Method for detecting activity of lipase in milk
By using 7-hydroxycoumarin-3-carboxylate succinimide ester as a substrate and combined with fluorescence detection methods, the problem of low accuracy of lipase activity detection in milk is solved, and efficient and sensitive lipase activity detection is achieved, which is suitable for production line monitoring and product quality control of dairy enterprises.
Patent Information
- Application Number
- CN202410994624.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-23
- Publication Date
- 2025-07-25
AI Technical Summary
In the prior art, the detection accuracy of lipase activity in milk is low and the sensitivity is not high, which affects the taste and shelf life of the milk.
The 7-hydroxycoumarin-3-carboxylic acid succinimide ester was used as the substrate to detect the content of 7-hydroxycoumarin-3-carboxylic acid by fluorescence to determine the activity of lipase in milk. The specific fluorescence signal generated by the decomposition of the substrate was used to combine clarification agents and reaction terminators to improve the accuracy and sensitivity of the detection.
It realizes efficient, sensitive and rapid detection of the activity of lipase in milk, reduces detection costs, and can effectively monitor products on the production line to ensure product quality.
Smart Images
Figure CN120369681A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of food detection, and particularly relates to a method for detecting the lipase activity in milk. Background Art
[0002] During the processes of milking cows, storing raw milk, and processing semi-finished products, milk is extremely vulnerable to microbial contamination. Milk is rich in nutrients, providing conditions for the growth and reproduction of microorganisms, which easily leads to the growth of microorganisms. These microorganisms are mainly psychrophilic bacteria that can produce highly heat-resistant lipases. These heat-resistant lipases can still maintain a certain activity after pasteurization or even ultra-high temperature sterilization. The residual lipases in milk will continuously decompose the fat in milk, destroying the taste of liquid milk, causing off-flavors, and shortening the shelf life of the product. There are many existing methods for measuring lipase activity, such as the national standard method "GB / T 23535-2009 Lipase Preparation", p-nitrophenol method, chromatography method, copper soap method, turbidity method, etc. However, these detection methods have problems of low accuracy and low sensitivity. Therefore, a highly sensitive detection method that can accurately measure the lipase activity in milk is needed. Summary of the Invention
[0003] The purpose of the embodiments of the present invention is to provide a method for detecting the lipase activity in milk to solve the problems of low detection accuracy and low sensitivity of the lipase activity in milk.
[0004] The embodiments of the present invention provide a method for detecting the lipase activity in milk, including:
[0005] Taking the milk to be detected;
[0006] Mixing and reacting the milk with a substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester;
[0007] Taking the reaction mixture and detecting the content of 7-hydroxycoumarin-3-carboxylic acid by fluorescence;
[0008] Determining the lipase activity in milk according to the content of 7-hydroxycoumarin-3-carboxylic acid.
[0009] Optionally, before the step of mixing and reacting the milk with a substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester, it further includes:
[0010] Defatting the milk to be detected.
[0011] Optionally, after defatting the milk to be detected and before the step of mixing and reacting the milk with a substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester, it further includes:
[0012] Add a clarifying agent to the milk to be detected, and centrifuge and clarify the milk to obtain clarified milk.
[0013] Optionally, the reaction temperature for mixing and reacting the milk with a substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester is 40 - 65°C.
[0014] Optionally, the reaction time for mixing and reacting the milk with a substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester is 5 - 15 min.
[0015] Optionally, the volume ratio of the substrate solution to the milk is 1:(1 - 10), and the concentration of 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester in the substrate solution is 0.5 - 1.5 mol / L.
[0016] Optionally, before the step of taking the reaction mixture and detecting the content of 7-hydroxycoumarin-3-carboxylic acid by fluorescence, it further includes:
[0017] Add a reaction terminator to the said mixture;
[0018] The step of taking the reaction mixture and detecting the content of 7-hydroxycoumarin-3-carboxylic acid by fluorescence includes:
[0019] Take the mixture after adding the reaction terminator and detect the content of 7-hydroxycoumarin-3-carboxylic acid by fluorescence.
[0020] Optionally, the reaction terminator is an acidic solution.
[0021] Optionally, the acidic solution includes at least one of hydrochloric acid solution, nitric acid solution and sulfuric acid solution.
[0022] Optionally, the excitation wavelength for fluorescence detection is 330 - 352 nm, and the emission wavelength is 435 - 457 nm.
[0023] The method for detecting the lipase activity in milk according to the embodiments of the present invention includes: taking the milk to be detected; mixing and reacting the milk with a substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester; taking the reacted mixture and detecting the content of 7-hydroxycoumarin-3-carboxylic acid by fluorescence; and determining the lipase activity in the milk according to the content of 7-hydroxycoumarin-3-carboxylic acid. During the detection process, lipase decomposes the substrate 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester to produce a substance 7-hydroxycoumarin-3-carboxylic acid with a specific fluorescence signal. By detecting the content of 7-hydroxycoumarin-3-carboxylic acid by fluorescence and determining the lipase activity in the milk according to the content of 7-hydroxycoumarin-3-carboxylic acid, it is possible to accurately detect the lipase activity at a low level in milk, and the detection method is efficient, sensitive and rapid. 7-Hydroxycoumarin-3-carboxylic acid belongs to the coumarin fluorescent group and is an ultraviolet-excitable blue fluorescent dye. By introducing an electron-donating group hydroxyl at the 7-position and an electron-withdrawing group carboxylic acid at the 3-position of the coumarin parent nucleus, a push-pull electron system is formed, which can enhance fluorescence. After the substrate is decomposed by lipase, the product 7-hydroxycoumarin-3-carboxylic acid itself has a hydroxyl group at the 7-position and a carboxylic acid group at the 3-position, which can enhance the fluorescence signal and thus improve the detection sensitivity. At the same time, lipase specifically recognizes the ester group in the substrate 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester and has high selectivity for the ester group in the substrate. In addition, the operation is simple, the reagents selected are cheap, environmentally friendly, and the detection cost is low. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 Schematic diagram of the decomposition of 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester by lipase;
[0025] Figure 2 Schematic diagram of the detection process;
[0026] Figure 3 Graph showing the effect of reaction temperature on lipase A;
[0027] Figure 4 Graph showing the effect of reaction time on lipase A;
[0028] Figure 5 Standard curve of 7-hydroxycoumarin-3-carboxylic acid;
[0029] Figure 6 Effect of the clarifying agent; DETAILED DESCRIPTION OF THE EMBODIMENTS
[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0031] The method for detecting the lipase activity in milk according to the embodiment of the present invention includes:
[0032] Taking the milk to be detected;
[0033] Mixing and reacting the milk with a substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester;
[0034] Taking the reaction mixture and detecting the content of 7-hydroxycoumarin-3-carboxylic acid by fluorescence;
[0035] Determining the lipase activity in the milk according to the content of 7-hydroxycoumarin-3-carboxylic acid.
[0036] Before mixing and reacting the milk with the substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester, the milk to be detected can be pretreated. For example, the milk to be detected can be defatted. For example, the milk can be centrifuged to remove milk fat, which can avoid competition between milk fat and the substrate 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester during the action of lipase. A clarifying agent can be added to the milk to clarify the milk. The milk after defatting can be clarified. The composition of the clarifying agent can be a phosphate buffer solution containing 1 mol / L sodium citrate. Sodium citrate chelates calcium ions in casein micelles, and centrifuging to remove casein micelles can achieve the clarifying effect.
[0037] During the detection process, the milk to be detected can be taken, and the milk can be centrifuged to remove milk fat. A clarifying agent can be added to the defatted milk to clarify the milk. Then, the clarified milk can be reacted with a substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester. The reaction can be carried out in a water bath. After reacting for a certain period of time, the reaction can be terminated by adding concentrated hydrochloric acid to denature and inactivate the enzyme. The reaction mixture after the reaction can be taken to detect the content of 7-hydroxycoumarin-3-carboxylic acid by fluorescence. The lipase activity in the milk can be determined according to the content of 7-hydroxycoumarin-3-carboxylic acid. In an acidic environment, 7-hydroxycoumarin-3-carboxylic acid, as one of the products, can emit fluorescence after an excitation wavelength of 342 nm and an emission wavelength of 447 nm, and the lipase activity can be quantified based on this. Among them, the enzyme activity is defined as that within 1 minute, 1 mL of lipase hydrolyzes 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester to produce 1 μmol of 7-hydroxycoumarin-3-carboxylic acid, which is 1 enzyme activity unit, expressed as U / mL. Lipase decomposes the substrate 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester to produce substances with specific fluorescence signals, 7-hydroxycoumarin-3-carboxylic acid and succinimide, specifically as Figure 1 shown.
[0038] For the method for detecting lipase activity in milk in the embodiments of the present invention, during the detection process, lipase is used to decompose the substrate 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester to produce a substance with a specific fluorescence signal, 7-hydroxycoumarin-3-carboxylic acid. The content of 7-hydroxycoumarin-3-carboxylic acid is detected by fluorescence, and the lipase activity in the milk is determined according to the content of 7-hydroxycoumarin-3-carboxylic acid, so as to accurately detect the lipase activity at a low level in the milk. The detection method is efficient, sensitive, and fast. 7-hydroxycoumarin-3-carboxylic acid belongs to the coumarin fluorescent group and is an ultraviolet-excitable blue fluorescent dye. An electron-donating group hydroxyl is introduced at the 7th position of the coumarin mother nucleus and an electron-withdrawing group carboxyl is introduced at the 3rd position to form a push-pull electron system, which can enhance fluorescence. After the substrate is decomposed by lipase, the product 7-hydroxycoumarin-3-carboxylic acid itself has a hydroxyl group at the 7th position and a carboxyl group at the 3rd position, which can enhance the fluorescence signal and thus improve the detection sensitivity. At the same time, lipase specifically recognizes the ester group in the substrate 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester and has high selectivity for the ester group in the substrate. In addition, the operation is simple, the reagents selected are cheap, environmentally friendly, and the detection cost is low.
[0039] The present invention is conducive to solving the pain points of dairy enterprises in the production line and supply chain. It can monitor products at any processing stage, which can not only help enterprises with upstream control, quickly classify raw milk, safely process raw milk with different lipase enzyme activities into high-quality products using different sterilization schemes, determine the level of residual enzyme in the finished product, and predict the shelf life of the product. Especially for ultra-high temperature sterilized milk, it can determine whether it can be sold as a long-term product, which has great practical application value.
[0040] In some embodiments, before the step of mixing and reacting the milk with the substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimide ester, it may further include:
[0041] Defat the milk to be detected. Defatting can avoid competition between milk fat and the substrate 7-hydroxycoumarin-3-carboxylic acid succinimide ester during the action of lipase and reduce the influence of milk fat.
[0042] In some embodiments of the present invention, after defatting the milk to be detected and before the step of mixing and reacting the milk with the substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimide ester, it may further include:
[0043] Add a clarifying agent to the milk to be detected and centrifuge the milk for clarification to obtain clarified milk. For example, the clarifying agent can be a phosphate buffer solution containing sodium citrate, the clarifying agent can be composed of bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane, sodium hydroxide, and disodium ethylenediaminetetraacetate, and the clarifying agent can also be composed of gelatin and chitosan. A clarifying agent can be added to the defatted milk for clarification. The clarifying agent can be a phosphate buffer solution containing 1 mol / L sodium citrate. Sodium citrate chelates calcium ions in casein micelles, and centrifugation removes casein micelles, thereby achieving the clarification effect. Using a clarifying agent can reduce the interference of the white background of milk on the fluorescence emission, accurately quantify the fluorescence intensity emitted from 7-hydroxycoumarin-3-carboxylic acid, and improve the accuracy of experimental detection results. As Figure 6 shown, from left to right, the volume ratios of defatted milk to the clarifying agent are 1:0, 1:1, 1:2, 1:3, 1:4, 1:5 in sequence. When the volume ratio of defatted milk to the clarifying agent is 1:2, a good clarification effect has been achieved.
[0044] In some embodiments, the reaction temperature for mixing and reacting the milk with the substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimide ester can be 40 - 65 °C. For example, the reaction temperature can be 40, 45, 50, 60, or 65 °C, etc. The specific reaction temperature can be selected according to the actual situation.
[0045] In an embodiment of the present invention, the reaction time for mixing the milk with the substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimide ester can be 5 - 15 min. For example, the reaction time can be 5, 10, or 15 min. The specific reaction temperature can be selected according to the actual situation.
[0046] Optionally, the volume ratio of the substrate solution to the milk is 1:(1 - 10), and the concentration of 7-hydroxycoumarin-3-carboxylic acid succinimide ester in the substrate solution can be 0.5 - 1.5 mol / L. For example, the concentration of 7-hydroxycoumarin-3-carboxylic acid succinimide ester in the substrate solution can be 1 mol / L.
[0047] According to some embodiments, before the step of taking the reaction mixture and detecting the content of 7-hydroxycoumarin-3-carboxylic acid by fluorescence, the following steps may further be included:
[0048] Adding a reaction terminator to the reaction mixture;
[0049] The step of taking the reaction mixture and detecting the content of 7-hydroxycoumarin-3-carboxylic acid by fluorescence may include:
[0050] Taking the reaction mixture after adding the reaction terminator and detecting the content of 7-hydroxycoumarin-3-carboxylic acid by fluorescence.
[0051] By adding a reaction terminator to the reaction mixture, the enzyme can be inactivated and the reaction can be terminated to facilitate accurate detection.
[0052] Optionally, the reaction terminator can be an acidic solution. 7-Hydroxycoumarin-3-carboxylic acid can stably emit fluorescence in an acidic environment (pH < 4), which meets the premise that the system becomes strongly acidic due to using concentrated hydrochloric acid to terminate the reaction in this detection process. After using concentrated hydrochloric acid to terminate the reaction, detection can be carried out, and it can be completed in one step without additional operations.
[0053] Optionally, the acidic solution can include at least one of hydrochloric acid solution, nitric acid solution, and sulfuric acid solution. For example, the reaction terminator can be an acidic solution, and the hydrochloric acid solution can be 1 mol / L hydrochloric acid.
[0054] Optionally, the excitation wavelength for fluorescence detection is 330 - 352 nm, and the emission wavelength is 435 - 457 nm. For example, the excitation wavelength for fluorescence detection is 342 nm, and the emission wavelength is 447 nm.
[0055] As Figure 2 shown, the steps of the detection method in the present invention are further described.
[0056] The specific detection process includes:
[0057] Step 1: Take 40 - 50 mL of milk sample and centrifuge it at a speed of 8000 - 10000 rpm for 20 - 30 min. Take the skim milk (the supernatant) for use.
[0058] Step 2: Take 30 mL of the skim milk (the supernatant) from Step 1 into a test tube, add 60 - 150 mL of clarifying agent (phosphate buffer solution containing 1 mol / L sodium citrate), centrifuge it at a speed of 8000 - 10000 rpm for 10 - 20 min, and take the supernatant for use.
[0059] Step 3: Take 200 μL of the supernatant from Step 2 into a small test tube, add 20 - 200 μL of substrate solution (1 mol / L 7 - hydroxycoumarin - 3 - carboxylic acid succinimidyl ester solution), vortex for 5 - 30 s, place it in a water bath at 40 - 65 °C and react for 5 - 15 min. Then add 120 - 200 μL of terminator. The terminator can be 1 mol / L hydrochloric acid solution. Then take 100 μL and put it into one well of a 96 - well plate. Design three parallel samples in a group and detect them on the machine. Conduct fluorescence intensity detection on an enzyme - linked immunosorbent assay (ELISA) reader. The excitation wavelength is 342 nm and the emission wavelength is 447 nm.
[0060] Step 4: Use the supernatant from Step 2 to establish an enzyme activity determination curve of 7 - hydroxycoumarin - 3 - carboxylic acid in the concentration range of 0 - 100 μmol / L. Complete the fluorescence intensity determination work on an ELISA reader at an excitation wavelength of 342 nm and an emission wavelength of 447 nm. Draw a standard curve with 7 - hydroxycoumarin - 3 - carboxylic acid (μmol / L) as the abscissa and relative fluorescence unit (RFU) as the ordinate, and complete the corresponding linear regression.
[0061] Step 5: Calculate the lipase activity according to the enzyme activity definition. The enzyme activity definition of the present invention is that within 1 min, 1 mL of lipase hydrolyzes 7 - hydroxycoumarin - 3 - carboxylic acid succinimidyl ester to produce 1 μmol of 7 - hydroxycoumarin - 3 - carboxylic acid, which is 1 enzyme activity unit, expressed as U / mL.
[0062] It is possible to set up experimental groups and control groups for detection. The difference between the control group and the experimental group is the addition order of the terminator and the substrate solution. The control group adds the terminator first and then the substrate solution. The concentrations and volumes of the terminator and the substrate solution used are the same as those in the experimental group.
[0063] During the detection process:
[0064] I. Selection of the optimal temperature of lipase A in the milk system
[0065] Step 1. Milk centrifugation: Take 50 mL of milk sample and centrifuge it at a speed of 8000 rpm for 30 min. Take the skim milk (the supernatant) for use.
[0066] Step 2. Clarify the skim milk: Take 30 mL of the skim milk (supernatant) from Step 1 in a test tube, add 60 mL of a clarifying agent, which is a phosphate buffer solution containing 1 mol / L sodium citrate, centrifuge at a speed of 8000 rpm for 10 min, and take the supernatant for use;
[0067] Step 3. Prepare 0.001 g / mL enzyme-containing supernatant: Weigh 0.0418 g of lipase A, take the supernatant from Step 2 and make up the volume to 40 mL to obtain 0.001 g / mL enzyme-containing supernatant;
[0068] Step 4. React and detect in the experimental group: Take 6 1.5-mL centrifuge tubes, add 200 μL of the 0.001 g / mL enzyme-containing supernatant from Step 3 to each tube, then add 20 μL of the substrate solution, where the substrate solution is 1 mol / L 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester solution, vortex for 5 s, place them in a water bath at 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, and 65 °C for 5 min respectively, and take them out after reacting for 5 min;
[0069] Add 120 μL of the terminator respectively, where the terminator is 1 mol / L hydrochloric acid, vortex for 5 s, take 100 μL and put it into one well of a 96-well plate. Design three parallel samples in one group and perform fluorescence intensity detection on an enzyme-labeled instrument with an excitation wavelength of 342 nm and an emission wavelength of 447 nm;
[0070] Step 5. React and detect in the control group: Take 6 1.5-mL centrifuge tubes, add 200 μL of the 0.001 g / mL enzyme-containing supernatant from Step 3 to each tube, then add 120 μL of the terminator respectively, where the terminator is 1 mol / L hydrochloric acid, vortex for 5 s, place them in a water bath at 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, and 65 °C for 5 min respectively, and take them out after reacting for 5 min;
[0071] Add 20 μL of the substrate solution respectively, where the substrate solution is 1 mol / L 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester solution, vortex for 5 s, take 100 μL and put it into one well of a 96-well plate. Design three parallel samples in one group and perform fluorescence intensity detection on an enzyme-labeled instrument with an excitation wavelength of 342 nm and an emission wavelength of 447 nm;
[0072] Step 6. Establish the standard curve of 7-hydroxycoumarin-3-carboxylic acid: Use the supernatant obtained in Step 2 to establish the enzyme activity determination curve of 7-hydroxycoumarin-3-carboxylic acid in the concentration range of 0-100 μmol / L; complete the fluorescence intensity measurement by a microplate reader at the excitation wavelength of 342 nm and the emission wavelength of 447 nm; plot the standard curve with 7-hydroxycoumarin-3-carboxylic acid (μmol / L) as the abscissa and relative fluorescence unit (RFU) as the ordinate. After completing the corresponding linear regression, the standard curve equation is y = 171.05x + 8917.7, R 2 = 0.995;
[0073] Step 7. Calculation results of enzyme activity: Calculate according to the enzyme activity definition of the present invention. The enzyme activity definition is that within 1 minute, 1 mL of lipase hydrolyzes 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester to produce 1 μmol of 7-hydroxycoumarin-3-carboxylic acid, which is 1 enzyme activity unit, expressed as U / mL. The measured enzyme activity is calculated with the maximum enzyme activity value as 100% to calculate the relative enzyme activity. Lipase A reacts at different temperatures, and its enzyme activity results are as Figure 3 shown. The maximum enzyme activity is obtained at 50 °C, so the optimal temperature of lipase A in the milk system is 50 °C.
[0074] II. Selection of the optimal reaction time of lipase A in the milk system
[0075] Modify the reaction time in the above reaction temperature optimization experiment detection process. The reaction temperature is set at 50 °C, and the reaction times are set at 5, 10, and 15 min. At the same time, take samples of the experimental group and the control group for detection and analysis. The measured enzyme activity is calculated with the maximum enzyme activity value as 100% to calculate the relative enzyme activity. Lipase A reacts at different reaction times, and its enzyme activity results are as Figure 4 shown. The maximum enzyme activity is obtained at 5 min, so the optimal reaction time of lipase A in the milk system is 5 min.
[0076] III. Research on the detection limit
[0077] Step 1. Centrifuge the milk: Take 50 mL of milk sample and centrifuge it at a speed of 8000 rpm for 30 min, and take the skim milk (supernatant) for use;
[0078] Step 2. Clarify the skim milk: Take 30 mL of the skim milk (supernatant) in Step 1 into a test tube, add a clarifying agent. The clarifying agent is 60 mL of a phosphate buffer solution containing 1 mol / L sodium citrate, and centrifuge it at a speed of 8000 rpm for 10 min, and take the supernatant for use;
[0079] Step 3: Prepare a series of enzyme-containing supernatant liquids: Weigh 0.0201 g of lipase A, and add the supernatant liquid from Step 2 to 2.0001 g to obtain the original enzyme-containing supernatant liquid with a mass fraction of 1.0%; Pipette 200 μL from the original enzyme-containing supernatant liquid and make up the volume to 2 mL with the supernatant liquid from Step 2 to obtain the enzyme-containing supernatant liquid 1 with a mass fraction of 1×10 -1 %; Pipette 200 μL from the enzyme-containing supernatant liquid 1 and make up the volume to 2 mL with the supernatant liquid from Step 2 to obtain the enzyme-containing supernatant liquid 2 with a mass fraction of 1×10 -2 %; Pipette 200 μL from the enzyme-containing supernatant liquid 2 and make up the volume to 2 mL with the supernatant liquid from Step 2 to obtain the enzyme-containing supernatant liquid 3 with a mass fraction of 1×10 -3 %; Pipette 200 μL from the enzyme-containing supernatant liquid 3 and make up the volume to 2 mL with the supernatant liquid from Step 2 to obtain the enzyme-containing supernatant liquid 4 with a mass fraction of 1×10 -4 %; Pipette 200 μL from the enzyme-containing supernatant liquid 4 and make up the volume to 2 mL with the supernatant liquid from Step 2 to obtain the enzyme-containing supernatant liquid 5 with a mass fraction of 1×10 -5 %; Pipette 200 μL from the enzyme-containing supernatant liquid 5 and make up the volume to 2 mL with the supernatant liquid from Step 2 to obtain the enzyme-containing supernatant liquid 6 with a mass fraction of 1×10 -6 %;
[0080] Step 4: React and detect for the experimental group: Take 6 1.5-mL centrifuge tubes, and add 200 μL of 1×10 -1 % enzyme-containing supernatant liquid 1, 1×10 -2 % enzyme-containing supernatant liquid 2, 1×10 -3 % enzyme-containing supernatant liquid 3, 1×10 -4 % enzyme-containing supernatant liquid 4, 1×10 -5 % enzyme-containing supernatant liquid 5 and 1×10 -6 % enzyme-containing supernatant liquid 6 respectively. Then, add 20 μL of the substrate solution. The substrate solution is 1 mol / L 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester solution. Vortex for 5 s, place it in a 50°C water bath for 5 min. After reacting for 5 min, take it out, add 120 μL of the terminator respectively. The terminator is 1 mol / L hydrochloric acid. Vortex for 5 s, take 100 μL and place it in one well of a 96-well plate. Design three parallel samples in a group and perform fluorescence intensity detection on an enzyme-labeled instrument. The excitation wavelength is 342 nm and the emission wavelength is 447 nm;
[0081] Step 5: React and detect for the control group: Take 6 1.5-mL centrifuge tubes, and add 200 μL of 1×10 -1 % enzyme-containing supernatant liquid 1, 1×10 -2 % enzyme-containing supernatant liquid 2, 1×10 -3 % enzyme-containing supernatant liquid 3, 1×10 -4 % enzyme-containing supernatant liquid 4, 1×10-5 % enzyme-containing supernatant 5 and 1×10 -6 % enzyme-containing supernatant 6. Then, 120 μL of a terminator was added to each. The terminator was 1 mol / L hydrochloric acid. The mixture was vortexed for 5 s and placed in a water bath at 50 °C for 5 min. After the 5-min reaction, the mixture was taken out and 20 μL of a substrate solution was added to each. The substrate solution was 1 mol / L 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester solution. The mixture was vortexed for 5 s, and 100 μL was taken and placed in a well of a 96-well plate. Three parallel samples were designed for each group. Fluorescence intensity was detected on a microplate reader with an excitation wavelength of 342 nm and an emission wavelength of 447 nm;
[0082] Step 6. Establish a standard curve for 7-hydroxycoumarin-3-carboxylic acid: Use the supernatant obtained in Step 2 to establish a curve for the determination of the enzyme activity of 7-hydroxycoumarin-3-carboxylic acid in the concentration range of 0 to 100 μmol / L. Fluorescence intensity determination was completed on a microplate reader at an excitation wavelength of 342 nm and an emission wavelength of 447 nm. A standard curve was plotted with 7-hydroxycoumarin-3-carboxylic acid (μmol / L) as the abscissa and relative fluorescence unit (RFU) as the ordinate. After performing the corresponding linear regression, the standard curve equation was y = 171.05x + 8917.7, R 2 = 0.995, as specifically shown in Figure 5 the following;
[0083] Step 7. Calculation results of enzyme activity: Calculate according to the enzyme activity definition of the present invention. The enzyme activity definition is that within 1 min, 1 mL of lipase hydrolyzes 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester to produce 1 μmol of 7-hydroxycoumarin-3-carboxylic acid, which is defined as 1 enzyme activity unit and is expressed as U / mL.
[0084] A series of enzyme activities of enzyme-containing skim milk supernatants with mass fractions of 1×10 -6 %, 1×10 -5 %, 1×10 -4 %, 1×10 -3 %, 1×10 -2 % and 1×10 -1 % are shown in Table 1. The detection limit is at an enzyme addition amount of 1×10 -4 % (0.00028 U / ml), and the results of the detection limit are shown in Table 1.
[0085] Table 1 Results of the detection limit
[0086] Enzyme mass fraction Enzyme activity (U / mL) <![CDATA[1×10 -1 %]]> 0.00661 <![CDATA[1×10 -2 %]]> 0.00554 <![CDATA[1×10 -3 %]]> 0.00159 <![CDATA[1×10 -4 %]]> 0.00028 <![CDATA[1×10 -5 %]]> -0.00021 <![CDATA[1×10 -6 %]]> -0.00155
[0087] Comparative Example 1
[0088] The national standard method "GB / T 23535-2009 Lipase Preparation" selects phenolphthalein as the chromogenic reagent, titrates free fatty acids with a standard sodium hydroxide solution, calculates the lipase activity based on the amount of sodium hydroxide used, and the detection limit is 0.09% of the enzyme addition amount.
[0089] Comparative Example 2
[0090] The p-nitrophenol method uses lipase to decompose p-nitrophenyl ester to release free fatty acids and the yellow substance p-nitrophenol, measures the absorbance of p-nitrophenol at a wavelength of 405 nm, calculates the lipase activity, and the detection limit is 0.0031% of the enzyme addition amount.
[0091] Comparative Example 3
[0092] The chromatography method uses tributyrin as the substrate, qualitatively and quantitatively analyzes the product n-butyric acid by gas chromatography, and the detection limit is 0.055% of the enzyme addition amount.
[0093] Comparative Example 4
[0094] The copper soap method is to add copper ions to form copper soap with free fatty acids, measure the absorbance at a wavelength of 435 nm, calculate the lipase activity, and the detection limit is 1.33% of the enzyme addition amount.
[0095] Comparative Example 5
[0096] The detection limit of the Ab102524 Lipase Activity Assay kit (Colorimetric) commercial kit is 0.0013% of the enzyme addition amount.
[0097] Comparative Example 6
[0098] The turbidimetry method uses the decrease in turbidity at 340 nm to be proportional to the activity of lipase in the sample, and the detection limit is 2% of the enzyme addition amount.
[0099] Comparative Example 7
[0100] The 4-methylumbelliferone method uses lipase to decompose the substrate 4-methylumbelliferyl butyrate, and 4-methylumbelliferone, one of the products, releases fluorescence at an excitation wavelength of 355 nm and an emission wavelength of 460 nm, calculates the lipase activity, and the detection limit is 0.0002% of the enzyme addition amount.
[0101] It can be seen that in the detection method of this application, the detection limit is 1×10 -4 %(0.00028 U / ml), which is lower than the detection limits of the methods in the comparative examples, and the detection is more sensitive.
[0102] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit of the present invention and the scope protected by the claims, and all of them fall within the protection scope of the present invention.
Claims
1. A method for detecting the lipase activity in milk, characterized in that, Including: Taking the milk to be detected; Mixing and reacting the milk with a substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimide ester; Taking the reacted mixture and detecting the content of 7-hydroxycoumarin-3-carboxylic acid by fluorescence; Determining the lipase activity in the milk according to the content of 7-hydroxycoumarin-3-carboxylic acid.
2. The detection method according to claim 1, characterized in that, Before the step of mixing and reacting the milk with a substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimide ester, it further includes: Defatting the milk to be detected.
3. The detection method according to claim 1, wherein After defatting the milk to be detected and before the step of mixing and reacting the milk with a substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimide ester, it further includes: Adding a clarifying agent to the milk to be detected and centrifuging and clarifying the milk to obtain clarified milk.
4. The detection method according to claim 1, wherein The reaction temperature for mixing and reacting the milk with a substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimide ester is 40 - 65 °C.
5. The detection method according to claim 1, characterized in that The reaction time for mixing and reacting the milk with a substrate solution containing 7-hydroxycoumarin-3-carboxylic acid succinimide ester is 5 - 15 min.
6. The detection method according to claim 1, wherein The volume ratio of the substrate solution to the milk is 1:(1 - 10), and the concentration of 7-hydroxycoumarin-3-carboxylic acid succinimide ester in the substrate solution is 0.5 - 1.5 mol / L.
7. The detection method according to claim 1, wherein Before the step of taking the reacted mixture and detecting the content of 7-hydroxycoumarin-3-carboxylic acid by fluorescence, it further includes: Adding a reaction terminator to the mixture; The step of taking the reacted mixture and detecting the content of 7-hydroxycoumarin-3-carboxylic acid by fluorescence includes: Taking the mixture after adding the reaction terminator and detecting the content of 7-hydroxycoumarin-3-carboxylic acid by fluorescence.
8. The detection method according to claim 7, wherein The reaction terminator is an acidic solution.
9. The detection method according to claim 8, wherein The acidic solution includes at least one of hydrochloric acid solution, nitric acid solution and sulfuric acid solution.
10. The detection method according to claim 1, wherein The excitation wavelength for fluorescence detection is 330 - 352 nm, and the emission wavelength is 435 - 457 nm.