Method for rapidly detecting enzyme activity of lipase in raw milk
The amount of change in fat and free fatty acids in raw milk was measured by a milk component analyzer, and the lipase activity was calculated based on the regression equation of the lipase standard working fluid. The problems of large detection errors and high costs in the existing technology were solved, and the rapid and accurate results were achieved. lipase activity detection is suitable for large-scale promotion.
Patent Information
- Application Number
- CN202411327291.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-23
- Publication Date
- 2024-12-27
AI Technical Summary
The method of detecting the activity of lipase in raw milk in the prior art has problems such as complex operational processes, large errors and high costs, and it is difficult to achieve rapid, accurate and economical testing.
The milk component analyzer is used to determine the change amount of fat and free fatty acids in raw milk. By setting the lipase standard working liquid to draw the working curve and obtain the regression equation, the lipase activity is accurately calculated based on the change value of the sample to be tested, and the raw milk is used to calculate the The corresponding relationship between fat and free fatty acids is proofread to reduce errors.
It realizes fast, accurate and low-cost lipase activity detection, which is suitable for large-scale promotion and application, and significantly improves the accuracy of the detection results.
Smart Images
Figure CN119193762A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biological enzyme activity detection, and in particular to a method for rapidly detecting lipase activity in raw milk. Background Art
[0002] UHT sterilized milk generally has a shelf life of up to 6 months. During storage, problems such as fat floating, protein coagulation, acid packing, and bag swelling may occur. Among them, fat floating is a common phenomenon of UHT sterilized milk during its shelf life, which has a serious impact on product quality. The phenomenon of fat floating in UHT sterilized milk is related to factors such as the physical and chemical composition of milk, residual lipase, homogenization conditions, and emulsifiers. In recent years, the impact of lipase content in raw milk and the quality of UHT sterilized milk has attracted more and more attention from dairy processing companies. Studies have shown that lipase in milk is mainly composed of endogenous (lipoprotein lipase) and exogenous (lipase produced by microorganisms). Endogenous lipoprotein lipase is secreted by mammary gland cells, and its level depends on the variety, lactation stage, and nutritional status. Lipoprotein lipase is heat unstable and can be completely inactivated by pasteurization; exogenous lipase is mainly heat-resistant lipase produced by psychrophilic bacteria, and some residues remain even after UHT treatment. The residual lipase can decompose fat and release free fat. These free fats can act as adhesives when fat globules collide with each other, thus forming fat globule clusters and causing fat to float. At the same time, after lipase destroys the fat globule membrane, fat is more easily oxidized or hydrolyzed, unsaturated fatty acids increase, and the acidity of milk increases, producing unpleasant flavors. Since milk fat contains more low-level fatty acids, even a slight hydrolysis will produce a pungent rancid taste.
[0003] Therefore, in order to control the influence of lipase in raw milk on the quality of UHT sterilized milk fat floating, flavor fission, etc., it is urgent to quickly detect the activity of lipase in raw milk. The existing methods for detecting lipase activity mainly include plate method, acid-base titration method, p-nitrophenol method, fluorescence method, chromatography method, etc. Plate method is mostly suitable for rapid and rough determination of exogenous lipase; acid-base titration method is mostly suitable for qualitative determination of endogenous and exogenous lipase in milk, but the detection process of this method has problems such as difficulty in judging the titration end point, large human error, low accuracy of the measured results, and poor stability; p-nitrophenol method is widely used, the method has good stability, and the enzyme activity data is highly accurate, but the operation is relatively complicated, the drug price is expensive, and some toxic substances are used in the test, which are easy to cause harm to the human body and the environment; fluorescence method and chromatography method require expensive substrate reagents or equipment, and are generally only used for qualitative and quantitative determination of exogenous lipase. It can be seen that there is an urgent need to provide a method for detecting lipase activity in raw milk with fast speed, good result accuracy, low cost and convenient operation. Summary of the invention
[0004] The purpose of the present invention is to address the problems of complex operation process, large error and high cost in the prior art method for determining lipase activity in raw milk, and to propose a method for rapidly detecting lipase activity in raw milk.
[0005] In order to achieve the above-mentioned object of the invention, the present invention provides a method for rapidly detecting the activity of lipase in raw milk, comprising the following steps: S1. Inactivate the raw milk at a temperature not lower than 90° C. for not lower than 10 seconds, and then cool it to 2-6° C. to obtain inactivated raw milk; add a lipase standard substance to the inactivated raw milk to prepare at least 5 lipase standard working solutions with different activity values; measure an appropriate amount of raw milk as a sample to be tested; S2. Determine the content of fat and / or free fatty acids in the lipase standard working solution before and after hydrolysis using a milk component analyzer, and calculate the content change value of fat and / or free fatty acids in the lipase standard working solution before and after hydrolysis; Determine the content of fat and / or free fatty acids in the sample to be tested before and after hydrolysis using a milk component analyzer, and calculate the content change value of fat and / or free fatty acids in the sample to be tested before and after hydrolysis; S3. Use the lipase activity value (U / mL) of the lipase standard working solution as the horizontal axis and the change value of the content of fat (g / 100g) and / or free fatty acid (mevk / L) in the lipase standard working solution before and after hydrolysis as the vertical axis to draw a corresponding working curve, and obtain the regression equation of the corresponding working curve; S4. According to the regression equation of the corresponding working curve, the lipase activity value in the sample to be tested is calculated using the change value of the content of fat and / or free fatty acids in the sample to be tested before and after hydrolysis.
[0006] The invention discloses a method for rapidly detecting the activity of lipase in raw milk. The method is based on the principle that lipase in raw milk can hydrolyze fat and produce free fatty acids under certain temperature conditions, thereby reducing the fat content and increasing the free fatty acid content in raw milk. A milk component analyzer is not only used to determine the change in fat and / or free fatty acids in raw milk before and after hydrolysis, but also a lipase standard working solution is set for comparison, a working curve is drawn, and a regression equation is obtained, so that the activity of lipase in raw milk can be accurately calculated according to the change in fat and / or free fatty acids in a sample to be tested and in combination with the regression equation. The milk component analyzer used in the method is a conventional instrument in dairy enterprises, so there is no need to purchase special detection equipment, the cost is low, and the operation is convenient. At the same time, the milk component analyzer can be used to rapidly and accurately obtain the change value of the components in raw milk, the detection speed is fast, the result accuracy is good, and the method is more suitable for large-scale promotion and application in the detection of lipase activity in raw milk.
[0007] Among them, in step S1, preferably, the temperature of the inactivation treatment is 92-95°C; if the temperature is too low, the bacteria and exogenous lipase will not be completely inactivated, which will decompose the milk components and cause inaccurate test results; and if the temperature is too high, the milk components will deteriorate and affect the hydrolysis effect, thereby causing inaccurate test results.
[0008] Preferably, the inactivation treatment time is 10-15s; if the time is too short, the bacteria and exogenous lipase will not be completely inactivated, which will decompose the milk components and cause inaccurate test results; if the time is too long, the milk components will deteriorate, affecting the hydrolysis effect, thereby causing inaccurate test results.
[0009] Preferably, the criterion for judging the effectiveness of the inactivation treatment is: keeping warm at 40±3°C for 1 hour, fat change <0.02g / 100g.
[0010] Preferably, the activity values of the different lipase standard working solutions are in a geometric progression; the preferred lipase standard working solution is required to make the working curve smoother, the characteristics more obvious, the obtained regression equation more accurate, and the test result more accurate.
[0011] Wherein, in step S2, preferably, the repeatability (%Cv) of the milk component analyzer is 2 )≤0.25%; Accuracy (%Cv) 2 )≤1.0 %; the optimal instrument precision and accuracy requirements can significantly improve the accuracy of the test results.
[0012] Preferably, before using the milk component analyzer for measurement, the milk component analyzer needs to be calibrated; calibration can make the test results of the milk component analyzer more accurate and reduce unnecessary errors.
[0013] Preferably, the hydrolysis method comprises: placing the lipase standard working solution and the sample to be tested under the condition of 40±3° C. for hydrolysis for 1 hour, taking them out, and cooling them to 2-6° C. within 10 minutes.
[0014] Preferably, in the hydrolysis method, water bath heating is adopted; water bath heating is more uniform and the hydrolysis conditions are more controllable.
[0015] Preferably, in the hydrolysis method, the temperature is cooled to 2-6° C. within 5 minutes; the faster the cooling speed, the lower the hydrolysis effect of lipase on fat, thereby preventing fat from being hydrolyzed by lipase after the hydrolysis is completed, thereby ensuring more accurate test results.
[0016] Among them, preferably, step S4, according to the regression equation of the corresponding working curve, uses the change values of the fat and free fatty acid contents in the sample to be tested before and after hydrolysis to respectively calculate the lipase activity value in the sample to be tested; the lipase activity value calculated by the change value of the fat content in the sample to be tested and the lipase activity value calculated by the change value of the free fatty acid content are differenced, if the absolute difference between the two is greater than 10% of the arithmetic mean of the two, the activity value of the lipase standard working solution is adjusted and re-tested; if the absolute difference between the two is less than or equal to 10% of the arithmetic mean of the two, the arithmetic mean of the two is used as the final measurement result; using the correspondence relationship between the changes in fat and free lipase acid in the raw milk, the results measured by different substances are mutually calibrated, which can effectively avoid errors and mistakes in the detection process, reduce the result deviation caused by instrument accuracy, etc., and significantly improve the accuracy of the detection results.
[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The method of the present invention uses a milk component analyzer to determine the change in fat and / or free fatty acids in raw milk before and after hydrolysis. The detection cost is low, the operation is convenient, and the detection speed is fast. It is more suitable for large-scale promotion and application in the detection of lipase activity in raw milk.
[0018] 2. The method of the present invention sets a lipase standard working solution for comparison, draws a working curve and obtains a regression equation, thereby accurately calculating the lipase activity in the raw milk according to the change in fat and / or free fatty acids in the sample to be tested in combination with the regression equation, and the detection result has high accuracy.
[0019] 3. The method of the present invention utilizes the corresponding relationship between the changes in fat and free lipase acid in raw milk to calibrate the results measured by different substances, which can effectively avoid errors and mistakes in the detection process, reduce the result deviation caused by instrument accuracy, etc., and significantly improve the accuracy of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 This is the fat-lipase activity working curve in Example 1 of the present invention.
[0020] Figure 2 This is the free fatty acid-lipase activity working curve in Example 2 of the present invention. DETAILED DESCRIPTION
[0021] The present invention is further described in detail below in conjunction with test examples and specific implementation methods. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.
[0022] Materials: Raw milk; Lipase standard (20000u / g, Sigma-Aldrich (Shanghai) Trading Co., Ltd.).
[0023] Equipment: FT1 milk composition analyzer; HHS-21-6 digital constant temperature water bath; 20~200μL pipette.
[0024] Embodiment 1: A method for rapidly detecting lipase activity in raw milk comprises the following steps: S1. Inactivate the raw milk at 95°C for 10 seconds, and then cool it to 6°C to obtain inactivated raw milk; add lipase standard to the inactivated raw milk to prepare 5 lipase standard working solutions with different activity values (0.25U / mL, 0.5U / mL, 1U / mL, 2U / mL, 4U / mL); measure 100mL of raw milk as the sample to be tested; S2. The fat content in the lipase standard working solution before and after hydrolysis (the lipase standard working solution and the sample to be tested were placed in a water bath at 40°C for hydrolysis for 1 hour, taken out, and cooled to 4°C within 5 minutes) was determined using a milk component analyzer (calibrated before testing), and the fat content change in the lipase standard working solution before and after hydrolysis was calculated (Table 1); the fat content in the sample to be tested before and after hydrolysis was determined using a milk component analyzer, and the fat content change in the sample to be tested before and after hydrolysis was calculated; S3. Use the lipase activity value (U / mL) of the lipase standard working solution as the horizontal axis and the change value of the fat content in the lipase standard working solution before and after hydrolysis (g / 100g) as the vertical axis to draw the corresponding working curve ( Figure 1 ), and the regression equation of the corresponding working curve was obtained (y = 0.0748x + 0.08; R2 = 0.9994); S4. According to the regression equation of the corresponding working curve, the lipase activity value in the sample to be tested was calculated using the change in fat content in the sample to be tested before and after hydrolysis (Table 2).
[0025] Table 1 Changes in fat content in lipase standard working solution before and after hydrolysis Table 2 Test results of samples before and after hydrolysis According to the test results, the fat content before and after hydrolysis was used as the standard to measure the lipase activity of raw milk. Its linearity met the requirements of GB / 27404 and satisfied the recovery rate range requirement of 70-110%. The detection time for each group was less than 30 minutes.
[0026] Example 2 A method for rapidly detecting lipase activity in raw milk comprises the following steps: S1. Inactivate the raw milk at 92°C for 15s, and then cool it to 2°C to obtain inactivated raw milk; add lipase standard to the inactivated raw milk to prepare 5 lipase standard working solutions with different activity values (0.25U / mL, 0.5U / mL, 1U / mL, 2U / mL, 4U / mL); measure 100mL of raw milk as the sample to be tested; S2. The free fatty acid content in the lipase standard working solution before and after hydrolysis (the lipase standard working solution and the sample to be tested were placed in a water bath at 43°C for hydrolysis for 1 hour, taken out, and cooled to 6°C within 8 minutes) was determined using a milk component analyzer (calibrated before testing), and the change in the free fatty acid content in the lipase standard working solution before and after hydrolysis was calculated; the free fatty acid content in the sample to be tested before and after hydrolysis was determined using a milk component analyzer, and the change in the free fatty acid content in the sample to be tested before and after hydrolysis was calculated (Table 3); S3. Use the lipase activity value (U / mL) of the lipase standard working solution as the horizontal axis and the change value of the free fatty acid content in the lipase standard working solution before and after hydrolysis (mevk / L) as the vertical axis to draw the corresponding working curve ( Figure 2 ), and the regression equation of the corresponding working curve was obtained (y = 0.5255x + 0.8854; R2 = 0.9768); S4. According to the regression equation of the corresponding working curve, the lipase activity value in the sample to be tested was calculated using the change in the content of free fatty acids in the sample to be tested before and after hydrolysis (Table 4).
[0027] Table 3 Changes in fatty acid content in lipase standard working solution before and after hydrolysis Lipase standard working liquid concentration U / mL 0.25 0.50 1.00 2.00 4.00 Increase of free fatty acids after hydrolysis mevk / L 0.85 1.16 1.53 2.05 2.91 Table 4 Test results of samples before and after hydrolysis According to the test results, the fatty acid content before and after hydrolysis was used as the standard to measure the lipase activity of raw milk. Its linearity met the requirements of GB / 27404 and satisfied the recovery rate range requirement of 70-110%. The detection time for each group was less than 30 minutes.
[0028] Example 3 A method for rapidly detecting lipase activity in raw milk comprises the following steps: S1. Inactivate the raw milk at 90°C for 20 seconds, and then cool it to 2°C to obtain inactivated raw milk; add lipase standard to the inactivated raw milk to prepare 5 lipase standard working solutions with different activity values (0.25U / mL, 0.5U / mL, 1U / mL, 2U / mL, 4U / mL); measure 100mL of raw milk as the sample to be tested; S2. Determine the contents of fat and free fatty acids in the lipase standard working solution before and after hydrolysis (the lipase standard working solution and the sample to be tested are placed in a water bath at 37°C for hydrolysis for 1 hour, taken out, and cooled to 2°C within 5 minutes) using a milk component analyzer (calibrated before testing), and calculate the change values of the contents of fat and free fatty acids in the lipase standard working solution before and after hydrolysis; determine the contents of fat and free fatty acids in the sample to be tested before and after hydrolysis using a milk component analyzer, and calculate the change values of the contents of fat and free fatty acids in the sample to be tested before and after hydrolysis (Table 1 and Table 3); S3. Use the lipase activity value (U / mL) of the lipase standard working solution as the horizontal axis, and the fat (g / 100g) and free fatty acid content mevk / L in the lipase standard working solution before and after hydrolysis as the vertical axis to draw the corresponding working curve, and obtain the regression equation of the corresponding working curve; S4. According to the regression equation of the corresponding working curve, the lipase activity value in the sample to be tested is calculated using the change values of the fat and free fatty acid contents in the sample to be tested before and after hydrolysis; the lipase activity value calculated by the change value of the fat content in the sample to be tested (Table 2) and the lipase activity value calculated by the change value of the free fatty acid content (Table 4) are differenced. If the absolute difference between the two is greater than 10% of the arithmetic mean of the two, the activity value of the lipase standard working solution is adjusted and the test is performed again; if the absolute difference between the two is less than or equal to 10% of the arithmetic mean of the two, the arithmetic mean of the two is used as the final measurement result (Table 5).
[0029] Table 5 Test results of the samples to be tested According to the test results, the activity values of the lipase standard working solutions of raw milk 2 and raw milk 4 were adjusted (the activity values of the standard solutions were 0.1U / mL, 0.2U / mL, 0.4U / mL, 0.8U / mL, and 1.6U / mL), and then retested. The test results are shown in Table 6: Table 6 Results of retesting of raw milk 2 and raw milk 4 Sample No. Spiking Lipase activity U / mL (fat as marker) Lipase activity U / mL (free fatty acids as marker) The percentage of the difference to the arithmetic mean (%) Final test result (U / mL) Raw milk 2 0.25 U / mL 0.24 0.23 4.2 0.24 Raw milk 4 1.0U / mL 0.96 0.98 2.1 0.97 According to the test results, by utilizing the corresponding relationship between the changes in fat and free lipase acid in raw milk and cross-checking the results measured by different substances, errors and mistakes in the detection process can be effectively avoided, the result deviation caused by instrument accuracy can be reduced, and the accuracy of the test results can be significantly improved.
Claims
1. A method for rapidly detecting lipase activity in raw milk, characterized in that: The following steps are involved: S1. Inactivate the raw milk at a temperature not lower than 90° C. for not lower than 10 seconds, and then cool it to 2-6° C. to obtain inactivated raw milk; add a lipase standard substance to the inactivated raw milk to prepare at least 5 lipase standard working solutions with different activity values; measure an appropriate amount of raw milk as a sample to be tested; S2. Determine the content of fat and / or free fatty acids in the lipase standard working solution before and after hydrolysis using a milk component analyzer, and calculate the content change value of fat and / or free fatty acids in the lipase standard working solution before and after hydrolysis; Determine the content of fat and / or free fatty acids in the sample to be tested before and after hydrolysis using a milk component analyzer, and calculate the content change value of fat and / or free fatty acids in the sample to be tested before and after hydrolysis; S3, using the lipase activity value of the lipase standard working solution as the horizontal axis and the change value of the content of fat and / or free fatty acids in the lipase standard working solution before and after hydrolysis as the vertical axis, drawing a corresponding working curve, and obtaining a regression equation for the corresponding working curve; S4. According to the regression equation of the corresponding working curve, the lipase activity value in the sample to be tested is calculated using the change value of the content of fat and / or free fatty acids in the sample to be tested before and after hydrolysis.
2. The method for rapid detection of lipase activity in raw milk according to claim 1, characterized in that: In step S1, the temperature of the inactivation treatment is 92-95°C.
3. The method for rapid detection of lipase activity in raw milk according to claim 1, characterized in that: In step S1, the inactivation treatment time is 10-15s.
4. The method for rapid detection of lipase activity in raw milk according to claim 1, characterized in that: In step S1, the criterion for judging the effectiveness of the inactivation treatment is: keeping warm at 40±3°C for 1 hour, and the fat change is less than 0.02g / 100g.
5. The method for rapid detection of lipase activity in raw milk according to claim 1, characterized in that: In step S1, the activity values of the different lipase standard working solutions are in a geometric progression.
6. The method for rapid detection of lipase activity in raw milk according to claim 1, characterized in that: In step S2, the repeatability of the milk component analyzer is ≤0.25%; the accuracy is ≤1.0%.
7. The method for rapid detection of lipase activity in raw milk according to claim 1, characterized in that: In step S2, before using the milk component analyzer for measurement, the milk component analyzer needs to be calibrated.
8. The method for rapid detection of lipase activity in raw milk according to claim 1, characterized in that: In step S2, the hydrolysis method includes: placing the lipase standard working solution and the sample to be tested under the condition of 40±3°C for hydrolysis for 1 hour, taking them out, and cooling them to 2-6°C within 10 minutes.
9. The method for rapid detection of lipase activity in raw milk according to claim 8, characterized in that: In the hydrolysis method, cooling to 2-6° C. is performed within 5 min.
10. The method for rapidly detecting lipase activity in raw milk according to any one of claims 1 to 9, characterized in that: Step S4, according to the regression equation of the corresponding working curve, the lipase activity value in the sample to be tested is calculated using the change values of the fat and free fatty acid contents in the sample to be tested before and after hydrolysis; the lipase activity value calculated by the change value of the fat content in the sample to be tested and the lipase activity value calculated by the change value of the free fatty acid content are differenced, if the absolute difference between the two is greater than 10% of the arithmetic mean of the two, the activity value of the lipase standard working solution is adjusted and retested; if the absolute difference between the two is less than or equal to 10% of the arithmetic mean of the two, the arithmetic mean of the two is used as the final measurement result.