Active powder for rapidly detecting concentration of succinic acid, detection device and detection method

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to active powder for rapidly detecting the concentration of succinic acid, a detection device and a detection method, the detection process is simplified through standardized addition of the active powder, online monitoring of the concentration of succinic acid can be realized in combination with a matched detection device, the accuracy of a detection result is improved, and the detection efficiency is improved. Rapid detection of the concentration of succinic acid can be achieved, the concentration of succinic acid can be monitored on line in combination with a matched device, and the production efficiency and the product quality control level are greatly improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to an active powder, a detection device and a detection method for rapid detection of succinic acid concentration. Background Art

[0002] Succinic acid (also known as butanedioic acid), as an important organic acid compound, plays an increasingly important role in the fields of food, medicine, chemical industry and energy. Succinic acid is not only a natural metabolic intermediate, but also a key C4 platform compound, which has key application value in the synthesis of renewable resource products such as biofuels and bioplastics. At the same time, succinic acid is also an important microbial fermentation product, and the accurate detection of its concentration is of great significance for the optimization of the production process, the control of product quality and the reduction of production costs. With the expansion of industrial production scale, although the traditional methods for detecting succinic acid concentration have high sensitivity and accuracy, they usually require complex experimental equipment, long analysis time and high operation cost, which makes these methods difficult to meet the requirements of real-time detection in the rapid, efficient and large-scale production process. Therefore, accurately and rapidly detecting the concentration of succinic acid is of great significance for aspects such as product quality control, production process optimization and environmental protection.

[0003] At present, the detection methods for succinic acid concentration mainly include chromatography, electrochemistry, enzyme-linked immunosorbent assay, etc. The existing technologies usually detect based on the physical and chemical properties of succinic acid or its biocatalytic characteristics in reaction. Among them, chromatography has problems such as complex operation, high equipment requirements and long time, especially the need for sample pretreatment, which limits the applicability of this method. There are also research reports on multi-enzyme coupling method, enzyme-linked immunosorbent assay and bioluminescence method. The multi-enzyme coupling method uses three reactions of succinyl-CoA synthetase (SCS), pyruvate kinase (PK) and lactate dehydrogenase (LDH) to finally detect the reduction of NADH to calculate the succinic acid content; the enzyme-linked immunosorbent assay coats a microplate with purified succinic acid antibody to make a solid-phase antibody, adds succinic acid and HRP-labeled succinic acid antigen into the microplate coated with monoclonal antibody, and makes them compete for binding. After thorough washing, substrate TMB is added for color development. The depth of the sample color is negatively correlated with the content of succinic acid (SUCN) in the sample. The absorbance is measured at a wavelength of 450 nm, and then the content of succinic acid (SUCN) in the sample is calculated through a standard curve; the bioluminescence method detects the newly formed ATP by adding luciferin and luciferase reagents to generate bioluminescence, and the amount of light generated is proportional to the activity of succinate and iron(II) / 2-oxoglutarate-dependent oxygenase.

[0004] The above methods mainly have problems such as poor timeliness and complex steps, and cannot truly achieve the rapid and immediate determination of succinic acid concentration, especially for the continuous detection of succinic acid during the production process. Therefore, it is of great significance to develop a rapid and accurate method for detecting succinic acid concentration and an on-line detection system. Summary of the Invention

[0005] To overcome the deficiencies of the above prior art, the present invention provides an active powder, a detection device and a detection method for rapid detection of succinic acid concentration. This technology can achieve rapid detection of succinic acid concentration in a short time, and can realize on-line monitoring of succinic acid concentration in combination with a supporting device, with the advantages of simple operation and accurate detection results.

[0006] To solve the above problems, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides an active powder for rapid detection of succinic acid concentration. In terms of parts by mass, the component content of the active powder is as follows: 50 - 80 parts of pH buffer salt, 0.1 - 2 parts of phenazine methosulfate (PMS), 0.01 - 0.5 parts of 2,6-dichlorophenolindophenol (DCPIP), 10 - 40 parts of polyol, 0.1 - 1 part of divalent metal ion, 1 - 5 parts of protective agent, and succinic dehydrogenase is 10 - 50 U / g of active powder.

[0008] Further, in terms of parts by mass, the component content of the active powder is as follows: 50 - 80 parts of pH buffer salt, 0.1 part of phenazine methosulfate (PMS), 0.01 part of 2,6-dichlorophenolindophenol (DCPIP), 10 parts of polyol, 0.1 - 1 part of divalent metal ion, 1 - 5 parts of protective agent, and succinic dehydrogenase is 10 - 50 U / g of active powder.

[0009] Further, the pH buffer salt is selected from one of phosphate buffer salt, sodium acetate buffer salt or citrate buffer salt. Preferably, the pH buffer salt is phosphate buffer salt;

[0010] Or, the polyol is selected from one or more of glycerol, sorbitol or erythritol. Preferably, the polyol is glycerol and sorbitol;

[0011] Or, the protective agent is selected from one or more of mercaptoethanol, bovine serum albumin or Phos-E. Preferably, the protective agent is mercaptoethanol and bovine serum albumin;

[0012] Or, the divalent metal ion is selected from one or more of calcium chloride, magnesium chloride, copper chloride or zinc chloride. Preferably, the divalent metal ion is a combination of calcium chloride and magnesium chloride.

[0013] In a second aspect, the present invention provides a detection device for rapidly detecting the concentration of succinic acid. The detection device comprises a control system, a sampling device, a rotating robotic arm device, a diluent storage device, and a testing device which are connected in sequence;

[0014] The sampling device consists of a sampler, a stop valve, a peristaltic pump, and a sample cell. Among them, the sampler is located at the front end of the sampling device, and the sampler is connected to the stop valve, the peristaltic pump, and the sample cell in sequence through a sample delivery pipe;

[0015] The testing device consists of a light source lamp, a monochromator, a test tube, a phototube dark box, an electronic system, a turntable, and a starting position which are connected in sequence;

[0016] The detection device is used in conjunction with the above-mentioned active powder.

[0017] Furthermore, the main function of the control system is to control the sampling device, the rotating robotic arm device, the testing device, and sample dilution. At the same time, the control system internally sets quantitative determination standard curve parameters, reaction types, reaction sample injection volume parameters, dilution parameters and programs, reagent and data analysis modules, and a display module. The concentration of succinic acid is calculated according to the test results, and dilution is carried out in real time according to the results, and finally the accurate concentration of succinic acid is measured.

[0018] Furthermore, the sample turntable is provided with 12 - 24 test holes. Disposable test tubes are placed inside the test holes, and the colorimetric tubes are pre-filled with active powder.

[0019] In a third aspect, the present invention provides a method for rapidly and online detecting the concentration of succinic acid, characterized in that the method is realized by the above-mentioned active powder and detection device.

[0020] Among them, the method comprises the following steps:

[0021] S1. Turn on the instrument switch, and the instrument will automatically perform a series of checks to prepare for the normal operation of the instrument;

[0022] S2. Assemble the active powder into the assay tube;

[0023] S3. Immerse the sampler into the fermentation broth in the bioreactor, take the fermentation supernatant into the sample cell for subsequent determination;

[0024] S4. Inject the sample into the test tube by the first rotating robotic arm, record the absorbance value at OD 600nm through the detection device, transmit the data back to the control system, calculate the concentration of succinic acid in the test tube according to the standard curve built in the control system, and display it on the screen.

[0025] Furthermore, in S2, the mass-volume ratio of the active powder to the sample to be tested added is (3 - 5):100.

[0026] Further, in S4, if the sample is measured after dilution, the dilution factor of the dilution operation in the dilution step is calculated before display, and the result is displayed on the screen and recorded after calculation. After each sample test is completed, the turntable rotates once, and the new test tube is moved into the starting position.

[0027] The above one or more technical solutions have the following beneficial effects:

[0028] (1) The present invention provides a method capable of quickly detecting the concentration of succinic acid in a short time, meeting the demand for real-time detection in large-scale production processes. By adding standardized active powder, the detection process is simplified, making the operation more convenient and rapid. Combining with the supporting device can realize the on-line monitoring of the succinic acid concentration, improving the accuracy of the detection result. Compared with the traditional detection method, the present invention reduces the need for complex experimental equipment, shortens the analysis time, and reduces the operating cost.

[0029] (2) The present invention uses a disposable test tube or test well plate pre-filled with active powder to realize the corresponding relationship between the succinic acid concentration and the absorbance value of the test tube or test well plate, improving the sample measurement speed. The active powder and the test device can meet the detection requirements of different succinic acid concentrations. By providing test tubes or well plates of various specifications, users can select the appropriate specifications according to different test scenarios.

[0030] (3) The present invention is applicable to the detection of a large number of samples, and is applicable to different test scenarios and equipment, including conventional spectrophotometers and microplate readers, as well as large-scale test scenarios. Through the rotary robotic arm and other automated components, manual operation is reduced, and the efficiency and accuracy of the test are improved. Different specifications of reagent tubes or well plates can be selected according to different test requirements and scenarios, and the standard curve can be preset in advance, simplifying the operation of users.

[0031] (4) The detection device described in the present invention can realize the real-time monitoring of the succinic acid concentration in the biological manufacturing process, support automatic sampling, automatic measurement and data processing, further improving the control ability of the production process and promoting the improvement of production efficiency.

[0032] (5) Compared with the traditional detection method, the system provided by the present invention has a simple structure, is easy to operate, and does not require complex experimental equipment and personnel training, reducing the production cost and time cost. Combining with automated equipment such as a rotary robotic arm, functions such as automatic sampling, dilution, and sample injection can be realized, greatly reducing manual intervention and improving work efficiency. It is applicable to fields such as biological manufacturing, fermentation broth detection, food, and chemical industry, especially in environments where high-efficiency and rapid monitoring of succinic acid concentration are required, and has a wide application prospect.

[0033] Generally speaking, through an innovative active powder formulation and an efficient testing device, the present invention solves the problems of poor timeliness and complex operation existing in traditional methods for detecting succinic acid concentration, and can achieve rapid, accurate, and automated detection of succinic acid concentration, meeting the requirements of modern production environments for high-efficiency detection technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings forming a part of this specification are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not unduly limit the present invention.

[0035] Figure 1 Schematic diagram of the on-line succinic acid detection system and device of the present invention;

[0036] Figure 2 Schematic diagram of the sampling device of the present invention;

[0037] Figure 3 Schematic diagram of the detection device of the present invention;

[0038] Figure 4 Schematic diagram of the rotating robotic arm device of the present invention;

[0039] Figure 5 Standard curve fitting curve and equation in Embodiment 2 of the present invention;

[0040] In the figures: 1, ceramic membrane tube; 2, sampler; 3, stop valve; 4, peristaltic pump; 5, sample cell; 6, light source lamp; 7, monochromator; 8, colorimetric tube; 9, phototube dark box; 10, electronic system; 11, turntable; 12, starting position; 13, sampling needle; 14, robotic arm; 15, base; 16, rotating shaft. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0042] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0043] The present invention will be further described with specific examples. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the sales company; the materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained through commercial channels.

[0044] A specific embodiment of the present invention provides an active powder for rapidly detecting the concentration of succinic acid. In terms of parts by mass, the component contents of the active powder are as follows: 50 - 80 parts of pH buffer salt, 0.1 - 2 parts of phenazine methosulfate (PMS), 0.01 - 0.5 parts of 2,6-dichlorophenol indophenol (DCPIP), 10 - 40 parts of polyol, 0.1 - 1 part of divalent metal ion, 1 - 5 parts of protective agent, and 10 - 50 U / g of succinic dehydrogenase in the active powder.

[0045] In one or more embodiments of the present invention, the pH buffer salt is selected from one of phosphate buffer salt, sodium acetate buffer salt, or citrate buffer salt; the diluent is mainly distilled water or tap water. Among them, the purpose of the phosphate is mainly to maintain the pH value of the whole solution after reconstitution. Preferably, the pH buffer salt is phosphate buffer salt, and calcium chloride, magnesium chloride, and sodium chloride are added as auxiliary regulators. Further preferably, the pH buffer salt is sodium chloride - disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0046] In one or more embodiments of the present invention, the polyol is selected from one or more of glycerol, sorbitol, or erythritol. Preferably, the polyol is glycerol and sorbitol.

[0047] In one or more embodiments of the present invention, the protective agent is selected from one or more of mercaptoethanol, bovine serum albumin, or Phos-E. Preferably, the protective agent is mercaptoethanol or bovine serum albumin.

[0048] In one or more embodiments of the present invention, the divalent metal ion is selected from one or more of calcium chloride, magnesium chloride, copper chloride, or zinc chloride. Preferably, the divalent metal ion is a combination of calcium chloride and magnesium chloride.

[0049] In one or more embodiments of the present invention, the succinic dehydrogenase mainly includes the following forms. The source of the succinic dehydrogenase can be succinic dehydrogenase extracted from animal and plant cells, such as bovine heart; it can also be succinic dehydrogenase extracted from microorganisms, such as Escherichia coli, Saccharomyces cerevisiae, etc.; it can also be the whole or part of the subunits of succinic dehydrogenase obtained by recombinant protein expression; furthermore, the succinic dehydrogenase involved in the present invention can be purified or unpurified crude enzyme, and no specific limitation is made here.

[0050] In one or more embodiments of the present invention, the content of succinate dehydrogenase is 10 - 50 U / g of active powder.

[0051] Another specific embodiment of the present invention provides a detection device for rapidly detecting the concentration of succinic acid. The detection device includes a control system, a sampling device, a rotating robotic arm device, a diluent storage device, and a testing device connected in sequence.

[0052] The detection device provided by the present invention needs to be paired with the disposable test tube provided by the present invention, and the test tube is pre-filled with the active powder provided by the present invention. Based on the active powder and the test tube, the detection device provided by the present invention can measure the succinic acid concentration in the range of 0 - 2 g / L. Preferably, the determination range of the succinic acid concentration of the on-line detection system and device provided by the present invention is 0 - 1.6 g / L. Further preferably, when the succinic acid concentration is in the range of 0.1 - 0.6 g / L, the linear range of the detection system and device provided by the present invention is above 0.99.

[0053] The main function of the control system is to control the sampling device, the rotating robotic arm device, the testing device, and sample dilution. At the same time, the control system is built-in with parameters of the quantitative determination standard curve, reaction type, reaction injection volume parameters, dilution parameters and procedures, reagent and data analysis modules, and a display module. It calculates the succinic acid concentration according to the test results, and dilutes in real time according to the results, and finally measures the accurate succinic acid concentration.

[0054] In one or more embodiments of the present invention, the standard curve built into the control system of the present invention is: Y = -0.0016X 4 +0.0487X 3 +0.0573X 2 -1.0208X + 1.5645, R 2 = 0.9979; where Y is the succinic acid concentration of the sample; X is the OD600 value measured by the test system; the range of X is 0.200 - 1.500 valid. The control system calculates the concentration of succinic acid according to the measured OD value and the dilution factor.

[0055] In one or more embodiments of the present invention, the sampling device is composed of a sampler 2, a stop valve 3, a peristaltic pump 4, and a sample cell 5. As Figure 2 shown, the sampler 2 is composed of a stainless steel skeleton and a ceramic membrane tube 1. The ceramic membrane tube 1 is nested at the front end of the stainless steel skeleton. The sampler 2 is connected to the stop valve 3, the peristaltic pump 4, and the sample cell 5 in sequence through a sample delivery tube.

[0056] The sampling device mainly takes the fermentation liquid in the bioreactor to the sample pool 5 through the sampler 2. The present invention provides a sampler 2 that can be directly inserted into the bioreactor for sterilization operation, wherein the sampler 2 is composed of a stainless steel frame and a ceramic membrane tube 1. The stainless steel frame is a stainless steel tube with a groove at the front end and a hole in the center, with a diameter of 10-15mm and a length of 150-400mm; the wall thickness of the ceramic membrane tube is 1-2mm, the diameter is 10-15mm, and the length is 50mm. The ceramic membrane tube 1 can isolate the microorganisms in the reactor, and the fermentation clear liquid can flow into the sample pool 5 through the sampler 2 for subsequent measurement.

[0057] In one or more embodiments of the present invention, the pore size of the ceramic membrane tube 1 is 100nm-500nm. Preferably, the pore size of the ceramic membrane tube is 200nm.

[0058] In one or more embodiments of the present invention, after receiving the sampling instruction from the control system, the sampling device opens the stop valve 3 and the peristaltic pump 4 to take the sample from the bioreactor into the sample pool 5 .

[0059] In one or more embodiments of the present invention, the rotating mechanical arm device includes a first rotating mechanical arm and a second rotating mechanical arm. The first rotating mechanical arm and the second rotating mechanical arm have the same structure and are both composed of a sampling needle 13, a mechanical arm 14, a base 15, and a rotating shaft 16. Figure 4 As shown, the base 15 is a rigid supporting component, and a fixed interface (bolt hole or snap-on structure) is provided at the bottom to connect with the platform. The base 15 has a built-in rotation drive unit, and precise start and stop and rotation angle control are achieved through an embedded controller; the rotating shaft 16 is coaxially connected to the drive output end at the upper end of the base 15 through a flange, and receives the torque input of the motor inside the base; the robotic arm is a multi-section connecting rod structure, which includes at least one active rotating joint, and its base end is fixedly connected to the output end of the rotating shaft 16, and a quick-change interface is provided at the end; the sampling needle 13 can be detachably installed through the quick-change interface at the end of the robotic arm 14, and the interface has a built-in gas / electric coupling channel to realize the transmission of the driving signal and negative pressure of the sampling needle. The sampling needle 13 adopts a modular design, supports rapid replacement of needles of different specifications, and is adapted to a variety of sampling scenarios. It can realize the absorption of 0-10mL samples and diluents to realize functions such as sample testing and sample dilution. The first rotating mechanical arm is mainly used in conjunction with the sampling device to suck the sample into the test tube. When executing the sample dilution instruction, the first rotating arm transfers the sample in the sample pool 5 to the sample pool of the diluent storage device. The second rotating mechanical arm is used in conjunction with the diluent storage device to dilute the sample. After the dilution is completed, the second rotating mechanical arm transfers the diluted sample to the test tube. In addition to the mechanical arm mentioned in the present invention, other forms of pipetting systems can also be used as tools for the present invention to achieve sample suction.

[0060] In one or more embodiments of the present invention, the diluent storage device includes 2 - 5 liquid storage bottles and a sample cell. Distilled water is stored in the liquid storage bottles; the sample cell of the diluent storage device is paired with a first robotic arm and a second robotic arm to achieve dilution, transfer, and removal of samples.

[0061] The dilution involved in the present invention is mainly achieved by adding the test sample and the diluent to the test tube in a certain proportion. The total volume of the diluent and the test sample remains unchanged, and the dilution purpose is achieved by adjusting the ratio of the diluent to the test sample. Taking the working volume of the test tube as 1 mL as an example, the following table shows the volume distribution to achieve dilution requirements of 2 - fold, 5 - fold, 10 - fold, 50 - fold, and 100 - fold. At the same time, other methods that can achieve sample dilution and sample transfer can also be used in the present invention.

[0062] Table 1 Sample Dilution Strategy

[0063] Total volume after dilution (mL) Volume of diluent added (mL) Volume of sample added (mL) Dilution factor Injection volume (μL) Dilution method 3 1.50 1.50 2 120 Diluted with diluent 3 2.40 0.60 5 120 Diluted with diluent 3 2.70 0.30 10 120 Diluted with diluent 3 2.94 0.06 50 120 Diluted with diluent 3 2.97 0.03 100 120 Diluted with diluent

[0064] Specifically, the control system provided by the present invention executes the following dilution program. When the measured OD value ranges from 0.000 to 1.500, the concentration of succinic acid is directly calculated according to the built - in function. When the OD value ranges from 1.500 to 1.800, the system executes a 2 - fold dilution command; when the OD value ranges from 1.800 to 2.000, the control system executes a 5 - fold dilution command; when the measured OD range is from 2.000 to 2.500, the system first executes a 10 - fold dilution command, and then determines whether to further dilute according to the measurement result of the diluted sample. If the measured OD value of the sample after 10 - fold dilution is still greater than 2.000, a 50 - fold dilution is performed. Both 10 - fold dilution and 50 - fold dilution are achieved by mixing; when the measured OD range is greater than 2.500, the system first executes a 50 - fold dilution command, and then determines whether to further dilute according to the measurement result of the diluted sample. If the measured OD value of the sample after 50 - fold dilution still does not meet the calculation requirements, the dilution multiple is continuously adjusted according to the measurement result, and a 100 - fold dilution is performed, or the diluted sample is further diluted 2 - fold, 5 - fold, or 10 - fold according to the diluted OD until the OD value is below 1.500; it should be noted that when the dilution multiple is greater than 100 - fold, the error between the measured result of the succinic acid concentration and the actual concentration will increase.

[0065] In one or more embodiments of the present invention, the test device is composed of a light source lamp 6, a monochromator 7, a test tube 8, a phototube cassette 9, an electronic system 10, a turntable 11, and a starting position 12 connected in sequence.

[0066] Among them, the turntable 11 is internally equipped with a matching test tube 8. The test tube 8 is a disposable test tube. According to different test scenarios or test equipment, the test tubes are sized from 0.1 mL to 5 mL, and the preferred size of the test tube is 1 - 5 mL. When in use, the test solution to be measured is directly added according to the size of the test tube. After reacting for 3 minutes, the absorbance value of the test sample at 600 nm is measured; further preferably, the test tube described in the present invention is a disposable colorimetric tube, which can be adapted to a conventional spectrophotometer. After the reaction ends, there is no need to transfer the liquid, and it can be directly placed in the spectrophotometer for measurement, and then the succinic acid concentration is calculated according to the measurement result.

[0067] In one or more embodiments of the present invention, according to the test requirements and scenarios for different concentrations of succinic acid, the test tube 8 can also be replaced with a test well plate. Different specifications of test tubes or well plates provided by the present invention will preset standard curves according to the filling amounts of different active powders to calculate the concentration of succinic acid. When in use, the test solution to be measured is directly added according to the instructions with a fixed volume of the test solution. After reacting for a period of time, the absorbance value at 600 nm is directly measured. There is no need for the user to separately make a standard curve, and the concentration of succinic acid can be directly calculated according to the preset standard curve of the present invention.

[0068] In one or more embodiments of the present invention, in order to meet the test requirements in a large - batch test scenario, the test well plate can be a 96 - well plate or a 24 - well plate suitable for an enzyme - linked immunosorbent assay (ELISA) reader. Each well plate is pre - filled with a fixed mass of active powder. During the test, a fixed volume of the test solution to be measured is added, and then the measurement is carried out by an ELISA reader. The concentration of succinic acid is calculated according to the preset standard curve.

[0069] In one or more embodiments of the present invention, the size of the turntable 11 can be selected according to actual needs. The preferred size of the turntable is 12 - 24 test wells. When testing a sample, a fixed volume of the sample is injected into the test tube by a rotating robotic arm, and then the instrument automatically records the absorbance value at OD 600 nm and transmits the data back to the control system. According to the standard curve built in the control system, the concentration of succinic acid in the test tube is calculated and displayed on the screen; if the sample is measured after dilution, the dilution factor of the dilution operation is calculated before display, and the result is displayed on the screen and recorded after calculation. After each sample test is completed, the turntable rotates once to move the new test tube to the starting position.

[0070] Another specific embodiment of the present invention provides a method for rapidly and on - line detecting the concentration of succinic acid, and the method is realized by the above - mentioned active powder and detection device.

[0071] Among them, the method includes the following steps:

[0072] S1. Turn on the instrument switch, and the instrument will automatically perform a series of checks to prepare for the normal operation of the instrument;

[0073] S2. Assemble the active powder into the measuring tube;

[0074] S3. Immerse the sampler into the fermentation broth in the bioreactor, take the clarified fermentation broth into the sample pool for subsequent measurement;

[0075] S4. Inject the sample into the test tube by the first rotating robotic arm, record the absorbance value at OD 600nm through the detection device, transmit the data back to the control system, calculate the succinic acid concentration in the test tube according to the standard curve built in the control system, and display it on the screen.

[0076] Further, in S2, the mass-volume ratio of the active powder to the test solution is (3 - 5):100, preferably 4:100.

[0077] In S4, if the sample is measured after dilution, calculate the dilution factor of the dilution operation in the dilution step before display, display the result on the screen after calculation and record it. After each sample test is completed, the turntable rotates once to move the new test tube to the starting position.

[0078] Example 1: Preparation of the active powder

[0079] Prepare the active powder for rapid detection of succinic acid concentration, and evaluate its stability and activity.

[0080] Steps:

[0081] 1) Raw material preparation: Succinate dehydrogenase (SDH): Extract succinate dehydrogenase from the broken Escherichia coli cell solution to make its enzyme activity greater than 200 U / mL; pH buffer salt: Select the combination of disodium hydrogen phosphate and sodium dihydrogen phosphate, purchased from Sinopharm Chemical Reagent Co., Ltd., to ensure that the solution pH is maintained at 7.5 ± 0.2; Phenazine methosulfate (PMS) is purchased from Shanghai Macklin Biochemical Co., Ltd. as an electron acceptor; 2,6-Dichlorophenolindophenol (DCPIP) is purchased from Shanghai Macklin Biochemical Co., Ltd. for color development in the succinic acid oxidation reaction; Polyhydric alcohol: Select glycerol and sorbitol as protective agents to maintain the stability of the enzyme; Divalent metal ions: Select calcium chloride and magnesium chloride as cofactors to enhance the catalytic activity of the enzyme; Protective agent: Select mercaptoethanol or bovine serum albumin (BSA) to protect the activity of succinate dehydrogenase.

[0082] 2) Preparation of the active powder:

[0083] Based on the mass of the added water, weigh the reagents according to the following mass fractions to prepare a solution with a pH value of 7.5 ± 0.2:

[0084] Buffer salt (disodium hydrogen phosphate and sodium dihydrogen phosphate): 60%;

[0085] Phenazine methosulfate (PMS): 0.1%;

[0086] 2,6 - Dichlorophenolindophenol (DCPIP): 0.01%;

[0087] Polyol (glycerol:sorbitol ratio is 1:1): 38.5%;

[0088] Protective agent (mercaptoethanol): 1%;

[0089] Metal ions (calcium chloride:magnesium chloride ratio is 1:1): 0.39%;

[0090] On this basis, add dehydrogenase according to the extracted succinate dehydrogenase enzyme activity to make it reach 50 U / g of dry powder for succinate dehydrogenase.

[0091] 3) Freeze - drying:

[0092] Carry out freeze - drying treatment on the prepared solution to remove the solvent and obtain a powdery active powder;

[0093] Carry out the freeze - drying process at low temperature to ensure that the enzyme activity is not damaged. The freeze - dried powder should maintain its activity, not less than 80% of the initial enzyme activity.

[0094] 4) Powder quality control:

[0095] Confirm the enzyme activity of each batch of powder through enzyme activity determination (using a standard succinate reaction system). The goal is to have more than 10 U of succinate dehydrogenase per gram of powder; evaluate the stability of the powder, store it at - 20 °C, and conduct a 6 - month stability test to ensure the stability of the succinate dehydrogenase activity during long - term storage.

[0096] Experimental results:

[0097] Table 2 Quality stability of active powders among different batches

[0098] Batch Enzyme activity of active powder U / g 1 40.1±4.1 2 47.2±3.5 3 50.2±5.1 4 43.6±4.5 5 42.6±6.3

[0099] After testing, the succinate dehydrogenase activities of the 5 batches of prepared active powders are all within the range of more than 10 U / g, meeting the requirements.

[0100] Table 3 Succinate dehydrogenase enzyme activity of active powder varying with time

[0101]

[0102]

[0103] The results showed that the initial enzyme activity after the preparation of the active enzyme solution was greater than 200 U / mL. After freeze-drying, when the active powder was restored to its original volume, the enzyme activity loss was about 25%. Subsequently, the active powder was placed in the refrigerator for cold storage. The results showed that after 6 months, more than 80% of the enzyme activity of the active powder could still meet the test requirements.

[0104] In this experiment, an active powder of succinate dehydrogenase with high activity and good stability was successfully prepared, which met the conditions for rapid detection of succinate concentration, could be stored for a long time, and was suitable for mass production and large-scale application.

[0105] Example 2: Standard curve of active powder

[0106] 1) Test tube assembly: A predetermined amount of the active powder prepared in Example 1 was filled into the test tubes to ensure that the content of the active powder in each test tube was the same. In this experiment, the size of the test tube was 5 mL, the effective working volume was 3 mL, and 120.0 mg of the active powder was pre-filled.

[0107] 2) Test operation: The tests in this experiment were divided into standard samples and fermentation broth. First, standard samples were prepared according to the following concentrations: 0.1 g / L, 0.2 g / L, 0.4 g / L, 0.8 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, and reacted at room temperature for 3 minutes.

[0108] 3) Measurement: The absorbance (OD value) of each test tube was measured using a spectrophotometer at a wavelength of 600 nm.

[0109] The experimental results are shown in Table 4.

[0110] Table 4 Test results of standard liquids

[0111]

[0112]

[0113] As Figure 4 shown, the OD600 measured for standard liquids of different concentrations showed a certain pattern. Different fitting methods (exponential fitting, linear fitting, logarithmic fitting, polynomial fitting) were used to fit the measurement results. The results showed that the equation fitting coefficient of the polynomial fitting was greater than 0.99, and the fitting equation was: Y = -0.0016X 4 + 0.0487X 3 + 0.0573X 2 - 1.0208X + 1.5645, R 2= 0.9979; where Y is the succinic acid concentration of the sample; X is the OD600 value measured by the test system; the range of X is effectively 0.200 - 1.500. Therefore, this fitted curve is used as the standard curve for the active powder provided by the present invention.

[0114] Example 3: Application of the active powder in a single test tube

[0115] 1) Test tube assembly: A predetermined amount of the active powder prepared in Example 1 was filled into the test tubes to ensure that the content of the active powder in each test tube was the same. In this experiment, the size of the test tube was 5 mL, the effective working volume was 3 mL, and 120 mg of the active powder was pre-filled. (Similarly, the present invention can provide test tubes of different specifications, and the mass of the pre-filled active powder is different according to the size of different test tubes.)

[0116] 2) Test operation: This experiment was divided into standard samples and fermentation broth. First, standard samples were prepared at the following concentrations: 0.1 g / L, 0.2 g / L, 0.4 g / L, 0.8 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L. The concentration of the fermentation broth varied from 0.1 - 100 g. The samples were added to different test tubes, 3 mL of the test solution to be measured was added according to the instructions, and the reaction was carried out at room temperature for 3 minutes. If the fermentation broth with a high succinic acid concentration needed to be diluted, an appropriate dilution factor was selected according to the measurement range.

[0117] 3) Measurement: The absorbance (OD value) of each test tube was measured using a spectrophotometer at a wavelength of 600 nm. By comparing with the preset standard curve (Y = -0.0016X 4 + 0.0487X 3 + 0.0573X 2 - 1.0208X + 1.5645 R2 = 0.9979, where Y is the succinic acid concentration of the sample; X is the OD600 value measured by the test system; the range of X is effectively 0.200 - 1.500), the succinic acid concentration in the sample was calculated.

[0118] Table 5 Succinic acid concentrations of different samples

[0119]

[0120] Results:

[0121] The measurement results showed that there was a good linear correlation between the succinic acid concentration and the measured absorbance value. The time required for each test was 5 minutes, the detection process was simple and fast, and the results were accurate.

[0122] This example verified the effectiveness of the active powder, which can quickly and accurately detect the succinic acid concentration and is suitable for single succinic acid concentration detection.

[0123] Example 4: 24-well Plate Detection System Suitable for High-throughput Detection

[0124] Evaluate the application effect of the active powder of the present invention and the test well plate in high-throughput detection scenarios.

[0125] 1) Test well plate assembly: Pre-load a fixed mass of the active powder prepared in Example 1 into each well of the 24-well plate to ensure that the components in each well of the well plate are consistent. In this experiment, the size of each well is 1000 microliters, and 5 mg of the active powder is pre-loaded. (Similarly, the present invention can provide test well plates of different specifications, and the mass of the pre-loaded active powder is different according to the size of different test well plates.)

[0126] 2) Test operation: The test in this experiment is a standard sample. Prepare standard samples according to the following concentrations, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, 2.0 g / L, 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L. Add the samples into different test tubes respectively, add 120 microliters of the test solution to be measured as required according to the instruction manual, and react at room temperature for 3 minutes.

[0127] 3) Measurement: Use a spectrophotometer to measure the absorbance (OD value) of each test tube at a wavelength of 600 nm. By comparing with the preset standard curve (Y = -0.0016X 4 + 0.0487X 3 + 0.0573X 2 - 1.0208X + 1.5645R 2 = 0.9979;; Y is the concentration of succinic acid in the sample; X is the size of OD600 measured by the test system; the range of X is 0.200 - 1.500 is valid), calculate the concentration of succinic acid in the sample and compare it with the actual concentration.

[0128] Table 6 Test Results of 24-well Plate

[0129]

[0130]

[0131] Results:

[0132] This test can complete the detection of 24 wells within 5 minutes. The high-throughput detection system has strong adaptability and can meet the rapid detection requirements of a large number of samples. When the sample concentration is less than 1.4 g / L, the detection results are in good agreement with the true values. When the concentration is greater than 1.5 g / L, the difference between the measured values of the samples and the true values gradually increases and shows no pattern, and dilution is required. This example shows that the active powder and the 24-well plate system can efficiently and accurately perform high-throughput detection of succinic acid concentration (0.1 - 1.5 g / L), meeting the rapid monitoring requirements in industrial production.

[0133] Example 5: Application of the on-line monitoring system to the real-time detection of succinic acid concentration during fermentation

[0134] Verify the effect of the on-line succinic acid detection system provided by the present invention on the real-time monitoring of succinic acid concentration during the biological manufacturing process.

[0135] 1. System configuration: Install the on-line succinic acid detection system provided by the present invention in a bioreactor, including a control system, a sampling device, a testing device, a rotating robotic arm device, and a diluent storage device. The testing device is pre-loaded with a fixed mass of active powder, and the preparation method of the active powder is the same as that in Example 1, except that the succinic acid dehydrogenase is 25 U / g.

[0136] 2. Sampling of the fermentation broth: Automatically sample from the fermentation broth through the sampling device (once every 30 minutes), and send the sample into the testing device for rapid detection.

[0137] 3. Testing process: After the sample enters the testing device, it reacts with the active powder for 3 minutes, and is measured at a wavelength of 600 nm using spectrophotometry. The control system automatically calculates the succinic acid concentration and compares it with the standard curve, and the detection results are displayed in real time. (Y = -0.0016X 4 +0.0487X 3 +0.0573X 2 -1.0208X + 1.5645; R 2 = 0.9979; Y is the succinic acid concentration of the sample; X is the OD600 value measured by the testing system; the range of X is 0.200 - 1.500 (valid))

[0138] 4. Result processing: The detection results are real-time fed back to the production management personnel through the control system for adjusting the fermentation conditions (such as temperature, pH value, nutrient supplementation, etc.) to optimize the production process of succinic acid.

[0139] Table 6 Succinic acid test results of the on-line monitoring system

[0140] Fermentation cycle (h) Succinic acid concentration (g / L) 0 0.000±0.03 8 3.211±0.05 16 7.564±0.11 24 9.211±0.12 32 18.366±0.11 40 25.632±0.24 48 35.988±0.36 56 52.212±0.62 64 45.322±0.41 72 36.477±0.25

[0141] In a bioreactor, the concentration of succinic acid changes with the fermentation process, and real-time data can accurately reflect the trend of concentration change. The system can continuously monitor the succinic acid concentration, helping operators adjust production parameters in real time and optimize the production efficiency of succinic acid. This embodiment shows that by combining a rapid detection method for succinic acid concentration and an on-line detection device, real-time monitoring of succinic acid in the biological manufacturing process can be achieved, greatly improving the production efficiency and the level of product quality control.

[0142] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An active powder for rapid detection of succinic acid concentration, characterized in that: The active powder comprises the following components in parts by mass: 50-80 parts of pH buffer salt, 0.1-2 parts of phenazine dimethyl sulfate (PMS), 0.01-0.5 parts of 2,6-dichlorophenol indophenol (DCPIP), 10-40 parts of polyol, 0.1-1 parts of divalent metal ions, 1-5 parts of protective agent, and 10-50 U / g of succinate dehydrogenase.

2. The active powder according to claim 1, characterized in that The active powder comprises the following components in parts by mass: 50-80 parts of pH buffer salt, 0.1 parts of phenazine dimethyl sulfate (PMS), 0.01 parts of 2,6-dichlorophenol indophenol (DCPIP), 10 parts of polyol, 0.1-1 parts of divalent metal ions, 1-5 parts of protective agent, and 10-50 U / g of succinate dehydrogenase.

3. The active powder according to claim 1, characterized in that The pH buffer salt is selected from one of phosphate buffer salt, sodium acetate buffer salt or citrate buffer salt, preferably, the pH buffer salt is phosphate buffer salt; Or, the polyol is selected from one or more of glycerol, sorbitol or erythritol, preferably, the polyol is glycerol or sorbitol; Or, the protective agent is selected from one or more of mercaptoethanol, bovine serum albumin or Phos-E, preferably, the protective agent is mercaptoethanol or bovine serum albumin; Alternatively, the divalent metal ion is selected from one or more of calcium chloride, magnesium chloride, copper chloride or zinc chloride. Preferably, the divalent metal ion is a combination of calcium chloride and magnesium chloride.

4. A detection device for rapidly detecting succinic acid concentration, characterized in that: The detection device comprises a control system, a sampling device, a rotating mechanical arm device, a diluent storage device and a testing device which are connected in sequence; The sampling device is composed of a sampler, a stop valve, a peristaltic pump and a sample pool, wherein the sampler is located at the front end of the sampling device, and the sampler is connected to the stop valve, the peristaltic pump and the sample pool in sequence through a sample delivery tube; The testing device is composed of a light source lamp, a monochromator, a testing tube, a photoelectric tube dark box and an electronic system, a rotating disk and a starting position which are connected in sequence; The detection device is used in conjunction with the active powder according to any one of claims 1 to 3.

5. The detection device according to claim 4, characterized in that: The main functions of the control system are to control the sampling device, the rotating mechanical arm device, the testing device and the sample dilution. At the same time, the control system has built-in quantitative determination standard curve parameters, reaction type, reaction injection volume parameters, dilution parameters and procedures, reagents and data analysis modules and display modules, calculates the succinic acid concentration according to the test results, and dilutes in real time according to the results, and finally measures the accurate succinic acid concentration.

6. The detection device according to claim 4, characterized in that: The sample turntable is provided with 12-24 test holes, each of which has a disposable test tube built in it, and the colorimetric tube is pre-filled with active powder.

7. A method for rapid online detection of succinic acid concentration, characterized in that: The method is achieved by the active powder according to any one of claims 1 to 3 and the detection device according to any one of claims 4 to 6.

8. The method according to claim 7, characterized in that The method comprises the following steps: S1. Turn on the instrument and it will automatically perform a series of checks to prepare for normal operation. S2, assembling active powder into the measuring tube; S3, the sampler is immersed in the fermentation liquid in the bioreactor, and the fermentation clear liquid is taken into the sample pool for subsequent measurement; S4. The first rotating mechanical arm injects the sample into the test tube, and the detection device records the absorbance value of OD 600nm, and transmits the data back to the control system. According to the standard curve built into the control system, the concentration of succinic acid in the test tube is calculated and displayed on the screen.

9. The method according to claim 8, characterized in that In S2, the mass volume ratio of the active powder to the sample to be tested is (3-5):

100.

10. The method according to claim 8, characterized in that In S4, if the sample is diluted for measurement, the operational dilution multiple of the dilution step is calculated before display, and the result is displayed on the screen and recorded after calculation. After each sample test is completed, the turntable rotates once to move a new test tube to the starting position.

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