Method for detecting plasmin in dairy product

Abstract

The invention provides a method for detecting plasmin in dairy products, which comprises the following steps: firstly dissociating plasmin in milk in a buffer solution (containing a dissociating agent), and then hydrolyzing non-fluorescent coumarin peptide by using the dissociated plasmin to release 7-amino-4-methyl coumarin (AMC). And determining the activity of plasmin in the sample by measuring the concentration change of the fluorescence product AMC. According to the present invention, the dissociation reagent, the detection mode (dynamic and non-dynamic), the detection instrument parameter, the specific detection process and the like are subjected to the experimental invention improvement, and the detection method suitable for the experimental matrix is established so as to provide the more accurate detection data; results show that the standard curve obtained by the method is excellent in linearity and relatively good in repeatability, and the detection result is not influenced by the milk turbidity and is stable.

Description

Technical Field

[0001] This invention relates to the technical field of plasmin detection, and specifically to a method for detecting plasmin in dairy products. Background Technology

[0002] The endogenous enzymes in milk that can cause protein hydrolysis mainly include plasmin, elastase, and cathepsin. Plasmin activity accounts for about 60% of the total protease activity and is an inherent natural protease in milk. It exists bound to casein micelles and is generally transported from the blood to the mammary glands and secreted into the milk. The plasmin system in milk is a complex system, including plasminogen, plasminogen activator, plasminogen activator inhibitor, and plasmin inhibitor. Plasminogen can be activated into plasmin by plasminogen activator. The casein hydrolysis caused by plasmin is an important reason for the gelation and bitterness of UHT milk during storage.

[0003] With the increasing emphasis on health and functionality in products, research on milk protein extracts has become a hot topic in the industry, and the purity and enzyme content of milk protein extracts are gradually attracting attention. However, there is currently no unified standard for the detection methods and procedures of plasmin in milk and milk protein extracts. Establishing an effective plasmin detection procedure is of great significance for studying the plasmin system components in milk and milk protein extracts, plasmin-induced milk aging gels, and factors affecting plasmin activity.

[0004] Currently, methods for determining plasmin in milk mainly include spectrophotometry, enzyme-linked immunosorbent assay (ELISA), SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and fluorescence methods. Among these, ELISA has relatively low accuracy due to interference from casein. While spectrophotometry has been improved, turbidity interference still exists, leading to significant errors in the results. Gel electrophoresis is more suitable for qualitative analysis. Fluorescence methods, due to their high sensitivity, have become an important method for enzyme research. Summary of the Invention

[0005] In view of this, the present invention provides a method for detecting plasmin in dairy products, which overcomes the problems of poor sensitivity and turbidity interference in existing methods, provides a technical means for the systematic study of plasmin, and lays the foundation for the study of enzymes in milk.

[0006] To address the technical problems mentioned in the background section, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for detecting plasmin in dairy products, the method comprising the following steps:

[0008] Add the dairy product to be tested to sodium citrate buffer and incubate to obtain the plasmin dissociation solution for later use;

[0009] The substrate solution was added to the fibrinolytic enzyme dissociation solution and the reaction was carried out. Samples were taken multiple times during the reaction. The sampling method was as follows: the mixture after the reaction was added to acetonitrile solution to terminate the enzymatic hydrolysis reaction, and the supernatant was taken as the sample after centrifugation. The substrate in the substrate solution was N-succinyl-L-alanyl-L-phenylalanyl-L-lysyl-7-amino-4-methylcoumarin peptide.

[0010] Standard working solutions of different concentrations were prepared using 7-amino-4-methylcoumarin as a standard substance;

[0011] The sample and standard working solutions of different concentrations were tested using an ELISA reader. A standard curve was plotted based on the test results. The relative fluorescence increment of the sample was calculated and substituted into the standard curve to calculate the activity of plasmin in the sample to be tested.

[0012] Optionally, the dairy product to be tested can be added to sodium citrate buffer and incubated at 37±1℃ for 15 min to 25 min.

[0013] Optionally, samples are taken four times during the reaction, with the first sample taken 3 to 6 minutes after the reaction, and then every 15 minutes thereafter.

[0014] Optionally, the relative fluorescence unit increment of the sampled sample is calculated by: calculating the relative fluorescence unit increment between the fourth sampled sample and the first sampled sample.

[0015] Optionally, the substrate solution includes a substrate and a substrate buffer, wherein the substrate buffer includes Tris-HCl buffer solution, dimethyl sulfoxide, and sodium chloride.

[0016] Optionally, the concentration of N-succinyl-L-alanyl-L-phenylalanyl-L-lysyl-7-amino-4-methylcoumarin peptide in the substrate solution is 1–2 mmol / L.

[0017] Optionally, the standard working solution includes an aqueous solution of 7-amino-4-methylcoumarin and acetonitrile; the concentrations of 7-amino-4-methylcoumarin in the standard working solution are 0.2 nmol / L, 0.5 nmol / L, 1 nmol / L, 2 nmol / L, 5 nmol / L, 10 nmol / L, 20 nmol / L, 50 nmol / L, 100 nmol / L, 200 nmol / L, 500 nmol / L, and 1000 nmol / L.

[0018] Optionally, the equation of the standard curve is y = 0.0041x - 1.4988(R). 2=0.9998), where x is the fluorescence value of 7-amino-4-methylcoumarin and y is the concentration of 7-amino-4-methylcoumarin.

[0019] Optionally, the enzyme-linked immunosorbent assay (ELISA) reader is a fluorescent ELISA reader.

[0020] Optionally, the instrument parameters of the fluorescence microplate reader are as follows: set temperature 25℃, excitation wavelength: 360nm, emission wavelength: 450nm, top measurement, extended gain, detection height 7mm, bandwidth 8nm.

[0021] Optionally, the dairy product to be tested includes at least one of sterilized milk, raw milk, skim milk, and milk protein extract.

[0022] The beneficial effects of the above-described technical solution of the present invention are as follows:

[0023] This invention provides a method for detecting plasmin in dairy products. First, the plasmin in milk is dissociated in a buffer solution (containing a dissociating agent). Then, the dissociated plasmin hydrolyzes non-fluorescent coumarin peptides to release 7-amino-4-methylcoumarin (AMC). The activity of plasmin in the sample is determined by measuring the concentration change of the fluorescent product AMC. This invention improves the dissociation reagent, detection mode (kinetic and non-kinetic), detection instrument parameters, and specific detection procedures through experimental invention, establishing a detection method suitable for experimental matrices to provide more accurate detection data. Results show that the standard curve obtained by this method has excellent linearity and good repeatability, and the detection results are not affected by milk turbidity, exhibiting stable results. Attached Figure Description

[0024] Figure 1 The standard curve for the standard substance 7-amino-4-methylcoumarin;

[0025] Figure 2 A comparison of the fibrinolytic activity of UHT sterilized milk, skim milk, and milk protein extracts before and after centrifugation;

[0026] Figure 3 This is a picture of the sample after centrifugation. Detailed Implementation

[0027] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the present invention.

[0028] The present invention will be further explained and described below with reference to the accompanying drawings.

[0029] In a first aspect, the present invention provides a method for detecting plasmin in dairy products, the method comprising the following steps:

[0030] Add the dairy product to be tested to sodium citrate buffer and incubate to obtain the plasmin dissociation solution for later use;

[0031] The substrate solution was added to the fibrinolytic enzyme dissociation solution and the reaction was carried out. Samples were taken multiple times during the reaction. The sampling method was as follows: the mixture after the reaction was added to acetonitrile solution to terminate the enzymatic hydrolysis reaction, and the supernatant was taken as the sample after centrifugation. The substrate in the substrate solution was N-succinyl-L-alanyl-L-phenylalanyl-L-lysyl-7-amino-4-methylcoumarin peptide.

[0032] Standard working solutions of different concentrations were prepared using 7-amino-4-methylcoumarin as a standard substance;

[0033] The sample and standard working solutions of different concentrations were tested using an ELISA reader. A standard curve was plotted based on the test results. The relative fluorescence increment of the sample was calculated and substituted into the standard curve to calculate the activity of plasmin in the sample to be tested.

[0034] According to some embodiments of the present invention, the dairy product to be tested is added to sodium citrate buffer and incubated at 37±1℃ for 15 min to 25 min.

[0035] According to some embodiments of the present invention, the dairy product to be tested includes at least one of sterilized milk, raw milk, skim milk, and milk protein extract.

[0036] In this invention, N-succinyl-L-alanyl-L-phenylalanyl-L-lysyl-7-amino-4-methylcoumarin peptide is used as a substrate. Plasmin reacts with the substrate N-succinyl-L-alanyl-L-phenylalanyl-L-lysyl-7-amino-4-methylcoumarin peptide to generate 7-amino-4-methylcoumarin. The content of 7-amino-4-methylcoumarin generated in this invention is calculated based on the change in fluorescence value before and after the reaction and the standard curve of 7-amino-4-methylcoumarin standard material. The plasmin activity is then calculated based on the content of 7-amino-4-methylcoumarin generated.

[0037] Specifically, the detection method provided in this invention may include the following steps:

[0038] (1) Sample pretreatment: The dairy product to be tested was incubated with sodium citrate buffer to obtain a plasmin dissociation solution for later use. Before testing, the dairy product to be tested needs to be pretreated to dissociate the plasmin. The pH value of the sodium citrate buffer is 7.0-8.5. The preparation method of the sodium citrate buffer is as follows: Prepare 100 mL each of 100 mM sodium dihydrogen phosphate solution and disodium hydrogen phosphate solution, take 50 mL of each solution, mix well, add 10.32 g of sodium citrate, dissolve and mix well, and store.

[0039] (2) Reaction with substrate: After adding substrate solution to the fibrinolytic enzyme dissociation solution, the reaction is carried out. During the reaction, samples are taken multiple times for testing. The sampling method is as follows: take the mixture after the reaction, add acetonitrile solvent to terminate the enzymatic hydrolysis reaction, centrifuge and take the supernatant as the sample.

[0040] The substrate solution described in this invention is an N-succinyl-L-alanyl-L-phenylalanyl-L-lysyl-7-amino-4-methylcoumarin peptide solution with a concentration of 1–2 mmol / L and a pH of 8.0. The substrate solution is prepared by mixing dimethyl sulfoxide and 60 mmol / L Tris-HCl buffer solution at a volume ratio of 1:4, adding sodium chloride and adjusting the sodium chloride concentration to 0.25 mol / L, then adding the substrate and adjusting the substrate concentration to 1–2 mmol / L.

[0041] During the reaction of the fibrinolytic enzyme dissociation solution with N-succinyl-L-alanyl-L-phenylalanyl-L-lysyl-7-amino-4-methylcoumarin peptide, the present invention repeatedly samples the reactants for testing. In each sampling process, 50 μL of the reaction mixture was rapidly mixed with 500 μL of acetonitrile and 500 μL of distilled water to stop the enzymatic reaction, centrifuged at 12000 rpm for 10 min, and the supernatant was collected for later use. The present invention found that if the sample is not centrifuged and the supernatant is not collected before testing, the turbidity of the sample will affect the test results of the fluorescent microplate reader, thus affecting the accuracy of the test.

[0042] (3) Prepare standard working solutions of different concentrations using 7-amino-4-methylcoumarin as the standard substance; the reagents are equal volumes of acetonitrile aqueous solution.

[0043] (4) Testing and Result Calculation: The sampled sample and standard working solution of different concentrations were tested using an enzyme-linked immunosorbent assay (ELISA) reader. Based on the test results, a standard curve of plasmin was plotted, the relative fluorescence unit increment of the sampled sample was calculated, and the plasmin activity in the sample to be tested was calculated by substituting the relative fluorescence unit increment of the sampled sample into the standard curve.

[0044] The fluorescence values ​​of the sampled specimens and standard working solutions of different concentrations were tested. A standard curve was plotted using the relationship between the fluorescence values ​​and concentrations of the standard working solutions of different concentrations. The relative fluorescence increment of the sampled specimens was then calculated and substituted into the standard curve to calculate the amount of 7-amino-4-methylcoumarin generated. Finally, the plasmin activity was calculated based on the amount of 7-amino-4-methylcoumarin generated.

[0045] According to some embodiments of the present invention, the volume ratio of the fibrinolytic enzyme dissociation solution to the substrate solution is (1-2):(2-1).

[0046] According to some embodiments of the present invention, the loading volume of the sample taken by the microplate reader and the standard working solution of different concentrations is 200 μL.

[0047] According to some embodiments of the present invention, samples are taken four times during the reaction process, with the first sample taken 3 to 6 minutes after the reaction, and then every 15 minutes thereafter.

[0048] According to some embodiments of the present invention, the relative fluorescence unit increment of the sampled sample is calculated by calculating the relative fluorescence unit increment between the fourth sampled sample and the first sampled sample.

[0049] According to some embodiments of the present invention, the substrate solution includes a substrate and a substrate buffer, wherein the substrate buffer includes Tris-HCl buffer solution, dimethyl sulfoxide and sodium chloride.

[0050] According to some embodiments of the present invention, the concentration of N-succinyl-L-alanyl-L-phenylalanyl-L-lysyl-7-amino-4-methylcoumarin peptide in the substrate solution is 1 to 2 mmol / L.

[0051] According to some embodiments of the present invention, the standard working solution comprises an aqueous solution of 7-amino-4-methylcoumarin and acetonitrile; the concentrations of 7-amino-4-methylcoumarin in the standard working solution are 0.2 nmol / L, 0.5 nmol / L, 1 nmol / L, 2 nmol / L, 5 nmol / L, 10 nmol / L, 20 nmol / L, 50 nmol / L, 100 nmol / L, 200 nmol / L, 500 nmol / L, and 1000 nmol / L, respectively.

[0052] According to some embodiments of the present invention, the equation of the standard curve is y = 0.0041x - 1.4988(R). 2 =0.9998), where x is the fluorescence value of 7-amino-4-methylcoumarin and y is the concentration of 7-amino-4-methylcoumarin.

[0053] According to some embodiments of the present invention, the enzyme-linked immunosorbent assay (ELISA) reader is a fluorescent ELISA reader.

[0054] According to some embodiments of the present invention, the instrument parameters of the fluorescence microplate reader are as follows: set temperature 25°C, excitation wavelength: 360nm, emission wavelength: 450nm, top measurement, extended gain, detection height 7mm, bandwidth 8nm.

[0055] This invention establishes a fluorescence-based detection procedure for plasmin in milk (raw milk, UHT milk, skim milk) and milk protein extracts through extensive experiments. The plasmin in milk dissociates in a buffer solution (containing a dissociating agent), hydrolyzing non-fluorescent coumarin peptides to release 7-amino-4-methylcoumarin (AMC). The reaction is terminated by adding a stop reagent at different time points, and the plasmin activity in the sample is determined by measuring the concentration change of the fluorescent product AMC. This invention improves the dissociation reagent, detection mode (kinetic and non-kinetic), detection instrument parameters, and specific detection procedure through experimental invention, establishing a detection method suitable for experimental matrices to provide more accurate detection data. Results show that the standard curve obtained by this method has excellent linearity and good repeatability, and the detection results are not affected by milk turbidity, exhibiting stable results. This method is an excellent method for studying plasmin in milk and milk protein extracts and is easy to promote.

[0056] The present invention will be further described below through some specific embodiments.

[0057] The materials and equipment used in the following embodiments and comparative examples are as follows:

[0058] Materials: 7-Amino-4-methylcoumarin (AMC), 1M Tris-HCl solution (pH=8.0), N-succinyl-L-alanyl-L-phenylalanyl-L-lysyl-7-amino-4-methylcoumarin peptide, dimethyl sulfoxide, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium citrate, 96-well microplate (black).

[0059] Equipment: water bath, pipette, vortex mixer, fluorescent microplate reader, 96-well microplate (black).

[0060] The instrument parameters of the fluorescent microplate reader used were as follows: set temperature 25℃, excitation wavelength: 360nm, emission wavelength: 450nm, top measurement, extended gain, detection height 7mm, bandwidth 8nm.

[0061] Reagent preparation:

[0062] PBS-sodium citrate buffer solution (first solution, pH = 7.0-8.5): Prepare 100 mL each of 100 mM sodium dihydrogen phosphate solution and disodium hydrogen phosphate solution. Take 50 mL of each solution, mix well, add 10.32 g of sodium citrate, dissolve and mix well, and store.

[0063] 1 mmol / L coumarin peptide solution (second solution): Take 3 mL of 1 mol / L Tris-HCl buffer solution (pH 8.0) and mix it with 47 mL of ultrapure water to obtain a 60 mmol / L Tris-HCl buffer solution. Take 4 mL of the 60 mmol / L Tris-HCl buffer solution and mix it with 1 mL of dimethyl sulfoxide. Weigh 0.07310 g of sodium chloride and dissolve it to make the final concentration of sodium chloride 0.25 mol / L. Prepare a 5 mL substrate buffer solution. Weigh 0.003108 g of N-succinyl-L-alanyl-L-phenylalanyl-L-lysyl-7-amino-4-methylcoumarin peptide into the above 5 mL solution to make the final concentration 1 mmol / L. Prepare fresh before use.

[0064] Standard substance solution: The standard substance is 7-amino-4-methylcoumarin. A 1 mmol / L stock solution is prepared using equal volumes of acetonitrile aqueous solution. The stock solution is then diluted to multiple standard curve working solution concentrations: 0.2 nmol / L, 0.5 nmol / L, 1 nmol / L, 2 nmol / L, 5 nmol / L, 10 nmol / L, 20 nmol / L, 50 nmol / L, 100 nmol / L, 200 nmol / L, 500 nmol / L, and 1000 nmol / L. Each solution is prepared fresh before use.

[0065] Example 1

[0066] (1) Pretreatment of plasmin samples: Take 1 mL each of cow's milk (commercially available UHT sterilized milk), raw milk, skim milk and milk protein extract into a 10 mL centrifuge tube, add 1 mL of PBS-sodium citrate buffer solution and incubate at 37℃ for 20 min, and take it out for use.

[0067] (2) Sample preparation: Specifically, taking UHT sterilized milk as an example, 200 μL of the prepared sample was mixed with 100 μL of the second solution. After incubating at 37°C for 5 min in a 10 mL centrifuge tube, 50 μL was quickly removed. 500 μL of acetonitrile and 500 μL of distilled water were added successively to terminate the reaction. The mixture was centrifuged at 12000 rpm for 10 min, and 200 μL of the supernatant was taken as UHT sterilized milk point 1. After incubating at 37°C for 15 min in a 10 mL centrifuge tube, 50 μL was removed. 500 μL of acetonitrile and 500 μL of distilled water were added successively to terminate the reaction. The mixture was centrifuged at 12000 rpm for 10 min, and 200 μL of the supernatant was taken as UHT sterilized milk point 2. The above steps were repeated after 30 min to obtain UHT sterilized milk point 3. The above steps were repeated after 45 min to obtain UHT sterilized milk point 4. Figure 3 The images show the centrifuged samples, demonstrating their clarity. The raw milk, skim milk, and milk protein extract were processed using the same method.

[0068] (3) On-machine testing: Take 200 μL of 50% acetonitrile aqueous solution as B0, and take 200 μL of the prepared standard curve working solution of 0.2 nmol / L (B12), 0.5 nmol / L (B1), 1 nmol / L (B2), 2 nmol / L (B3), 5 nmol / L (B4), 10 nmol / L (B5), 20 nmol / L (B6), 50 nmol / L (B7), 100 nmol / L (B8), 200 nmol / L (B9), 500 nmol / L (B10), 1000 nmol / L (B11). Add the reaction sample prepared in step (2) to a 96-well microplate. The order of spotting the 96-well microplate is shown in Table 1. The microplates are tested simultaneously. The instrument parameters of the fluorescent microplate reader are as described above.

[0069] Table 1

[0070]

[0071] (4) The test results of the fluorescence microplate reader are shown in Table 2.

[0072] Table 2

[0073]

[0074]

[0075] (5) Calculation of results:

[0076] AMC Standard Curve: Table 3 lists the concentrations and fluorescence values ​​of the AMC standard curve. The AMC standard curve was plotted based on the AMC concentration and fluorescence value (RFU), with the x-axis representing RFU value and the y-axis representing concentration. (See also...) Figure 1 .

[0077] Table 3

[0078]

[0079] The standard curve for plasmin, obtained by fitting the data in Table 3, is y = 0.0041x - 1.4988 (R²). 2 =0.9998), x is the AMC fluorescence value, y is the concentration, and the obtained curve has a good linear relationship.

[0080] Calculation of test results:

[0081] Based on the test results in Table 2, the ΔRFU values ​​were calculated as follows: In the example, based on the data in Table 2, ΔRFU for sterilized milk = RFU4 – RFU1 = 31199 - 29859 = 1340; ΔRFU for skim milk = RFU4 – RFU1 = 34245 - 32907 = 1338; ΔRFU for milk protein extract = RFU4 – RFU1 = 195316 - 48794 = 146522; ΔRFU for raw milk = RFU4 – RFU1 = 71780 - 34365 = 37415.

[0082] Referring to the AMC standard curve and curve equation (y = 0.0041x - 1.4988), the concentration of AMC produced after the reaction of the enzyme and substrate (B nmole) was calculated from the ΔRFU of the sterilized milk sample as 0.0041 × 1340 - 1.4988 = 3.9952 nmole.

[0083] The formula for calculating plasmin activity is:

[0084] Fibrinolytic activity nmole / min / mL = B × dilution factor / reaction time (One unit of fibrinolytic activity refers to the amount of enzyme that produces 1.0 nmol of coumarin from the substrate per minute at 37°C.)

[0085] B = the amount of AMC generated between the initial T and the final T (nmole), such as B = 3.9952 for the UHT sterilized milk sample in the above example.

[0086] Reaction time = Tfinal – Tinitial (minutes) = 45 min;

[0087] Dilution factor = dilution factor of pre-treated milk = 2 × 1.5 × 21 = 63;

[0088] The calculated fibrinolytic activity of UHT sterilized milk was milliunit / min / mL = 3.9952 × 63 / 45 = 5.59.

[0089] The fibrinolytic activity of several other dairy products was calculated using the same method and recorded in Table 4.

[0090] Table 4

[0091] Serial Number name ΔRFU concentration active milliunit / mL 1 UHT sterilized milk 1340 4.00 5.59 2 Skim milk 1338 3.99 5.58 3 milk protein extract 146522 599.24 839 4 Raw milk 37415 151.90 213

[0092] (6) Sample repeatability test

[0093] The UHT sterilized milk, skim milk, and milk protein extract were tested six times using the same test method described above, and the results are recorded in Table 5.

[0094] Table 5

[0095]

[0096]

[0097] As can be seen from Table 5, the RSDs of UHT sterilized milk, skim milk, and milk protein extract, which were tested 6 times using the method provided by this invention, were 9.6%, 9.67%, and 3.00%, respectively, all <10%, indicating that the repeatability of this method is good.

[0098] (7) Accuracy verification

[0099] Raw milk was used as the dairy product to be tested. Before the test, the raw milk was subjected to high temperature treatment. Specifically, it was treated at 80°C for different times. After treatment, the same test and calculation methods as in (1) to (5) above were used. The treatment time and test results are recorded in Table 6.

[0100] Table 6

[0101] Serial Number Processing time ΔRFU concentration active milliunit / mL 1 0 37415 151.90 213 2 15S 30530 123.67 173 3 1min 28512 115.40 162 4 3min 25599 103.46 145 5 6min 17300 69.43 97 6 20min 3361 12.28 17 7 36min 2750 9.78 14 8 60min 332 -0.14 0

[0102] As can be seen from the test results in Table 6, the longer the high-temperature treatment time, the lower the plasmin activity. In fact, plasmin is sensitive to temperature, indicating that the test results of this invention are consistent with the theory, and that the test method of this invention is accurate.

[0103] Comparative Example 1

[0104] This comparative example provides a method for detecting plasmin, which is basically the same as the detection method in Example 1. The only difference is that 1 mL of sample is taken into a 10 mL centrifuge tube, 1 mL of PBS-sodium citrate buffer solution is added and incubated at 37°C for 20 min. After incubation, the sample is centrifuged (12000 rpm, 10 min). Subsequent steps are the same as in Example 1.

[0105] See attached graph for a comparison of the activity of UHT sterilized milk, skim milk, and milk protein extract before and after centrifugation. Figure 2 As shown in the figure, if centrifugation is performed after pretreatment dissociation, the detected plasmin activity decreases. This may be because plasmin is affected by centrifugal force and settles, thus reducing the detection result.

[0106] Comparative Example 2

[0107] This comparative example provides a method for detecting plasmin, which is basically the same as the detection method in Example 1. The only difference is that the dissociation solution in the pretreatment step is different. Specifically, in this comparative example, 1 mL of skim milk sample is placed in a 10 mL centrifuge tube, and 1 mL of 100 mmol / L Tris-HCl buffer solution (pH 8.0, containing 50 mmol / L 6-aminocaproic acid and 0.4 mol / L sodium chloride) is added. This dissociation solution is used to perform two repeated tests.

[0108] The test results are shown in Table 7.

[0109] Table 7

[0110]

[0111] As shown in Table 7, the dissociation experiment using 6-aminohexanoic acid reagent was unstable and the results were low.

[0112] Comparative Example 3

[0113] This comparative example provides a method for detecting plasmin, which is basically the same as the method in Example 1, except that: 750 μL of sample is placed in a 10 mL centrifuge tube, 250 μL of PBS-sodium citrate buffer solution is added, and the mixture is incubated at 37°C for 20 min, followed by subsequent procedures. It was found that after adding acetonitrile and water and centrifuging, the liquid became turbid, affecting the detection results.

[0114] Comparative Example 4

[0115] This comparative example provides a method for detecting plasmin, which is basically the same as the method in Example 1, except that the instrument parameters of the fluorescent microplate reader are adjusted so that the excitation and emission wavelengths are 340 nm and 440 nm, respectively. It is then compared with a milk protein extract sample with excitation and emission wavelengths set to 360 nm and 450 nm, respectively.

[0116] The test results are shown in Table 8.

[0117] Table 8

[0118] Serial Number sample RF(360, 450) RF(340,440) 1 milk protein extract 142141 103894 2 milk protein extract 150373 124318 3 milk protein extract 141894 91480 4 milk protein extract 144144 94767 5 milk protein extract 148095 109145 6 milk protein extract 138721 87500

[0119] As can be seen from Table 8, the response value using an excitation wavelength of 340 nm and an emission wavelength of 440 nm is lower than that using an excitation wavelength of 360 nm and an emission wavelength of 450 nm. Therefore, in this invention, an excitation wavelength of 360 nm and an emission wavelength of 450 nm are selected as the test parameters for the fluorescence microplate reader.

[0120] Comparative Example 5

[0121] This comparative example provides a method for detecting plasmin, which is basically the same as the method in Example 1, except that the detection is performed using the kinetic mode of a fluorescence microplate reader. For pretreatment, 1 mL of sample (e.g., milk protein extract) is placed in a 10 mL centrifuge tube, and 1 mL of PBS-sodium citrate buffer is added. The tube is incubated at 37°C for 20 min before loading and measurement (a blank is required for all sample detections). Specifically, the sample is loaded into two wells of a 96-well plate (one well serves as a blank control, and the other well is used for testing). For the blank control, 70 μL of the sample preparation solution is loaded, and 30 μL of coumarin peptide buffer (without substrate) is added to bring the volume to 100 μL. For the test sample, 70 μL of sample is loaded, and 30 μL of coumarin peptide buffer (containing substrate) is added to bring the volume to 100 μL. The kinetic mode was selected for measurement at 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min, and calculations were performed according to the measurement mode. Compared with Example 1, the sampled samples automatically sampled using the kinetic mode of the fluorescence microplate reader were not centrifuged.

[0122] The final calculation results are shown in Table 9:

[0123] Table 9

[0124] Serial Number name ΔRFU concentration active milliunit / mL 1 milk protein extract 323 318.10 151

[0125] As can be seen from Table 9, the detection results are lower than those of Example 1. The sample turbidity of this detection method affects the fluorescence value release and is not suitable for sample detection.

[0126] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning understood by one of ordinary skill in the art to which this invention pertains. The terms "first," "second," and similar terms used in this invention do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Terms such as "connected" or "linked" are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. Terms such as "upper," "lower," "left," and "right" are used only to indicate relative positional relationships; when the absolute position of the described object changes, the relative positional relationship also changes accordingly.

[0127] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting plasmin in dairy products, characterized in that, The method includes the following steps: Add the dairy product to be tested to sodium citrate buffer and incubate to obtain the plasmin dissociation solution for later use; The substrate solution was added to the fibrinolytic enzyme dissociation solution and the reaction was carried out. Samples were taken multiple times during the reaction. The sampling method was as follows: the mixture after the reaction was added to acetonitrile solution to terminate the enzymatic hydrolysis reaction, and the supernatant was taken as the sample after centrifugation. The substrate in the substrate solution was N-succinyl-L-alanyl-L-phenylalanyl-L-lysyl-7-amino-4-methylcoumarin peptide. Standard working solutions of different concentrations were prepared using 7-amino-4-methylcoumarin as a standard substance; The sample and standard working solutions of different concentrations were tested using an ELISA reader. A standard curve was plotted based on the test results. The relative fluorescence increment of the sample was calculated and substituted into the standard curve to calculate the activity of plasmin in the sample to be tested.

2. The detection method according to claim 1, characterized in that, Add the dairy product to be tested to sodium citrate buffer and incubate at 37±1℃ for 15 min to 25 min.

3. The detection method according to claim 1, characterized in that, Four samples were taken during the reaction. The first sample was taken 3 to 6 minutes after the reaction, and then every 15 minutes thereafter.

4. The detection method according to claim 3, characterized in that, The relative fluorescence increment of the sampled sample is calculated as follows: calculate the relative fluorescence increment between the 4th sampled sample and the 1st sampled sample.

5. The detection method according to claim 1, characterized in that, The substrate solution includes a substrate and a substrate buffer, wherein the substrate buffer includes Tris-HCl buffer solution, dimethyl sulfoxide, and sodium chloride.

6. The detection method according to claim 1, characterized in that, The concentration of N-succinyl-L-alanyl-L-phenylalanyl-L-lysyl-7-amino-4-methylcoumarin peptide in the substrate solution is 1–2 mmol / L.

7. The detection method according to claim 1, characterized in that, The standard working solution comprises an aqueous solution of 7-amino-4-methylcoumarin and acetonitrile; the concentrations of 7-amino-4-methylcoumarin in the standard working solution are 0.2 nmol / L, 0.5 nmol / L, 1 nmol / L, 2 nmol / L, 5 nmol / L, 10 nmol / L, 20 nmol / L, 50 nmol / L, 100 nmol / L, 200 nmol / L, 500 nmol / L, and 1000 nmol / L; and / or The equation of the standard curve is y = 0.0041x - 1.4988(R). 2 =0.9998), where x is the fluorescence value of 7-amino-4-methylcoumarin and y is the concentration of 7-amino-4-methylcoumarin.

8. The detection method according to claim 1, characterized in that, The enzyme-linked immunosorbent assay (ELISA) reader is a fluorescent ELISA reader.

9. The detection method according to claim 8, characterized in that, The instrument parameters of the fluorescent enzyme-linked immunosorbent assay (ELISA) reader are as follows: Temperature set at 25℃, excitation wavelength at 360nm, emission wavelength at 450nm, top measurement, extended gain, detection height at 7mm, bandwidth at 8nm.

10. The detection method according to claim 1, characterized in that, The dairy products to be tested include at least one of sterilized milk, raw milk, skim milk, and milk protein extract.

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