Urease activity detection method based on non-reversible reaction of dopamine and resorcinol

Abstract

The invention discloses a urease activity detection method based on a non-reversible reaction of dopamine and resorcinol. The urease activity detection method comprises the following steps: mixing a sample to be detected, a buffer solution with the pH value of 7.0 and a urea solution with a certain concentration; adding a certain amount of dopamine and resorcinol into the mixed solution, under the condition of 25-45 DEG C, catalyzing urea hydrolysis by urease, causing the pH value of the surrounding area of the urease to rise so as to form a local alkaline environment, further inducing the reaction of the dopamine and the resorcinol to generate a blue fluorescent substance, and adding excessive hydrochloric acid to terminate the reaction after the reaction is carried out for 40-120 minutes; and detecting the fluorescence intensity of the reaction liquid by using a fluorospectro photometer, and calculating the urease activity in the sample according to a fluorescence intensity-urease concentration standard curve. The urease activity detection method based on the reaction of dopamine and resorcinol has the advantages of high detection speed, high sensitivity, good selectivity and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fluorescence detection, and particularly relates to a urease activity detection method based on non-reversible reaction of dopamine and resorcinol. BACKGROUND

[0002] Urease is widely present in bacteria, fungi, algae and plant seeds, especially in soybeans and sword beans. However, urease also brings negative effects to people's life. In the medical field, urease can be used as a diagnostic tool for detecting and measuring the urea level in human blood, so the detection of urease activity can determine whether there is a pathological change. Urease activity can detect whether it is helicobacter pylori infection. Most ureases function in the cytoplasm, and helicobacter pylori urease is the only urease found to be secreted outside the cell. Helicobacter pylori can produce high-activity urease, and rapid detection of urease activity can further speed up the judgment of helicobacter pylori infection, while avoiding the side effects of conventional urease detection methods. Urease also plays an important role in the pathogenesis of infectious stones, urinary tract infections, hepatic encephalopathy and pyelonephritis.

[0003] The fluorescence detection method of urease activity can not only be applied to the medical field, but also to environmental science, agricultural science, food science and other fields. In the field of agriculture, urease is also contained in extracted soybean meal and raw soybeans. If raw soybeans or extracted soybean cake meal without heat treatment are fed to ruminants, the microorganisms in the animal's rumen will synthesize their own microbial proteins using the hydrolysis products of urea, while feeding urea. Although it can save protein feed costs, urea will quickly decompose, and excessive ammonia will be absorbed by the blood of ruminants, reducing the utilization rate of nitrogen, causing ammonia poisoning in ruminants, and also polluting the environment. In the natural environment, if the urease activity of microorganisms in the soil is high, and urea is used for fertilization, a large amount of ammonia will be released, which not only inhibits the growth of plants, but also causes serious environmental problems. Therefore, the amount of urea can be adjusted according to the detection of urease activity.

[0004] In the field of food, the determination of urease activity in food can provide a basis for the quality evaluation of food. Legumes and cereals contain a large number of anti-nutritional factors such as urease, and urease activity can be used as a detection index for the heat-sensitive anti-nutritional factor inactivation method to improve the nutritional utilization rate of food. In the aspect of food processing technology optimization, the determination of urease activity during processing can provide a reference for the optimization of processing technology. For example, the magnetic biocatalyst of urease activity can still maintain this activity even after being reused 10 times. Urease is an enzyme present in many microorganisms, and its activity can reflect the freshness of food. Urease activity detection can also be used to detect harmful substances in food, such as urea, heavy metals, etc., which may have adverse effects on human health, so their detection is of great significance.

[0005] The research of urease inhibitors cannot be separated from the detection of urease activity, and rapid detection helps to solve a series of problems caused by excessively high or low urease activity, such as threatening the safety of the human body, polluting the ecological environment, or reducing production efficiency. The traditional detection method is often based on the decomposition of urea catalyzed by urease, which causes the pH of the whole solution to change, and the detection is carried out by using the color development of pH color reagent. The color change reaction of the existing pH color reagent is mostly reversible, that is, the color deepens after the pH increases, and the color becomes lighter after the pH decreases, which easily affects the detection sensitivity.

[0006] In view of this, the purpose of the present application is to provide a urease activity detection method with fast detection speed, high sensitivity and good selectivity. SUMMARY

[0007] The technical problem to be solved by the present application is to provide a urease activity detection method based on the non-reversible reaction of dopamine and resorcinol, which has the advantages of fast detection speed, high sensitivity, good selectivity and the like.

[0008] The technical solution of the present application is: A urease activity detection method based on the non-reversible reaction of dopamine and resorcinol, comprising the following steps: Step S1, mixing the sample to be tested, PBS 7.0 buffer solution and a certain concentration of urea solution; Step S2, adding a certain amount of dopamine and resorcinol to the mixed solution of step S1, under the condition of 25-45℃, urease catalyzing urea hydrolysis, causing the pH of the surrounding area of urease to rise and forming a local alkaline environment, the local alkaline environment inducing the reaction of dopamine and resorcinol to generate blue fluorescent substance, after 40-120min, adding excess hydrochloric acid to terminate the reaction; Step S3, using a fluorescence spectrophotometer to detect the fluorescence intensity of the reaction solution, and calculating the urease activity in the sample according to the fluorescence intensity-urease concentration standard curve.

[0009] Further, in step S1, the mass concentration of urea is 0.02-0.1%.

[0010] Further, the mass concentration of urea is 0.06-0.1%.

[0011] Further, in step S2, the molar ratio of dopamine to resorcinol is 1:1.

[0012] Further, in step S2, the reaction temperature is 35℃, and the reaction time is 60min.

[0013] Further, in step S3, when the urease concentration is 40-120 U / L, the fluorescence intensity-urease concentration standard curve is F=1.99U-70.35, and the correlation coefficient R²=0.9978; When the urease concentration is 120-200 U / L, the fluorescence intensity-urease concentration standard curve is F=0.53U+108.29, and the correlation coefficient R²=0.9803.

[0014] Compared with the prior art, the urease activity detection method based on the non-reversible reaction of dopamine and resorcinol provided by the application has the beneficial effects that: First, the urease activity detection method based on the non-reversible reaction of dopamine and resorcinol provided by the application has the principle that in a reaction system stabilized by PBS 7.0 buffer, urease catalyzes the hydrolysis of urea to produce NH3 and CO2, causing the pH in the surrounding area of the urease to rise to form a local alkaline environment (pH>8), thereby inducing resorcinol and dopamine to react around the urease to generate blue fluorescent substance Azamonardine (Aza), and then realizing urease activity detection by measuring the fluorescence intensity at 455 nm. The urease activity detection method based on the reaction of dopamine and resorcinol provided by the application has a good linear relationship between the fluorescence intensity and the urease activity in the range of 40-200 U / L, wherein the linear equation fitted in the range of 40-120 U / L is F=1.99U-70.35, R 2 =0.9978; and the linear equation fitted in the range of 120-200 U / L is F=0.53U+108.29, R 2 =0.9803, and the detection limit is 0.106 U / L, and the sensitivity is high.

[0015] Second, the urease activity detection method based on the non-reversible reaction of dopamine and resorcinol provided by the application can quickly and efficiently detect the urease activity in a sample, and has good selectivity.

[0016] Third, the urease activity detection method based on the non-reversible reaction of dopamine and resorcinol provided by the application is carried out in a PBS buffer system at pH 7.0, the reaction is non-reversible, and the influence of pH change on detection can be well excluded, for example, the phenomenon of protein precipitation caused by pH change can be well inhibited, thereby improving the detection sensitivity. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to make the technical solutions in the embodiments of the present application clearer, the accompanying drawings needed in the embodiment description will be briefly introduced. Obviously, the accompanying drawings in the following description are only some embodiments of the present application, and other accompanying drawings can be obtained by those skilled in the art without any creative effort.

[0018] Figure 1 is the reaction principle diagram of the urease activity detection method based on the reaction of dopamine and resorcinol of the present application; Figure 2 is the comparison diagram of dopamine and resorcinol under different reaction systems; Figure 3 is the ultraviolet-visible absorption spectrum diagram of the reaction solution of dopamine and resorcinol under the reaction system added with urease and urea; Figure 4 is the fluorescence excitation spectrum and emission spectrum diagram of the reaction solution of dopamine and resorcinol under different reaction systems; Figure 5 is the influence of the urea concentration on the fluorescence intensity in the present application; Figure 6 is the influence of the reaction temperature on the fluorescence intensity in the present application; Figure 7 is the influence of the reaction time on the fluorescence intensity in the present application; Figure 8 is the fluorescence spectrum diagram under different urease concentrations; Figure 9 is the standard curve of different urease concentrations and fluorescence intensity; Figure 10 is the fluorescence intensity comparison diagram of 100 U / L urease concentration and anti-interference solution. DETAILED DESCRIPTION

[0019] In order to make the technical solutions in the embodiments of the present application clearer, the accompanying drawings needed in the embodiment description will be briefly introduced. Obviously, the accompanying drawings in the following description are only some embodiments of the present application, and other accompanying drawings can be obtained by those skilled in the art without any creative effort.

[0020] The endpoints of the ranges and any values in the ranges disclosed herein are not limited to the precise values stated. The ranges and values should be construed to be approximations that allow for significant variation. Various ranges of values are stated in terms of being wider than the ranges and endpoints. Each narrower range can be a new value that is separately and specifically disclosed herein. These narrower ranges are not to be taken as an implied disavowal of the broader ranges or values. EMBODIMENT

[0021] A urease activity detection method based on the reaction of dopamine and resorcinol, comprising the following steps: Step S1, mix the sample to be tested, PBS 7.0 buffer solution and a certain concentration of urea solution; Step S2, add a certain amount of dopamine and resorcinol to the mixed solution of step S1, under the condition of 25-45℃, urease catalyzes the hydrolysis of urea, causing the pH of the surrounding area of urease to rise to form a local alkaline environment, which induces the reaction of dopamine and resorcinol to generate blue fluorescent substance, after 40-120min, add excess hydrochloric acid to terminate the reaction; Step S3, use a fluorescence spectrophotometer to detect the fluorescence intensity of the reaction solution, and calculate the urease activity in the sample according to the fluorescence intensity-urease concentration standard curve.

[0022] The detection method of the present application has the following principles: Dopamine and resorcinol generate blue fluorescent substance Aza under alkaline conditions, which is a pH-sensitive fluorescent substance and can be used as a pH indicator. Urease catalyzes the accelerated hydrolysis of urea into ammonia and carbamic acid, which is then further converted into ammonia and carbon dioxide, causing a change in the local pH of the solution, and the pH in the surrounding area of urease rises to alkaline (pH>8), which induces the reaction of dopamine and resorcinol to generate fluorescent substance, as shown in Figure 1 Due to the high selectivity and specificity of urease in catalyzing urea, different urease activity intensities result in different pH values of the surrounding environment during urea decomposition, thereby affecting the blue fluorescence intensity of the product Aza, which in turn reflects the activity of urease. In the reaction process, hydrochloric acid is added to reduce the pH value of the reaction system (pH<7) to maintain the fluorescence intensity at the end of the reaction. The detection method of the present application is carried out in a PBS buffer system with pH 7.0, which is stable and can eliminate the influence of pH change on detection.

[0023] Feasibility analysis of the detection method of the present application: In the reaction system with added urease and urea, after a long time of reaction, the solution of resorcinol and dopamine gradually turns into light yellow under natural light, and exhibits strong bright blue fluorescence under ultraviolet light, as shown in Figure 2 The left side is the reaction system without adding urease and urea (blank sample), and the right side is the reaction system with added urease and urea. Blue fluorescence is Aza, and light yellow is caused by the polymerization of dopamine due to the increase of pH value. As shown in Figure 3 In the reaction system with added urease and urea, the reaction solution has a strong absorption peak of π-π* transition in the UV-Vis spectrum at 420 nm; as shown in Figure 4As shown, the optimal excitation wavelength is 390 nm and the optimal emission wavelength is 455 nm, determined by the determination of the fluorescence spectrophotometer. Ex represents the fluorescence excitation spectrum of the reaction solution under the reaction system with the addition of urease and urea, Em represents the fluorescence emission spectrum of the reaction solution under the reaction system with the addition of urease and urea, and Blank represents the emission spectrum of the blank sample under the reaction system without the addition of urease and urea.

[0024] The detection method of the present application can produce obvious blue fluorescence under the irradiation of an ultraviolet lamp after the addition of urease in a stable pH reaction system containing a fluorescent substrate and urea, and can be used for detecting the content of urease.

[0025] In order to obtain better analysis performance in the analysis of urease activity, the urea concentration, temperature and reaction time are optimized. Specifically as follows: Example 2 Optimization of urea concentration Take 5 clean test tubes, respectively using the pipette to take 0.1% urea solution 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 0.1 mL, in turn into 5 test tubes, then use the pipette to take 0.8 mL, 0.6 mL, 0.4 mL, 0.2 mL, 0 mL of ultrapure water, add to the corresponding test tube, to make 1 mL 0.02%, 0.04%, 0.06%, 0.08%, 0.1% urea solution. Each test tube is in turn using the pipette to take 0.5 mL 0.1 U / mL urease solution, 2.5 mL PBS 7.0 buffer, 0.5 mL 0.1 mM resorcinol solution and 0.5 mL 0.1 mM hydrochloric acid dopamine solution, mixed reaction. The reaction condition is 25℃ for 60 min, after the reaction time, add 0.2 mL 1 mol / L hydrochloric acid, the pH value of the reaction system is less than 7, become acidic solution to terminate the reaction, then use the fluorescence spectrophotometer to test the fluorescence intensity. All the tests and analysis are repeated three times.

[0026] The effect of different urea concentrations on fluorescence intensity is shown in Figure 5 As shown in Figure 5It can be seen that the fluorescence intensity increases with the increase of urea concentration, and then the trend gradually flattens out, reaching a maximum at a urea concentration of 0.1%. Therefore, this urea concentration is selected for further study. When discussing the kinetics of enzymatic reactions, it is generally observed that the relationship between substrate concentration and reaction rate follows the Michaelis-Menten kinetic model. In this model, as the substrate concentration increases, the reaction rate also increases, until a plateau is reached, at which point the reaction rate no longer changes significantly even if the substrate concentration continues to rise, reflecting the saturation state of the enzyme. In the study of the effect of urea substrate concentration on urease activity, it was observed that the relationship generally conforms to the above theoretical framework. In the experiment, when the substrate concentration was increased by 0.02% increments, the change in fluorescence intensity (as a measure of urease activity) showed a gradually increasing trend. When the substrate concentration increased to 0.1%, the urease activity reached a maximum. Further analysis of the substrate concentration range of 0.06% to 0.1% showed a stable trend in fluorescence intensity measurements, indicating that the enzyme reaction rate tended to stabilize in this concentration range. Therefore, the concentration of urea is preferably 0.06-0.1%, more preferably 0.1%.

[0027] Example 3 Optimization of reaction temperature Take 5 clean test tubes, accurately pipette 0.5 mL 0.1 U / mL urease solution and 1 mL 0.1% urea solution, 2.5 mL PBS 7.0 buffer solution into the test tubes, then pipette 0.5 mL 0.1 mM dopamine hydrochloride and 0.5 mL 0.1 mM resorcinol into the 5 test tubes, respectively. Place the test tubes in 15°C, 25°C, 35°C, 45°C, 55°C, respectively, and react for 60 min. After the reaction is completed, add 0.2 mL 1 mol / L hydrochloric acid to make the pH value of the reaction system less than 7, and terminate the reaction as an acidic solution. Then use a fluorescence spectrophotometer to test the fluorescence intensity. All tests and analyses are repeated three times.

[0028] The effect of different reaction temperatures on fluorescence intensity is shown in Figure 6 As can be seen from Figure 6 With the gradual increase of environmental temperature from 15°C to 55°C, the fluorescence intensity indicator shows a trend of first increasing and then decreasing, with a peak at 35°C. This result points to 35°C as the optimal reaction temperature for this enzymatic reaction.

[0029] Example 4 Optimization of reaction time Take a clean test tube, accurate 0.5 mL 0.1 U / mL urease solution, respectively with 1 mL 1% urea solution, 2.5 mL PBS 7.0 buffer mixed in the test tube, and then in turn 0.5 mL 0.1 mM dopamine hydrochloride and 0.5 mL 0.1 mM resorcinol in the test tube. Under the condition of 35℃, respectively, every 5 min to measure the fluorescence intensity until the fluorescence intensity is no longer increased. All tests and analysis are repeated three times.

[0030] The effect of different reaction times on fluorescence intensity is shown in Figure 7 As can be seen from Figure 7 When the fluorescence intensity is taken as the ordinate, the reaction curve gradually flattens out within 3 h. When the urea concentration remains constant, as the reaction progresses, the urea molecules involved in the reaction are gradually consumed, leading to a decrease in their concentration, and thus causing the reaction rate to slow down. In addition, during the reaction process for a long time, due to the accumulation of products or by-products, the change of fluorescence intensity may reach a plateau, so that the difference in fluorescence intensity between different samples is reduced, which is not conducive to the accurate determination of urease activity. In order to ensure the accuracy of enzyme activity determination, it is usually required to retain a certain amount of unreacted substrate in the reaction conditions, so that the reaction approaches zero-order reaction kinetics, i.e. the reaction rate is independent of time. By analyzing the reaction rate-time graph, it can be determined that at 60 min, the reaction rate reaches a maximum. Therefore, 60 min is selected as the optimal reaction time point for determining urease activity, to ensure that near this time point, the reaction rate can accurately reflect the activity level of urease.

[0031] Therefore, finally, the reaction time of 60 min, the reaction temperature of 35℃, and the urea concentration of 0.1% are selected for urease activity detection determination. Through these optimization measures, the detection range is expanded and the sensitivity of the reaction system is improved.

[0032] Example 5 Preparation of standard curve Take 11 clean test tubes and accurately pipette 0 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, 4 mL, 4.5 mL, and 5 mL of 200 U / L urease solution. Add 4.5 mL, 4 mL, 3.5 mL, 3 mL, 2.5 mL, 2 mL, 1.5 mL, 1 mL, 0.5 mL, and 0 mL of ultrapure water respectively to dilute the solutions to urease concentrations of 0 U / L, 20 U / L, 40 U / L, 60 U / L, 80 U / L, 100 U / L, 120 U / L, 140 U / L, 160 U / L, 180 U / L, and 200 U / L. The 0 U / L solution is ultrapure water used as a blank sample to replace urease. Take 0.5 mL of each of the above diluted solutions, mix 1 mL of 0.1% urea solution with 0.5 mL of solutions of different concentrations of urease, and 2.5 mL of PBS 7.0 buffer. Then, add 0.5 mL of 0.1 mM dopamine hydrochloride and 0.5 mL of 0.1 mM resorcinol as fluorescent reactants. After the optimal reaction time at the optimal temperature, add 0.2 mL of 1 mol / L hydrochloric acid to terminate the reaction. The study was conducted on urease concentrations ranging from 0 to 200 U / L.

[0033] The ability of the fluorescence method to detect urease activity was evaluated under the conditions of 60 min reaction, 35℃ temperature, and 0.1% urea concentration. Figure 8 It can be seen that as the amount of urease increased from 0 U / L to 200 U / L, the fluorescence intensity at the excitation wavelength of 455 nm gradually increased.

[0034] Please see Figure 9 This is a standard curve showing the relationship between different urease concentrations and fluorescence intensity. Figure 9Based on the increasing trend of urease activity, it can be divided into two ranges: 40-120 U / L and 120-200 U / L. In the lower urease concentration range (40-120 U / L), a good linear relationship was observed between fluorescence intensity and urease concentration. Within the 40-120 U / L range, the fitted linear equation was F = 1.99U - 70.35, with a correlation coefficient R² = 0.9978. In the higher urease concentration range (120-200 U / L), the linear equation of the standard curve was F = 0.53U + 108.29, with a correlation coefficient R² = 0.9803. In this linear equation, U represents the x-axis (urease concentration), and F represents the y-axis (fluorescence intensity). These data indicate that the linear relationship between fluorescence intensity and urease concentration is significant within their respective concentration ranges. The limit of detection (LOD) was calculated to be 0.106 U / L according to the standard method recommended by the International Union of Pure and Applied Chemistry (IUPAC).

[0035] The urease-catalyzed hydrolysis of urea is localized, and the resulting hydroxyl ions (OH-) - This process directly serves as a condition for the chemical oxidation reaction of resorcinol and dopamine, generating the fluorescent compound Aza. This demonstrates that even at extremely low urease concentrations, urease activity can be quantitatively analyzed by monitoring the fluorescence intensity of Aza over extended reaction times. Notably, this series of chemical reactions is designed to be irreversible, ensuring the stability and accuracy of the detection process.

[0036] The detection method of the present invention is compared with existing detection methods, as shown in Table 1: Table 1: Comparison of LOD and detection range between this invention, the standard method, and other fluorescence methods. ; in: [1] National Food Safety Standard: Determination of Urease in Infant Foods and Dairy Products: [S]. 2013: [2] LI S, XIAO L, XIAO L, et al. Coordination polymer nanoprobeintegrated carbon dot and phenol red for turn-on fluorescence detection ofurease activity [J]. Mikrochimica Acta, 2023, 190(2): 79. [3] SHI F, SHANG D, WANG Z. An rGQD / chitosan nanocomposite-based ph-sensitive probe: Application to sensing in urease activity assays [J]. NewJournal of Chemistry, 2019, 43. [4] LIU J, ZHANG J, ZHANG Y, et al. A ph-responsive fluorometric and colorimetric system based on silicon quantum dots and 4-nitrophenol forurease activity detection [J]. Talanta, 2022, 237: 122956. The detection method of this invention is applicable to the detection of low concentrations of urease activity and has high sensitivity. Compared with other urease detection methods, this invention has a lower detection limit, more sensitive reaction, and simpler reactant addition, requiring no complex preparation. Furthermore, since the fluorescence intensity of this reaction is based on the yield of Aza, the sensitivity can be improved by extending the reaction time to allow resorcinol and dopamine to react fully. Regarding reaction time, since the fluorescence reaction will not occur at pH values ​​below 8, the reaction time is longer than other methods to obtain a more pronounced fluorescence signal.

[0037] Example 6: Selectivity Analysis To investigate selectivity, 0.5 mL of α-amylase (AM), wasabi root peroxidase (PH), glucose oxidase (GO), bovine liver catalase (CO), and Aspergillus glucose oxidase (GOA) were used at concentrations 10 times higher than urease activity. Common metal salts such as CaCl, NaCl, and MgSO4 were selected to replace urease in the assay. 0.5 mL of anti-interference solution was used instead of urease, followed by 1 mL of urea solution at the optimal concentration, 2.5 mL of PBS 7.0 buffer, 0.5 mL of 0.1 mM dopamine hydrochloride, and 0.5 mL of 0.1 mM resorcinol. The reaction was carried out at the optimal temperature and time, and 0.2 mL of 1 mol / L hydrochloric acid was added to terminate the reaction. Fluorescence intensity was then measured. All experiments and analyses were repeated three times.

[0038] Because urease can catalyze the hydrolysis of urea to produce OH... - Other enzymes convert urea to produce OH- -There is no catalytic effect; therefore, the analytical method of this invention has good selectivity for determining urease activity, such as... Figure 10 As shown. Under the same experimental conditions, α-amylase, wasabi root peroxidase, glucose oxidase, bovine liver catalase, Aspergillus glucose oxidase, CaCl, NaCl, and MgSO4 did not induce fluorescence. The experimental results indicate that a significant change in fluorescence intensity was observed only in the presence of urease, thus confirming the high selectivity of this detection system for the determination of urease activity.

[0039] Example 7: Analysis of Actual Samples To evaluate the practical application of the assay method, the urease content in two samples—a live-culture lactic acid beverage and a plant-based protein beverage—was quantitatively determined. The samples were purchased from a local supermarket. Because the beverages were opaque, turbid liquids, direct measurement of the plant protein would affect the fluorescence intensity, necessitating dilution.

[0040] Take 1 g (i.e., pipette 1 mL) of sample and add it to an Erlenmeyer flask containing 249 mL of ultrapure water. Dilute 250 times, then pipette 0.5 mL of the sample to replace urease. Add 1 mL of urea solution of the optimal concentration, 2.5 mL of PBS 7.0 buffer, 0.5 mL of 0.1 mM dopamine hydrochloride, and 0.5 mL of 0.1 mM resorcinol. React at the optimal temperature and time, then stop the reaction by adding 0.2 mL of 1 mol / L hydrochloric acid. Measure the fluorescence intensity. Based on the above experiments, spiked experiments were performed, adding 40 U / L, 100 U / L, and 200 U / L of urease respectively, and measuring the fluorescence intensity. All experiments and analyses were repeated three times. The detection results are shown in Table 2. Table 2: Determination of urease concentration in actual samples ; Urease cannot be found in live-culture lactic acid beverages, and the urease activity should be negative. Plant-based protein beverages, whose main ingredient is soybeans, are products that do not require heat treatment to inactivate enzymes before consumption; therefore, their urease activity should also be negative. GB20371—2016, "Plant Protein for Food Processing," specifies that urease activity should be detected using a negative quantitative method, and the urease activity index of a negative product should be ≤0.02 U / g. The urease activity in the selected samples was analyzed using standard methods. The results showed that neither sample contained any urease, and the urease activity index of both products was less than 0.02 U / g, both meeting the requirements of Chinese standards.

[0041] In summary, the detection method of the present invention has the advantages of convenient operation, high sensitivity, and good selectivity.

[0042] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations made to these embodiments without departing from the principles and spirit of the present invention still fall within the protection scope of the present invention.

Claims

1. A method for detecting urease activity based on the irreversible reaction of dopamine and resorcinol, characterized in that, Includes the following steps: Step S1: Mix the sample to be tested, PBS 7.0 buffer, and a urea solution of a certain concentration. Step S2: Add a certain amount of dopamine and resorcinol to the mixed solution in step S1. Under the condition of 25-45℃, urease catalyzes the hydrolysis of urea, causing the pH of the area around urease to rise and form a local alkaline environment. The local alkaline environment induces dopamine and resorcinol to react and generate a blue fluorescent substance. After reacting for 40-120 min, add excess hydrochloric acid to terminate the reaction. Step S3: Use a fluorescence spectrophotometer to detect the fluorescence intensity of the reaction solution, and calculate the urease activity in the sample based on the fluorescence intensity-urease concentration standard curve.

2. The method for detecting urease activity based on the reaction of dopamine and resorcinol according to claim 1, characterized in that, In step S1, the mass concentration of urea is 0.02-0.1%.

3. The method for detecting urease activity based on the reaction of dopamine and resorcinol according to claim 2, characterized in that, The mass concentration of urea is 0.06-0.1%.

4. The method for detecting urease activity based on the reaction of dopamine and resorcinol according to claim 1, characterized in that, In step S2, the molar ratio of dopamine to resorcinol is 1:

1.

5. The method for detecting urease activity based on the reaction of dopamine and resorcinol according to claim 1, characterized in that, In step S2, the reaction temperature is 35℃ and the reaction time is 60 min.

6. The method for detecting urease activity based on the reaction of dopamine and resorcinol according to claim 1, characterized in that, In step S3, when the urease concentration is 40-120 U / L, the standard curve of fluorescence intensity-urease concentration is F = 1.99U - 70.35, and its correlation coefficient R² = 0.9978. When the urease concentration is 120-200 U / L, the standard curve of fluorescence intensity-urease concentration is F = 0.53U + 108.29, and the correlation coefficient R² = 0.9803.

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