluctuation of the microbial ATP synthesis rate to <5%; after the preset fermentation time, a metal ion mixture is added, wherein a magnesium source is added between the preset time and 24 hours to control the Mg content in the fermentation broth. 2+ The concentration linearly increased to 5 mmol / L~10 mmol / L, and manganese source was added between 24h and 36h to control Mn in the fermentation broth. 2+ The concentration increased linearly to 1 mmol / L~3 mmol / L. Zinc source was added between 36 h and the fermentation endpoint to increase the Zn concentration in the fermentation broth. 2+ The concentration increased linearly from 0.5 mmol / L to 1.5 mmol / L; And a purification device, which purifies the fermentation products from the fermenter through a nine-stage filtration purification process. The purification device includes a plate and frame filter, a polypropylene pleated filter element, a polyethersulfone pleated microfiltration membrane module, a hydrophilic modified polysulfone ultrafiltration membrane module, a polyethersulfone ultrafiltration membrane module, a polyamide composite nanofiltration membrane module, a hydrophilic polyvinylidene fluoride membrane module, an aromatic polyamide reverse osmosis membrane module, and a polytetrafluoroethylene sterile pleated filter element.

2. The high-activity dextranase preparation system according to claim 1, characterized in that, It also satisfies at least one of the following conditions: (1) The fermentation tank is equipped with a stirring mechanism, which can control the rotation speed from 100 rpm to 500 rpm; (2) The monitoring mechanism includes at least one of a dissolved oxygen sensor, a pH sensor, a temperature sensor and a metabolic heat sensor. The control mechanism is electrically connected to the dissolved oxygen sensor, the pH sensor, the temperature sensor and the metabolic heat sensor. The parameter values ​​include at least one of dissolved oxygen, pH value, temperature and metabolic heat. The control mechanism is used to acquire parameter values ​​including dissolved oxygen, pH value, temperature and metabolic heat. (3) The feeding mechanism includes a multi-channel peristaltic pump, and each channel of the multi-channel peristaltic pump is connected to a carbon source, a nitrogen source and a metal ion source respectively; (4) The continuous flow circulation mechanism is equipped with a double-screw peristaltic pump.

3. The high-activity dextranase preparation system according to claim 2, characterized in that, It also satisfies at least one of the following conditions: (1) The inlet end of the dual-screw peristaltic pump is connected to the bottom of the fermenter, and the outlet end of the dual-screw peristaltic pump is connected to the top of the fermenter; (2) The inlet diameter of the dual-screw peristaltic pump is 70mm~90mm; (3) The diameter of the outlet end of the dual-screw peristaltic pump is 110mm~130mm.

4. The high-activity dextranase preparation system according to any one of claims 1 to 3, characterized in that, The purification apparatus also satisfies at least one of the following conditions: (1) The filter media surface of the plate and frame filter press has a diatomaceous earth filter aid layer with a thickness of 1 mm to 2 mm; (2) The pore size of the polypropylene pleated filter element is 5μm~6μm; (3) The polyethersulfone folded microfiltration membrane module meets the following conditions: the operating pressure range is 0.1MPa~0.4MPa, and the continuous filtration flux decay rate is ≤5% / 10h; (4) The molecular weight cutoff of the hydrophilic modified polysulfone ultrafiltration membrane module is 800KD~820KD; (5) The molecular weight cutoff of the polyethersulfone ultrafiltration membrane module is 5KD~6KD; (6) The molecular weight cutoff of the polyamide composite nanofiltration membrane module is 1KD~2KD; (7) The pore size of the hydrophilic polyvinylidene fluoride membrane module is 0.4 μm to 0.5 μm; (8) The aromatic polyamide reverse osmosis membrane module meets the following conditions: it can withstand operating pressures ranging from 1.5 MPa to 2 MPa, has a rejection rate of ≥99.2% for monovalent ions in the feed solution, and a water permeability ≥1.2 m. 3 / (m 2 •d); (9) The pore size of the polytetrafluoroethylene sterile pleated filter element is 0.22μm~0.25μm.

5. The high-activity dextranase preparation system according to any one of claims 1 to 3, characterized in that, It also includes a drying device connected to the purification device, which is used to perform low-temperature drying on the target enzyme purified from the purification device.

6. The high-activity dextranase preparation system according to claim 5, characterized in that, It also satisfies at least one of the following conditions: (1) The drying apparatus includes a freeze dryer; (2) The drying device is capable of gradient drying, and the gradient drying process includes: Pre-freezing: -45℃ to -37℃, drying for 1.5h to 2.5h; Main drying: -35℃~-25℃, 8Pa~12Pa pressure drying for 8h~12h; Drying: Dry at -20℃ to -15℃ and 4Pa to 6Pa pressure for 12 to 15 hours.

7. A method for preparing a high-activity dextranase, characterized in that, Includes the following steps: Activate the bacterial strain to obtain seed liquid; The seed culture was fermented to obtain fermentation products. During fermentation, the fermentation broth was circulated, at least one parameter value of the fermentation broth was monitored, and metabolic flow characteristic spectrum was collected. An adaptive pulse feeding algorithm was executed and feeding was performed based on the parameter value and the metabolic flow characteristic spectrum. When the 1.2 kHz component of the microfluidic impedance spectrum decayed by more than 15%, a 0.5-second nitrogen source pulse injection was triggered, and carbon source was simultaneously supplemented to maintain a dynamic stable carbon-nitrogen ratio of (10~15):

1. When the 3.8 kHz component of the microfluidic impedance spectrum increased by more than 20%, the circulation flow rate was reduced by 15%~25% to control the fluctuation of microbial ATP synthesis rate to <5%. After a preset fermentation time, a metal ion mixture was added. Among them, a magnesium source was added between the preset time and 24 hours to control the Mg content in the fermentation broth. 2+ The concentration linearly increased to 5 mmol / L~10 mmol / L, and manganese source was added between 24h and 36h to control Mn in the fermentation broth. 2+ The concentration increased linearly to 1 mmol / L~3 mmol / L. Zinc source was added between 36 h and the fermentation endpoint to increase the Zn concentration in the fermentation broth. 2+ The concentration increased linearly from 0.5 mmol / L to 1.5 mmol / L; Furthermore, the fermentation products are purified by sequentially performing the following processes: first, plate and frame pretreatment; second, pre-filtration; third, microfiltration; fourth, selective ultrafiltration; fifth, fine ultrafiltration; sixth, nanofiltration; seventh, fine filtration and polishing; eighth, reverse osmosis; and ninth, aseptic terminal filtration.

8. The method for preparing high-activity dextranase according to claim 7, characterized in that, It also satisfies at least one of the following conditions: (1) The fungal species include *Chaetoceros spp.*; (2) The inoculation amount of the seed liquid relative to the fermentation medium is 0.8%~1.2% (v / v).

9. The method for preparing high-activity dextranase according to any one of claims 7-8, characterized in that, It also satisfies at least one of the following conditions: (1) During the fermentation process, the following conditions must be met: Fermentation temperature: 25℃~30℃; Stirring speed: 100 rpm to 500 rpm; Ventilation rate is 0.8 vvm to 1.2 vvm; The total fermentation time is 40-42 hours. (2) During the recycling process of the fermentation broth, the following conditions must be met: The circulation flow rate is 1.8 L / h to 2.2 L / h; The dissolved oxygen concentration in the fermentation broth is 20%~40%.

10. The method for preparing high-activity dextranase according to any one of claims 7-8, characterized in that, It also satisfies at least one of the following conditions: (1) The carbon source includes a glucose solution with a concentration of 200 g / L to 220 g / L; (2) The nitrogen source includes soybean meal hydrolysate with a concentration of 50 g / L to 60 g / L; (3) The preset time is any time point between 12h and 15h; (4) The magnesium source includes MgSO4•7H2O solution; (5) The manganese source includes MnSO4•H2O solution; (6) The zinc source includes ZnSO4•7H2O solution.

11. The method for preparing high-activity dextranase according to any one of claims 7-8, characterized in that, It also meets the following conditions: (1) The plate and frame pretreatment meets the following conditions: the fermentation product is introduced into a plate and frame filter press, the filter medium of the plate and frame filter press has a diatomaceous earth filter aid layer with a thickness of 1 mm to 2 mm, and is filtered at 0.3 MPa to 0.4 MPa and 25℃ to 28℃ to obtain crude enzyme solution. (2) The pre-filtration treatment meets the following conditions: 0.1% (w / v) to 0.3% (w / v) of flocculant is added to the crude enzyme solution, and the mixture is stirred at 180 rpm to 220 rpm for 15 min to 20 min. The solution is then filtered through a polypropylene pleated filter with a pore size of 5 μm to 6 μm to obtain the filtrate. (3) The microfiltration process meets the following conditions: the filtrate is introduced into a polyethersulfone pleated microfiltration membrane module and cross-flow filtration is performed at 0.15MPa~0.2MPa, 18℃~20℃, and cross-flow velocity of 1.5m / s~2m / s to obtain the permeate; (4) The selective ultrafiltration process satisfies the following conditions: the permeate is introduced into a hydrophilic modified polysulfone ultrafiltration membrane module with a molecular weight cutoff of 800KD~820KD, and ultrafiltration is performed at 0.2MPa~0.3MPa, 18℃~20℃, and cross-flow velocity of 1.5m / s~2m / s to obtain the first ultrafiltrate. (5) The fine ultrafiltration process meets the following conditions: the first ultrafiltrate is introduced into a polyethersulfone ultrafiltration membrane module with a molecular weight cutoff of 5KD~6KD, and concentration and dialysis are carried out alternately under the conditions of 0.3MPa~0.4MPa and 18℃~20℃; Concentrate the first ultrafiltrate to 1 / 10 to 1 / 8 of its original volume, add an equal volume of phosphate buffer with a pH of 5.5 to 6.5, dialyze for 20 to 30 minutes, repeat 2 to 3 times to obtain the second ultrafiltrate, and control the conductivity of the second ultrafiltrate to ≤5 mS / cm. (6) The nanofiltration process meets the following conditions: the second ultrafiltrate is introduced into a polyamide composite nanofiltration membrane module with a molecular weight cutoff of 1KD~2KD, and the pH value is adjusted to 5~6. The solution is concentrated to 1 / 20~1 / 18 of the original volume under the conditions of 0.7MPa~0.8MPa and 18℃~20℃ to obtain nanofiltrate. (7) The fine filtration and polishing process meets the following conditions: the nanofiltration liquid is introduced into a hydrophilic polyvinylidene fluoride membrane module with a pore size of 0.4μm~0.5μm, and filtered at 0.1MPa~0.15MPa and 18℃~20℃ to obtain fine filtrate. The fine filtrate is controlled to meet the following conditions: the number of particles greater than or equal to 0.5μm is ≤10 particles / mL; (8) The reverse osmosis treatment meets the following conditions: the fine filtrate is introduced into an aromatic polyamide reverse osmosis membrane module and subjected to reverse osmosis treatment at 1.1MPa~1.2MPa and 18℃~20℃ to obtain a concentrate; (9) The aseptic terminal filtration process meets the following conditions: the concentrate is filtered through a polytetrafluoroethylene aseptic pleated filter cartridge with a pore size of 0.22μm~0.25μm, and the aseptic assurance level is controlled to reach 10. -6 .

12. The method for preparing high-activity dextranase according to claim 11, characterized in that, It also meets the following conditions: (1) The flocculant includes a chitosan-magnesium silicate composite flocculant; (2) After every 30 to 40 minutes of filtration, backwash for 1 to 2 minutes to control the turbidity of the permeate to ≤0.1 NTU; (3) The permeate from the reverse osmosis treatment is returned to the fine ultrafiltration process as dialysis water.

13. The method for preparing highly active dextranase according to any one of claims 7-8 and 12, characterized in that, It also includes the following steps: drying the purified target enzyme; The drying process meets the following conditions: The target enzyme is subjected to gradient drying. The gradient drying procedure includes: Pre-freezing: Drying at -45℃ to -37℃ under 15Pa to 25Pa pressure for 1.5h to 2.5h; Main drying: -35℃~-25℃, 8Pa~12Pa pressure for 24h~28h; Drying: Dry at -20℃ to -15℃ and 4Pa to 6Pa pressure for 12 to 15 hours.

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