Method and kit for rapidly detecting aconitic acid

Abstract

The invention relates to a method for rapidly detecting aconitic acid in a liquid sample and a kit invented according to the method, so that the detection of the aconitic acid is expanded from the original high-pressure liquid chromatography to the principle of fading potassium permanganate by using the aconitic acid, and the method for simply and rapidly detecting the concentration of the aconitic acid is invented. And the aconitic acid detection kit is successfully developed. The invention also discloses an application of the method for rapidly detecting aconitic acid in the liquid sample in the aspect of improving the overall detection accuracy, the experimental result shows that the detection time is within 20 minutes, and the experimental result shows that the accuracy is 98-100% compared with that of high pressure liquid chromatography (HPLC) detection.

Description

Technical Field

[0001] The invention relates to the technical field of rapid analysis and detection of organic acid aconitic acid, in particular to a detection method and a detection kit for rapid detection of aconitic acid content. Background Art

[0002] Aconitic acid is a tricarboxylic acid with a double bond. It can be used in emerging nematicide biopesticides, food preservatives, pharmaceutical precursors, green and degradable plasticizers and other fields, and has become a very important bio-based chemical. Due to the progress of biological science and genetic engineering, the research on increasing the production of aconitic acid by using molecular biological methods to modify the genes related to microbial strains has been widely carried out, and large-scale fermentation production of aconitic acid has been achieved. However, how to accurately and quickly detect the content of aconitic acid is of great significance.

[0003] At present, the determination methods of aconitic acid include acetic anhydride pyridine colorimetric method, high performance liquid chromatography, etc. However, the reaction time of the acetic anhydride pyridine colorimetric method is 30 minutes, the reaction heat is very high, and both pyridine and acetic anhydride are toxic, flammable and explosive substances, making this detection method difficult to implement; the high performance liquid chromatography method requires samples to be prepared in advance, and the detection cycle of a single sample is relatively long, and the detection equipment is demanding, and real-time rapid detection cannot be achieved.

[0004] In addition, microbial strains produce aconitic acid through industrial fermentation. In addition to aconitic acid, there are also some other miscellaneous acids such as malic acid, succinic acid, citric acid, etc. in the fermentation broth. Among them, citric acid is the main residual miscellaneous acid in the fermentation broth, and it is also a miscellaneous acid mixed from plants such as sugarcane. It has a great influence on the detection of aconitic acid. Therefore, it is necessary to eliminate the interference of citric acid and other miscellaneous acids on the detection of aconitic acid, so as to improve the accuracy of the overall detection. Therefore, it is necessary to develop a fast, simple and efficient aconitic acid detection kit for the detection of fermentation production of aconitic acid, and also provide an efficient method for screening strains that accumulate aconitic acid. Summary of the invention

[0005] The object of the present invention is to provide a method for rapid detection of aconitic acid and a detection kit thereof, so as to solve the problems raised in the above background technology.

[0006] To achieve the above objectives, the present invention provides the following solutions: Testing process: (1) Sample pretreatment (2) Sample color reaction (3) Detection calculation.

[0007] 2. Reagent Configuration Solution A: Sample pretreatment solution 40%~60% CaCl2 solution.

[0008] Preparation method of sample pretreatment solution (40%~60% CaCl2 solution): Take 40 g~60 g of anhydrous CaCl2, stir and dissolve it, and then dilute it to 100 mL with deionized water to obtain the sample pretreatment solution.

[0009] Solution B: The color developing solution is 2 g / L potassium permanganate solution.

[0010] Preparation method of color developing solution (2 g / L potassium permanganate): Accurately weigh 2 g of potassium permanganate powder, stir to dissolve, and then dilute to 100 mL with deionized water to obtain the color developing solution, which should be stored in a dark place.

[0011] 3. Rapid detection method of aconitic acid 1. Sample pretreatment: Take 10 mL of the sample to be tested and adjust its pH to 6-7 with sodium hydroxide; take 1 mL from it and put it in a 5 mL EP tube, add 2 mL of solution A (sample pretreatment solution), shake well, and react in a 70 ℃ water bath for 5 min; centrifuge at 8000 r / min for 5 min, obtain the supernatant for use, and complete the sample pretreatment.

[0012] 2. Sample color reaction Take 1 mL of the supernatant obtained after the above sample pretreatment in a 10 mL EP tube, add 5 mL of dilute sulfuric acid, and react at room temperature for 5 min; centrifuge at 8000 r / min for 5 min, take 1 mL of the supernatant in a 25 mL colorimetric tube, then add 2 mL of potassium permanganate standard working solution, and react in a water bath at 35 ℃ for 8 min; quickly dilute to 25 mL with room temperature or low temperature deionized water and shake to terminate the reaction. Take 1 mL of the reaction solution in a colorimetric dish, place it in a UV-visible spectrophotometer, and measure the absorbance of the reaction system at 410 nm.

[0013] 3. Detection calculation The absorbance values ​​detected above were substituted into the standard working curve to calculate the measurement results; and the data were analyzed.

[0014] The present invention further discloses the application of a method for rapidly detecting aconitic acid in a liquid sample in improving the accuracy of overall detection. The detection time is within 20 minutes, and the experimental results show that the accuracy is 98% to 100% compared with high pressure liquid chromatography (HPLC) detection.

[0015] Compared with the prior art, the method for rapid detection of aconitic acid and the kit disclosed in the present invention have the following positive effects: The present invention reduces the interference of various miscellaneous acids on aconitic acid detection by pre-treating the sample, thereby improving the accuracy. The present invention creates a suitable reaction system, causes aconitic acid to be oxidized and faded by potassium permanganate, and simultaneously constructs a corresponding equation between the fading effect of potassium permanganate and the content of aconitic acid, thereby realizing the detection of aconitic acid. The method is fast and convenient, does not require complex and expensive instruments and equipment, can complete the detection of aconitic acid in a relatively short time, is suitable for application scenarios such as rapid detection of a large number of samples, and has good application prospects and promotion value.

[0016] The research process of the present invention is briefly described as follows: 1. Sample pretreatment solution In actual production, whether detecting aconitic acid in microbial fermentation broth or extracting aconitic acid from plant stems, there will be interference from citric acid or trace amounts of malic acid and succinic acid. Therefore, it is necessary to remove the interference of these organic acids. Choosing calcium chloride to precipitate organic acids such as citric acid has the following advantages: (1) High reaction efficiency: The calcium ions in calcium chloride have a strong binding ability with organic acid ions such as citric acid, which can quickly form precipitation, achieve a better precipitation effect in a shorter time, and improve the treatment efficiency.

[0017] (2) Complete precipitation: As long as the reaction conditions are well controlled, such as appropriate temperature and pH value, calcium chloride can almost completely precipitate organic acids such as citric acid, which is beneficial for subsequent separation and purification, and improves the purity and quality of the product.

[0018] (3) Easy operation: When using calcium chloride to precipitate organic acids, just dissolve it and add it to the solution containing the organic acid. Stir evenly to allow the precipitation reaction to occur. No complicated equipment or cumbersome operating steps are required. It is easy to master and prepare the kit as a product for aconitic acid detection.

[0019] (4) Low cost: Calcium chloride is a common chemical raw material with a relatively low price. When precipitating a large amount of organic acids such as citric acid, it can effectively reduce production costs and improve economic benefits.

[0020] (5) Good stability: Calcium chloride is stable at room temperature and pressure, and is not easy to decompose, deteriorate or volatilize. It is easy to store and transport. It is also relatively stable during the precipitation reaction and is less affected by external environmental factors, which can ensure the consistency and reliability of the precipitation effect.

[0021] (6) Wide range of applications: In addition to citric acid, calcium chloride can also be used to precipitate a variety of other organic acids. It has a wide range of applicability and can meet the needs of organic acid precipitation in different fields and samples.

[0022] 2. Sample pretreatment process The reaction parameters of the sample pretreatment process were experimentally optimized, including: sample pH adjustment, calcium chloride solution concentration, pretreatment system, pretreatment temperature and time.

[0023] Adjustment of sample pH: By measuring the solubility of calcium citrate under different pH conditions, it was found that the solubility of calcium citrate dropped significantly under pH 7.5, with only 0.031 g dissolved in 100 mL of water. Figure 1 Therefore, the pretreatment sample was selected to adjust the pH to around 7~7.5.

[0024] The solubility of calcium aconitate has been studied in the previous period, and it is known that the solubility of calcium aconitate increases with the increase of temperature. Figure 2 As shown. Therefore, compared with calcium aconitate at the same concentration, calcium citrate has a higher solubility, and the interference of citric acid in the sample can be removed by calcium citrate precipitation.

[0025] Determination of the concentration of calcium chloride solution: By mixing different concentrations of calcium chloride and citric acid, the optimal concentration of calcium chloride solution is determined by the final precipitation amount, as shown in Table 1; taking into account the solubility of calcium chloride itself, the concentration of calcium chloride solution is selected to be 40%~60%.

[0026] Table 1. The amount of precipitation generated by the reaction of different concentrations of calcium chloride solution with citric acid Calcium chloride solution concentration (%) Citric acid concentration (%) Sedimentation amount (g) 20 0.2 0.000 30 0.2 0.000 40 0.2 0.012 50 0.2 0.022 60 0.2 0.030 Pretreatment system: The reaction system ratio of citric acid and calcium chloride has a great influence on the results. By studying different system ratios, the optimal ratio system was determined based on the final precipitation amount, as shown in Table 2. 1 ml of sample solution reacts with 2 ml of calcium chloride solution.

[0027] Table 2. The amount of precipitate generated by the reaction of citric acid and calcium chloride in different ratios Calcium chloride solution dosage (mL) Amount of citric acid solution added (mL) Sedimentation amount (g) 0.5 0.5 0.000 0.5 1 0.000 1 0.5 0.002 1 1 0.000 1 2 0.000 2 1 0.028 Pretreatment temperature: by reacting different concentrations of citric acid and calcium chloride at different temperatures, Figure 3 It can be seen that the precipitate generated by the reaction reaches a peak at 70 °C, so the pretreatment temperature is selected to be 70 °C.

[0028] Pretreatment time: After mixing citric acid and calcium chloride solution, the reaction was carried out at 70 °C. The amount of precipitation generated at different reaction times showed that after 5 min of reaction, the amount of precipitation generated remained basically stable. Therefore, the pretreatment time was selected to be 5 min. Figure 4 .

[0029] Conclusion: Take 10 mL of the sample to be tested and adjust its pH to 7-7.5 with sodium hydroxide; take 1 mL from it and put it in a 5 mL EP tube, add 2 mL of solution A (sample pretreatment solution), shake well, react in a 70 ℃ water bath for 5 min; centrifuge at 8000 r / min for 5 min, obtain the supernatant for use, and complete the sample pretreatment.

[0030] 3. Principle of selecting potassium permanganate color developing solution Potassium permanganate has strong oxidizing property, and color change will occur in redox reaction, such as under acidic conditions, after it reacts with reducing substances, the purple permanganate ion will be converted into colorless manganese ion, and this characteristic makes it have the potential as a color development detection reagent, and potassium permanganate is decolorized by spectrophotometer and applied to material detection. The present invention has strong reducibility according to the carbon-carbon double bond in aconitic acid molecule, and utilizes its reducing ability in potassium permanganate solution to make potassium permanganate decolorize, and its reaction conditions are studied in depth. Rapid detection can be performed by spectrophotometry while observing the color change of potassium permanganate. Through a large number of experiments, the reaction conditions are continuously optimized, and the accuracy and sensitivity of detection are improved, and a method for detecting aconitic acid with potassium permanganate color developing solution is finally established.

[0031] 4. Aconitic acid detection reaction system For the fading reaction of potassium permanganate, the following research has been done: A: Determination of fading reaction products: Add 2 mL of potassium permanganate standard working solution, 1 mL of aconitic acid standard solution of different dilutions, and 50 µL of 2 mol / L dilute sulfuric acid to a 25 mL colorimetric tube, respectively. After reacting at 35 °C for 8 min, dilute to the scale with deionized water and shake well. Take 1 mL into a cuvette and place it in a UV-Vis spectrophotometer. Measure the absorbance of the reaction system at 410 nm with the deionized water blank as the reference. At the same time, measure the absorbance of the sample solution in the above reaction system with potassium permanganate replacing the blank.

[0032] B: Selection of absorption wavelength: Add 2 mL of potassium permanganate standard working solution, 1 mL of aconitic acid standard solution of different dilutions, and 50 µL of 2 mol / L dilute sulfuric acid to a 25 mL colorimetric tube, react at 30 °C for 2 min, dilute to 25 mL with deionized water and shake well, take 1 mL into a colorimetric dish, place it in a UV-visible spectrophotometer, perform full wavelength scanning, and draw an absorption spectrum, see Figure 5 . The measurement wavelength was determined to be 410 nm.

[0033] C: Determination of reaction time: Add 2 mL of potassium permanganate standard working solution, 1 mL of aconitic acid standard solution, and 50 µL of 2 mol / L dilute sulfuric acid to a 25 mL colorimetric tube, shake well, and react at 30 °C. Fix the concentration of aconitic acid and investigate the effect of different reaction times (2-11 min) on the absorbance of the system. Figure 6 The reaction time was determined to be 8 min.

[0034] D: Determination of reaction temperature: Add 2 mL potassium permanganate standard working solution, 1 mL aconitic acid standard solution, and 50 µL 2 mol / L dilute sulfuric acid into a colorimetric tube, shake well, and investigate the effect of different reaction temperatures (25, 30, 35, 40, 45, 50°C) on the absorbance of the system, see Figure 7 The reaction temperature was determined to be 35°C E: Determination of potassium permanganate concentration: Potassium permanganate undergoes redox reaction with aconitic acid. The concentration of potassium permanganate is the key factor affecting the absorbance change of the system. Therefore, when the concentration of aconitic acid is fixed at 0.6 g / L, the effect of potassium permanganate concentration (1, 1.5, 2, 2.5, 3 g / L) on the absorbance of the system is investigated. See Figure 8 . Determine the potassium permanganate reaction concentration to be 2 g / L F: Plotting the standard working curve: Accurately pipette different volumes of 5 g / L aconitic acid standard solution and add it to a 25 mL colorimetric tube to make up to volume. A series of aconitic acid solutions with different concentrations are obtained. The absorbance A at 410 nm is measured. At the same time, the absorbance A0 of the corresponding reagent blank is measured. The difference ∆A = A0-A is calculated and the standard working curve is plotted. See Fig. 9 .

[0035] Investigate whether the sample pretreatment solution can reduce the content of impurities in the aconitic acid sample. The detection method is as above. Take 100 uL of the supernatant after the reaction, dilute it ten times, and use potassium permanganate spectrophotometry to detect the content of aconitic acid: Aconitic acid content of sample / (g / L) Citric acid content of sample / (g / L) Absorbance value (410nm Abs) Initial potassium permanganate absorbance (525nm Abs) ∆(Abs) Total amount of aconitic acid measured / (g / L) Recovery rate / % 0.4 10 0.569 1.024 0.455 0.36 90% 0.4 5 0.531 1.024 0.493 0.36 90% 0.4 1 0.459 1.024 0.565 0.38 95% Aconitic acid was then added to the blank medium to detect its recovery rate: Aconitic acid content of sample / (g / L) Absorbance value (410 nmAbs) Initial potassium permanganate absorbance (525 nm) ∆ Total amount of aconitic acid measured / (g / L) Recovery rate / % 0.4 1.014 1.471 7 0.457 7 0.36 90 0.5 0.309 1.471 7 1.162 7 0.48 96 Subsequently, Aspergillus niger strain was selected to ferment aconitic acid, and the absorbance at 410 nm of the fermentation broth supernatant was measured. At the same time, high performance liquid chromatography was used to detect the aconitic acid content in the fermentation broth. The comparison results are shown in the table below.

[0036] sample ∆ HPLC method to determine the content / (g / L) Content determined by spectrophotometry / (g / L) Recovery rate / % 1 0.606 1 0.39 0.38 98 2 1.267 4 0.48 0.48 100 In summary, using this detection method, the recovery rate can reach 98%~100%, and the error is within an acceptable range. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Solubility curves of calcium citrate at different temperatures; Figure 2 Solubility curves of calcium aconitate at different temperatures; Figure 3 Reaction curves of different concentrations of citric acid and calcium chloride with temperature; Figure 4 Reaction time curve of 2g / L citric acid and calcium chloride; Figure 5 Full wavelength scanning absorption spectra of the reaction between different concentrations of TAA and potassium permanganate; Figure legend: a, b, c are 0.75, 0.80, and 0.85 g / L aconitic acid solutions, respectively; Figure 6 Effect of reaction time on absorbance; Figure 7 Effect of reaction temperature on absorbance; Figure 8 Color development results of different concentrations of potassium permanganate and TAA; Figure caption: From left to right, the concentrations of potassium permanganate are 1.0, 1.5, 2.0, 2.5, and 3.0 g / L; Fig. 9 Working curves of different concentrations of TAA; Fig.10 Reaction results of mixed acid with different concentrations and calcium chloride. DETAILED DESCRIPTION

[0038] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention. The raw materials and reagents used in the present invention are all commercially available. Example

[0039] Solution A: Sample pretreatment solution: 50% CaCl2 solution.

[0040] Preparation method of sample pretreatment solution 50% CaCl2: Take 50 g of anhydrous CaCl2, stir and dissolve it, and then dilute it to 100 mL with deionized water to obtain the sample pretreatment solution.

[0041] The standard citric acid solution and the standard aconitic acid solution were mixed for the experiment. The pH was adjusted to 6-7 with sodium hydroxide, and then calcium chloride solution was added. The mixture was reacted in a 70 ℃ water bath for 5 min and centrifuged at 8000 r / min for 5 min. A large amount of precipitation was found after centrifugation. The supernatant was measured and found to be alkaline. The experimental results are shown in Fig.10 .

[0042] Solution B: The color developing solution is 2 g / L potassium permanganate solution.

[0043] Preparation method of color developing solution (2 g / L potassium permanganate): Accurately weigh 2 g of potassium permanganate powder, stir to dissolve, and then dilute to 100 mL with deionized water to obtain the color developing solution, which should be stored in a dark place.

[0044] Preparation method of aconitic acid (5 g / L) standard solution: Accurately weigh aconitic acid powder, stir to dissolve, and then dilute to 100 mL with deionized water to obtain the aconitic acid standard solution.

[0045] The spectrophotometric method for rapid detection of aconitic acid is characterized by comprising the following steps: 1: Determination of fading reaction products: Add 2 mL of potassium permanganate standard working solution, 1 mL of aconitic acid standard solution of different dilutions, and 50 µL of 2 mol / L dilute sulfuric acid to a 25 mL colorimetric tube, react at 35 °C for 8 min, dilute to the scale with deionized water and shake well, take 1 mL into a colorimetric dish, place it in a UV-visible spectrophotometer, and measure the absorbance of the reaction system at 410 nm using the deionized water blank as a reference, and simultaneously measure the absorbance of the corresponding reagent blank.

[0046] B: Selection of absorption wavelength: Add 2 mL of potassium permanganate standard working solution, 1 mL of aconitic acid standard solution of different dilutions, and 50 µL of 2 mol / L dilute sulfuric acid to a 25 mL colorimetric tube. After reacting at 30 °C for 2 min, dilute to 25 mL with deionized water and shake well. Take 1 mL in a colorimetric dish and place it in a UV-visible spectrophotometer for full wavelength scanning. At the same time, measure the absorbance of the culture medium itself and draw an absorption spectrum, such as Figure 5 As shown in the figure, the highest absorption peak of the reaction between different concentrations of aconitic acid and potassium permanganate is around 400 nm. Taking 0.85 g / L aconitic acid solution as an example, the absorbance after the reaction was measured at 400, 410, and 420 nm, respectively, and it was found that it showed the maximum absorbance at 410 nm. Therefore, 410 nm was selected as the measurement wavelength after the reaction was terminated.

[0047] C: Determination of reaction time: Add 2 mL of potassium permanganate standard working solution, 1 mL of aconitic acid standard solution, and 50 µL of 2 mol / L dilute sulfuric acid to a 25 mL colorimetric tube, shake well, and react at 30 °C. Fix the concentration of aconitic acid and investigate the effect of different reaction times (2-11 min) on the absorbance of the system. Figure 6 As shown in the figure, when the reaction temperature is 30 ℃ and the reaction time is 2 min, the absorbance is high. As time goes on, aconitic acid and potassium permanganate further undergo redox reaction, causing the color of the reaction system to become lighter and the absorbance to gradually decrease. The absorbance tends to be stable at 8-10 min, indicating that the reaction has been completed and the absorbance is stable without obvious changes. Considering the purpose of rapid detection, the reaction time is selected as 8 min.

[0048] D: Determination of reaction temperature: Add 2 mL of potassium permanganate standard working solution, 1 mL of aconitic acid standard solution, and 50 µL of 2 mol / L dilute sulfuric acid into a colorimetric tube, shake well, and examine the effect of different reaction temperatures (25, 30, 35, 40, 45, 50 °C) on the absorbance of the system. Figure 7 As shown in the figure, when the reaction time is 8 min, at 25 ℃, the absorbance is high. As the reaction temperature increases, the reaction speed increases and the absorbance gradually decreases. At 35-40 ℃, the absorbance shows an inflection point platform, and the absorbance is basically stable without obvious changes. When the temperature is higher than 40 ℃, aconitic acid and potassium permanganate further undergo redox reaction, making the color of the reaction system lighter and the absorbance gradually decreases. Considering the purpose of rapid detection, the reaction temperature is selected as 35 ℃.

[0049] E: Determination of potassium permanganate concentration: Potassium permanganate undergoes a redox reaction with aconitic acid. The concentration of potassium permanganate is the key factor affecting the absorbance change of the system. Therefore, when the concentration of aconitic acid is fixed at 0.6 g / L, the effect of potassium permanganate concentration (1, 1.5, 2, 2.5, 3 g / L) on the absorbance of the system is investigated. Figure 8 As shown in the figure, with the increase of potassium permanganate concentration, the color of the system deepens and the absorbance of the system increases. When the concentration of potassium permanganate is 2.5~3.0 g / L, the color of the solution is too dark, exceeding the maximum value of the measurable range of the UV-visible spectrophotometer, so the concentration of potassium permanganate in the system is selected to be 2.0 g / L.

[0050] F: Plotting the standard working curve: Accurately pipette different volumes of 5 g / L aconitic acid standard solution into a 25 mL colorimetric tube, measure the absorbance A at 410 nm, and simultaneously measure the absorbance A0 of the corresponding reagent blank, calculate the difference ∆A=A0-A, and plot the standard working curve, such as Fig. 9 shown. Example

[0051] Solution A: Sample pretreatment solution 60% CaCl2 solution.

[0052] Preparation method of 60% CaCl2 sample pretreatment solution: Take 60 g of anhydrous CaCl2, stir and dissolve it, and then dilute it to 100 mL with deionized water to obtain the sample pretreatment solution.

[0053] Solution B: The color developing solution is 2 g / L potassium permanganate solution.

[0054] Preparation method of color developing solution (2 g / L potassium permanganate): Accurately weigh potassium permanganate powder, stir to dissolve, and then dilute to 100 mL with deionized water to obtain the color developing solution, which should be stored in a dark place.

[0055] A colorimetric solution detection method for rapid detection of aconitic acid, characterized in that it comprises the following steps: 1. Sample pretreatment: Take 10 mL of the sample to be tested and adjust its pH to 6-7 with sodium hydroxide; take 1 mL from it and put it in a 5 mL EP tube, add 2 mL of solution A (sample pretreatment solution), shake well, and react in a 70 ℃ water bath for 5 min; centrifuge at 8000 r / min for 5 min, obtain the supernatant for use, and complete the sample pretreatment.

[0056] 2. Sample color reaction Take 100 uL of the supernatant obtained after the above sample pretreatment in a 1.5 mL EP tube, add 500 uL of dilute sulfuric acid, react at room temperature for 5 min; centrifuge at 8000 r / min for 5 min, take 1 mL of the supernatant in a 25 mL colorimetric tube, then add 2 mL of potassium permanganate standard working solution, react in a water bath at 35 ℃ for 8 min; quickly dilute to 25 mL with room temperature or low temperature deionized water and shake to terminate the reaction. Take 1 mL of the reaction solution in a colorimetric dish, place it in a UV-visible spectrophotometer, and measure the absorbance of the reaction system at 410 nm.

[0057] 3. Detection calculation The absorbance values ​​detected above were substituted into the standard working curve to calculate the measurement results; and the data were analyzed.

[0058] Take 100 uL of the supernatant after the sample pretreatment reaction, dilute it ten times, and use potassium permanganate spectrophotometry to detect the aconitic acid content as shown in the following table: Aconitic acid content of sample / (g / L) Citric acid content of sample / (g / L) Absorbance value (410nm Abs) Initial potassium permanganate absorbance (525nm Abs) ∆(Abs) Total amount of aconitic acid measured / (g / L) Recovery rate / % 0.4 10 0.569 1.024 0.455 0.36 90% 0.4 5 0.531 1.024 0.493 0.36 90% 0.4 1 0.459 1.024 0.565 0.38 95% Aconitic acid was then added to the blank medium, and the recovery rate was detected as shown in the following table: Aconitic acid content of sample / (g / L) Absorbance value (410 nmAbs) Initial potassium permanganate absorbance (525 nm) ∆ Total amount of aconitic acid measured / (g / L) Recovery rate / % 0.4 1.014 1.471 7 0.457 7 0.36 90 0.5 0.309 1.471 7 1.162 7 0.48 96 Subsequently, Aspergillus niger strain was selected to ferment aconitic acid, and the absorbance at 410 nm of the fermentation broth supernatant was measured. At the same time, high performance liquid chromatography was used to detect the aconitic acid content in the fermentation broth. The comparison results are shown in the table below.

[0059] sample ∆ HPLC method to determine the content / (g / L) Content determined by spectrophotometry / (g / L) Recovery rate / % 1 0.606 1 0.39 0.38 98 2 1.267 4 0.48 0.48 100 In summary, using this detection method, the recovery rate can reach 98%~100%, and the error is within an acceptable range.

Claims

1. A method for rapid detection of aconitic acid in a liquid sample: characterized in that Proceed as follows: (1) The sample is treated with certain reagents and temperatures, the reaction system of the sample with potassium permanganate and the fading reaction conditions, and the calculation of the aconitic acid concentration in the sample; (2) The composition and concentration of the prepared test kit and the reaction conditions of each test process, wherein the test kit includes: Solution A: Sample pretreatment solution 40%~60% CaCl2 solution; Solution B: The color developing solution is 2 g / L potassium permanganate solution; (3) Sample pretreatment method: Take 10 mL of the sample to be tested and adjust its pH to 6-7 with sodium hydroxide; take 1 mL from it and put it in a 5 mL EP tube, add 2 mL of sample pretreatment solution A, shake well, and react in a 70 ℃ water bath for 5 min; centrifuge at 8000 r / min for 5 min, and obtain the supernatant for use, thus completing the sample pretreatment; (4) Sample color reaction: Take 1 mL of the supernatant obtained after pretreatment and place it in a 10 mL EP tube. Add 5 mL of dilute sulfuric acid and react at room temperature for 5 min. Centrifuge at 8000 r / min for 5 min. Take 1 mL of the supernatant and place it in a 25 mL colorimetric tube. Then add 2 mL of potassium permanganate standard working solution and react in a water bath at 35 °C for 8 min. Quickly dilute to 25 mL with room temperature or cold deionized water and shake to terminate the reaction. Take 1 mL of the reaction solution and place it in a colorimetric dish. Place it in a UV-visible spectrophotometer and measure the absorbance of the reaction system at 410 nm.

2. The method for rapid detection of aconitic acid in a liquid sample as described in claim 1 completes the detection within 20 minutes, and has an accuracy of 98% to 100% compared with high pressure liquid chromatography (HPLC) detection, and is used to improve the accuracy of overall detection.

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