A rapid method and reagent kit for determining the sterol content of vegetable oils.

Abstract

The present invention relates to a rapid testing method and a rapid testing kit for the sterol content of plant oils and fats. Rapid testing method: providing a sterol extraction solution of a plant oil and fat sample, diluting the sterol extraction solution with a buffer solution to obtain a sample solution, adding the sample solution to a sterol test plate, adding sterol oxidase for oscillation reaction, and obtaining the sterol content of the plant oil and fat based on the color depth of the solution of the sterol test plate being positively correlated with the sterol content. The method of the present invention is simple to operate, has a fast testing speed, and is low in cost. It is suitable for the rapid detection of the sterol content in plant oils and fats, and provides technical support for the screening of high sterol materials, variety breeding, and quality control of raw materials and products of processing enterprises.

Description

Technical Field

[0001] The invention belongs to the field of agricultural product quality and safety detection, and specifically relates to a rapid detection method and a rapid detection kit for the sterol content of vegetable oil. Background Art

[0002] China is a major producer and consumer of oils and fats. Plant oils and fats are the main source of dietary sterols for humans, and the plant sterols they provide account for 40% of the total dietary sterol intake. Therefore, it is of great significance to determine the sterol content in plant oils and fats.

[0003] Phytosterols are triterpenoid compounds with cyclopentane polyhydrophenanthrene as the skeleton. They are a group of specific secondary metabolites of oil crops and are widely found in the roots, stems, leaves, fruits and seeds of plants. At present, more than 200 types of phytosterols have been reported, among which β-sitosterol, stigmasterol, campesterol and rapeseed sterol are the most common. Phytosterols are a secondary metabolite produced by plants during their growth. They have the effects of lowering cholesterol, anti-oxidation, anti-inflammatory, and enhancing immunity. They have good preventive and therapeutic effects on chronic diseases such as cardiovascular disease and cancer, and have attracted widespread attention from scholars at home and abroad. The content and composition of phytosterols in vegetable oils can reflect the type of oil. The composition and content of phytosterols in different oil crops and vegetable oils are different, so they can be used as specific markers to measure the quality of vegetable oils.

[0004] At present, the determination of sterol content in oils and fats mostly uses detection technologies such as GC, GC-MS, HPLC or HPLC-MS to achieve accurate determination of sterol fraction and total amount of sterol. The above methods are accurate and sensitive, and are widely used in qualitative and quantitative analysis of sterols in oils and fats, as well as identification of adulteration of edible oils based on sterol composition. However, these methods require the use of large instruments, are time-consuming, costly, and cannot achieve rapid detection. Therefore, there is an urgent need for a method for rapid on-site detection of sterols in plant oils and fats. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a rapid testing method and a rapid testing kit for the sterol content of vegetable oils and fats.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] A rapid test kit for sterol content in plant oil comprises a buffer for diluting a sterol extract, a sterol oxidase and a sterol test plate. The sterol oxidase is used for catalyzing sterols in the extract to produce hydrogen peroxide. A freeze-dried color developer is fixed on the sterol test plate. The freeze-dried color developer comprises horseradish peroxidase, citric acid, a freeze-dried protective agent and 3,3',5,5'-tetramethylbenzidine (TMB), and the mass ratios are: 8-250; 3.5-10.5; 0.6-3.0; 15-50. The freeze-dried protective agent consists of sucrose, bovine serum albumin, and Tween 20 or sodium dodecylbenzene sulfonate (SDS).

[0008] According to the above scheme, the freeze-drying protective agent includes: sucrose; bovine serum albumin; Tween 20 or sodium dodecylbenzene sulfonate (SDS) at 0.3-1.5; 0.15-0.75; 0.15-0.75.

[0009] According to the above scheme, the buffer used for diluting the sterol extract is: 0.05-0.10 mol / L pH 7.0 2-(N-morpholino)ethanesulfonic acid aqueous solution.

[0010] According to the above scheme, the freeze-dried color developing agent is obtained by freeze-drying a mixture of horseradish peroxidase aqueous solution, citric acid aqueous solution, 3,3',5,5'-tetramethylbenzidine (TMB) anhydrous ethanol solution and a solution containing sucrose, bovine serum albumin, Tween 20 or sodium dodecylbenzene sulfonate (SDS).

[0011] The specific preparation method is as follows: prepare horseradish peroxidase aqueous solution, citric acid aqueous solution, freeze-drying protection solution, 3,3',5,5'-tetramethylbenzidine (TMB) ethanol solution, mix, and freeze-dry. In the mixed system, the concentration range of each substance is: 8-250 mg / mL, 3.5-10.5 mg / ml, 0.6-3.0 mg / mL, 15-50 mg / mL; the concentration of each component in the freeze-drying protection solution is: 0.3-1.5 mg / mL sucrose, 0.15-0.75 mg / mL bovine serum albumin, 0.15-0.75 mg / mL Tween 20 or 0.15-0.75 mg / mL sodium dodecylbenzene sulfonate.

[0012] According to the above scheme, the sterol test plate is provided with micropores; the vacuum freeze drying is: freeze drying is directly placed in the micropores on the sterol test plate to obtain a sterol test plate loaded with a freeze-dried color developer.

[0013] A rapid method for measuring the sterol content of a plant oil comprises the following steps: providing a sterol extraction solution of a plant oil sample, diluting the sterol extraction solution with a buffer solution to obtain a sample solution, adding the sample solution to a sterol test plate for the sterol content of the plant oil, adding sterol oxidase to perform an oscillation reaction, and obtaining the sterol content of the plant oil based on the fact that the color depth of the solution of the sterol test plate is positively correlated with the sterol content;

[0014] Wherein: the sterol test plate is fixed with a freeze-dried color developer, which includes horseradish peroxidase, citric acid, a freeze-dried protective agent and 3,3',5,5'-tetramethylbenzidine, with a mass ratio of 8-250:3.5-10.5:0.6-3.0:5-50; the freeze-dried protective agent is composed of sucrose and bovine serum albumin and Tween 20 or sodium dodecylbenzene sulfonate.

[0015] According to the above scheme, the oscillation reaction time is 30 to 60 minutes, and the oscillation reaction temperature is 37 to 42°C.

[0016] According to the above scheme, the amount of the sample solution used is 100-150 μL.

[0017] According to the above scheme, the dosage of the sterol oxidase is 10-50 μL, and the concentration is 15-25 U / mL.

[0018] According to the above scheme, the vegetable oil and fat samples include: rapeseed, peanut, soybean and other vegetable oil samples and edible vegetable oil samples.

[0019] According to the above scheme, the sample extraction method can be selected according to the detection needs.

[0020] Specifically, in the detection of sterol content in vegetable oils and fats:

[0021] Extract the total sterols in the vegetable oil samples and detect the total amount of sterols in the samples;

[0022] Or extract free sterols from vegetable oil and fat samples and detect the free sterol content in the samples.

[0023] According to the above scheme, the sterol oxidase is cholesterol oxidase, and the enzyme activity is ≥25U / mg. Specifically, cholesterol oxidase expressed by recombinant Escherichia coli can be used.

[0024] According to the above scheme, the quantitative detection is to establish a standard working curve using the concentration of the standard β-sitosterol, and the sterol content in the sample can be directly obtained by substituting the detection signal of the sample to be tested into the standard curve.

[0025] According to the above scheme, the quantitative detection is specifically as follows: using an enzyme-labeled instrument to detect, drawing a standard curve based on the OD value detected by the enzyme-labeled instrument and the sterol concentration of the standard, and obtaining the sterol content based on the quantitative detection of the standard curve.

[0026] According to the above scheme, the characteristic wavelength of the microplate reader detection is 450nm.

[0027] The determination of sterol content in oils and fats mostly uses detection technologies such as GC, GC-MS, HPLC or HPLC-MS to determine the sterol fraction and total sterol content, but these methods require the use of large instruments, are time-consuming, costly, and cannot achieve rapid detection. The structure of sterol is similar to that of cholesterol. There are occasional reports of methods for detecting sterol using cholesterol oxidase in the prior art, but the reaction steps are many, the time is long, and the detection reagents must be prepared and used immediately, which cannot meet the needs of rapid on-site detection.

[0028] The invention prepares a buffer reaction system by combining horseradish peroxidase, 3,3',5,5'-tetramethylbenzidine with citric acid aqueous solution, sucrose, bovine serum albumin, Tween 20 or sodium dodecylbenzene sulfonate (SDS), and freeze-drying the mixture to obtain a freeze-dried color developer. Multi-component and multi-system reagents coexist efficiently. Sterol oxidase, specifically cholesterol oxidase (the structure of plant sterol is similar to that of cholesterol), is used to catalyze plant sterols such as beta-sitosterol, stigmasterol and campesterol to produce hydrogen peroxide. The hydrogen peroxide catalyzes the color developer horseradish peroxidase and 3,3',5,5'-tetramethylbenzidine in a sterol test plate to produce a soluble blue product. The color depth of the solution based on the sterol test plate is positively correlated with the sterol content, thereby achieving the purpose of rapid detection. The invention can also improve the storage stability of horseradish peroxidase and TMB color development components, and finally realize the rapid detection of the sterol content of oil and fat, and solve the problems of multiple color development agent preparation components, mutual interference, instability, immediate preparation and use, low detection efficiency, etc. in the prior art.

[0029] Beneficial effects of the present invention:

[0030] 1. The present invention mixes horseradish peroxidase, 3,3',5,5'-tetramethylbenzidine with citric acid aqueous solution, sucrose, bovine serum albumin, Tween 20 or sodium dodecylbenzene sulfonate (SDS) and other components for freeze-drying to obtain a freeze-dried color developer, and multi-component and multi-system reagents coexist efficiently. The rapid test kit provided is used for sterol content detection, which improves the detection efficiency and the storage stability of the detection reagent.

[0031] 2. The enzymatic detection method for sterols in oils and fats established by the present invention is simple to operate, fast in testing speed, and low in cost. It is suitable for the rapid detection of sterol content in plant oils and fats, and provides technical support for the screening of high sterol materials, variety breeding, and quality control of raw materials and products of processing enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the standard curve of sterol content in plant oils. The horizontal axis is the concentration of the standard, and the vertical axis is the OD value detected by the microplate reader. DETAILED DESCRIPTION

[0033] Example 1 Rapid test kit for sterol content in vegetable oils and fats:

[0034] A rapid test kit for sterol content in vegetable oil comprises a buffer for diluting sterol extract, sterol oxidase and a sterol test plate.

[0035] The sterol oxidase is used to catalyze the sterol in the extract to produce hydrogen peroxide, and can be specifically cholesterol oxidase, with an enzyme activity of ≥25 U / mg, and cholesterol oxidase expressed by recombinant Escherichia coli can be used.

[0036] The buffer solution for diluting the sterol extract is: 0.10 mol / L 2-(N-morpholino)ethanesulfonic acid aqueous solution with a pH of 7.0.

[0037] The sterol test plate is loaded with a freeze-dried color developer; the freeze-dried color developer is obtained by freeze-drying a mixture of a horseradish peroxidase aqueous solution, a citric acid aqueous solution, a 3,3',5,5'-tetramethylbenzidine anhydrous ethanol solution, and a solution containing sucrose, bovine serum albumin, Tween 20 or sodium dodecylbenzene sulfonate, wherein the mass ratio of horseradish peroxidase (CAS: 9003-99-0, 15KU), citric acid, a freeze-dried protective agent, and 3,3',5,5'-tetramethylbenzidine (TMB) (CAS: 54827-17-7) is 90, 7.5, 2.5, 40; in the freeze-drying protective agent: the mass ratio of sucrose, bovine serum albumin, and sodium dodecylbenzene sulfonate (SDS) is 0.7, 0.55, and 0.5. The preparation method is as follows: prepare horseradish peroxidase aqueous solution, citric acid aqueous solution, freeze-drying protective solution, 3,3',5,5'-tetramethylbenzidine (TMB) ethanol solution, mix, freeze-dry, in the mixed system: the concentration range of each substance is: 50mg / mL, 5.5mg / ml, 1.5mg / mL, 25mg / mL; its freeze-drying protective solution is composed of 0.5mg / mL sucrose, 0.75 mg / mL bovine serum albumin, and 0.75mg / mL Tween 20 aqueous solution. The color developer obtained by freeze drying is white sponge-like, loose and porous. It dissolves rapidly within 5 seconds after adding the sample solution. The solution before and after freeze drying is tested with a gradient hydrogen peroxide solution. The OD value correlation coefficient R of the color developer solution before and after freeze drying 2 The value is greater than 0.98, indicating that the freeze-dried protective agent of the present invention can realize the efficient coexistence of multi-component and multi-system reagents, and can ensure the activity stability of components such as horseradish peroxidase.

[0038] Prepare a gradient β-sitosterol (0.136 mg / mL to 13.6 mg / mL) standard solution with ethanol, take 5 μL of the gradient standard solution and add it to 200 μL of 0.10 mol / L pH 7.0 2-(N-morpholino)ethanesulfonic acid aqueous solution and mix well to prepare a gradient sample solution. Take 100 μL of the sample and add it to the microwells of the sterol test plate, add 25 μL of sterol oxidase at a concentration of 25 U / mL, and react at 42°C for 30 minutes to form a blue colored product. Use an enzyme reader at 450nm to detect, and draw a standard curve with the OD value detected by the enzyme reader and the concentration of the standard. The signal is positively correlated with the concentration, and the linear equation is: Y = 0.1557X + 0.0231 correlation coefficient R 2 =0.9925, such as Figure 1 shown.

[0039] Example 2 Rapid test kit for sterol content in vegetable oils and fats:

[0040] The difference from Example 1 is:

[0041] The buffer solution for diluting the sterol extract is: 0.05 mol / L pH 7.0 2-(N-morpholino)ethanesulfonic acid aqueous solution.

[0042] The freeze-dried color developer is obtained by freeze-drying a mixture of horseradish peroxidase aqueous solution, citric acid aqueous solution, 3,3',5,5'-tetramethylbenzidine anhydrous ethanol solution and a solution containing sucrose, bovine serum albumin, Tween 20 or sodium dodecylbenzene sulfonate, wherein the mass ratio of horseradish peroxidase (CAS: 9003-99-0, 15KU), citric acid, freeze-drying protective agent and 3,3',5,5'-tetramethylbenzidine (TMB) (CAS: 54827-17-7) is: 90, 7.5, 2.5, 40; in the freeze-drying protective agent: the mass ratio of sucrose, bovine serum albumin, and sodium dodecylbenzene sulfonate (SDS) is 0.7, 0.55, and 0.5. The preparation method is as follows: prepare horseradish peroxidase aqueous solution, citric acid aqueous solution, freeze-drying protective solution, 3,3',5,5'-tetramethylbenzidine (TMB) ethanol solution, mix, freeze-dry, and in the mixed system: the concentration range of each substance is: 90mg / mL, 7.5mg / ml, 2.5mg / mL, 40mg / mL; its freeze-drying protective solution is composed of 0.7mg / mL sucrose, 0.55mg / mL bovine serum albumin, and 0.5mg / mL Tween 20 aqueous solution. The color developer obtained by freeze drying is white sponge-like, loose and porous. It dissolves rapidly 3s after adding the sample solution. The solution before and after freeze drying is tested with a gradient hydrogen peroxide solution. The OD value correlation coefficient R of the color developer solution before and after freeze drying 2 The value is greater than 0.97. This indicates that the freeze-dried protective agent of the present invention can realize the efficient coexistence of multi-component and multi-system reagents and ensure the activity stability of components such as horseradish peroxidase.

[0043] Standard curve establishment: Use ethanol to prepare a gradient β-sitosterol (0.136 mg / mL to 13.6 mg / mL) standard solution, take 5 μL of the gradient standard solution and add it to 200 μL of 0.05 mol / L pH 7.0 2-(N-morpholino)ethanesulfonic acid aqueous solution and mix well to prepare a gradient sample solution. Take 150 μL of the sample and add it to the microwells of the sterol test plate, add 25 μL of sterol oxidase, the enzyme concentration is 25 u / mL, and react at 42°C constant temperature and oscillate for 30 minutes to form a blue colored product. Use an enzyme reader at 450nm to detect, and use the enzyme reader to detect the OD value and the standard concentration to draw a standard curve. The correlation coefficient R 2 =0.9809.

[0044] Example 3

[0045] 1. Detection of sterol content in rapeseed

[0046] Ten samples of rapeseed were selected and the total amount of rapeseed sterols was determined by rapid test method and GC-MS method respectively.

[0047] Extraction of total amount of rapeseed sterols: Take 200 mg of ground sample in a test tube, add 2 mL of prepared 90% ethanol solution containing 2 mol / L KOH, vortex oscillate for 1 min to mix, and place it in 75°C ultrasonic treatment for 30 min, then cool to room temperature, add 1 mL of n-hexane and 2 mL of ultrapure water, mix evenly, take the supernatant into a 10 mL test tube, repeat the extraction three times, blow nitrogen to dryness, add 150 μL of anhydrous ethanol to re-dissolve, and obtain sterol extract.

[0048] Total amount of rapeseed sterols detection: Take 20 μL of sterol extract and mix it with 480 μL of 0.05 mol / L pH 7.0 2-(N-morpholino)ethanesulfonic acid aqueous solution to obtain a test solution. Take 150 μL of sample and add it to the microwells of the sterol test plate in Example 1, add 30 μL of sterol oxidase, the enzyme concentration is 25 U / mL, and react at 42°C constant temperature oscillation for 45 minutes to form a blue colored product, add 50 μL of 2 mol / L concentrated sulfuric acid to terminate the reaction to form a yellow colored product, and use an enzyme reader at 450nm to detect and obtain the OD value of the sample solution. Substitute it into the standard working curve to calculate the sterol content of the sample. .

[0049] The comparison results of the two methods are shown in Table 1. The relative errors of the detection results of the two methods are within ±10%. Compared with the GC-MS method, the rapid test method does not require derivatization. With the help of the sterol rapid test plate, a large number of samples can be detected simultaneously, which is time-saving, efficient, easy to operate, and low-cost. It is more suitable for on-site rapid detection of sterols in oils and fats.

[0050] Table 1 Comparison between the rapid determination method and GC-MS method for total sterols in rapeseed

[0051]

[0052] 2. Detection of free sterol content in rapeseed oil

[0053] Ten portions of rapeseed oil were selected, and the free sterol content in the rapeseed oil was determined by rapid test method and GC-MS method respectively.

[0054] Extraction of free sterols: Accurately weigh 0.05 g of oil sample, dissolve it in 5 mL of n-hexane solution, vortex and oscillate evenly, and set aside.

[0055] Activation of the SPE column: weigh 1.0g of anhydrous sodium sulfate and add it to the top of the silica solid phase extraction column (0.5g / 6mL), then activate it with 10mL of n-hexane solution, the flow rate is 1.5mL / min, and the effluent is discarded; loading: inject 5mL of sample solution into the SPE column, control the flow rate to 1.2mL / min, and discard the effluent; elution: elute with 10mL of 5% ether-n-hexane solution to remove triglycerides and sterol ester compounds, the flow rate is controlled at 1.2mL / min, and the effluent is discarded; elution: elute free sterols with 10mL of 20% ether-n-hexane solution and collect them in a clean and dry test tube, the flow rate is controlled at 1.5mL / min.

[0056] Detection of free sterol content: Take 20μL of sterol extract and mix it with 380μL of 0.05mol / L pH 7.0 2-(N-morpholino)ethanesulfonic acid aqueous solution to prepare the test solution. Take 100μL of sample and add it to the microwell of the sterol test plate, add 30μL of sterol oxidase, the enzyme concentration is 25u / mL, and react at 42℃ constant temperature oscillation for 45min to form a blue colored product. Add 50μL of 2mol / L concentrated sulfuric acid to terminate the reaction to form a yellow colored product. Use an enzyme reader at 450nm to detect and obtain the OD value of the sample solution. At the same time, use β-sitosterol to establish a standard working curve, and substitute the sample solution OD into the standard working curve to calculate the sample sterol content.

[0057] The comparison results of the two methods are shown in Table 2. The relative errors of the detection results of the two methods are within ±10%. The rapid test method is accurate, simple and efficient, and can realize the synchronous rapid detection of large quantities of samples. It is suitable for high sterol material screening, variety breeding and product quality control of processing enterprises.

[0058] Table 2 Comparison between rapid determination method and GC-MS method for free sterol content in rapeseed oil

[0059]

Claims

1. A rapid test kit for the sterol content of vegetable oils and fats, characterized in that: The invention comprises a buffer for diluting a sterol extract, a sterol oxidase and a sterol test plate, wherein the sterol oxidase is used for catalyzing the sterol in the extract to produce hydrogen peroxide, and the sterol test plate is loaded with a freeze-dried color developer, wherein the freeze-dried color developer comprises horseradish peroxidase, citric acid, a freeze-dried protective agent and 3,3',5,5'-tetramethylbenzidine, and the mass ratio is 8-250:3.5-10.5:0.6-3.0:5- 50; the lyophilization protective agent is composed of sucrose and bovine serum albumin and Tween 20 or sodium dodecylbenzene sulfonate, and is obtained by mixing and lyophilizing a horseradish peroxidase aqueous solution, a citric acid aqueous solution, a 3,3',5,5'-tetramethylbenzidine anhydrous ethanol solution and a lyophilization protective solution containing sucrose, bovine serum albumin, Tween 20 or sodium dodecylbenzene sulfonate. The preparation method is as follows: prepare a horseradish peroxidase aqueous solution, a citric acid aqueous solution, a lyophilization protective solution, and a 3,3',5,5'-tetramethylbenzidine ethanol solution, mix them, and lyophilize them. In the mixed system, the concentration range of each substance is: 8 ~ 250 mg / mL, 3.5 ~ 10.5 mg / ml, 0.6 ~ 3.0 mg / mL, and 15 ~ 50 mg / mL; the lyophilization protective solution is composed of 0.3 ~ 1.5 mg / mL sucrose, 0.15 ~ 0.75 mg / mL bovine serum albumin, 0.15 ~ 0.75 It is composed of 0.15 ~ 0.75 mg / mL Tween 20 or 0.15 ~ 0.75 mg / mL sodium dodecylbenzene sulfonate aqueous solution.

2. The rapid test kit according to claim 1, characterized in that: The freeze-drying protective agent comprises sucrose, bovine serum albumin, Tween 20 or sodium dodecylbenzene sulfonate in a ratio of 0.3 to 1.5: 0.15 to 0.75: 0.15 to 0.

75.

3. The rapid test kit according to claim 1, characterized in that: The buffer solution for diluting the sterol extract is: 0.05-0.10 mol / L 2-(N-morpholino)ethanesulfonic acid aqueous solution with a pH of 7.

0.

4. The rapid test kit according to claim 1, characterized in that: The sterol test plate is provided with micropores; the freeze-dried protective solution is directly placed in the micropores on the sterol test plate for freeze-drying, thereby obtaining a sterol test plate loaded with a freeze-dried color developer.

5. A method for rapidly measuring the sterol content of vegetable oils and fats, characterized in that: The steps are as follows: providing a sterol extraction solution of a plant oil sample, diluting the sterol extraction solution with a buffer solution to obtain a sample solution, adding the sample solution to a sterol test plate, adding sterol oxidase to perform an oscillation reaction, and obtaining the sterol content of the plant oil based on the fact that the color depth of the solution of the sterol test plate is positively correlated with the sterol content; The sterol oxidase is used to catalyze the sterol in the extract to produce hydrogen peroxide. The sterol test plate is fixed with a freeze-dried color developer, which includes horseradish peroxidase, citric acid, a freeze-dried protective agent and 3,3',5,5'-tetramethylbenzidine in a mass ratio of 8 ~ 250:3.5 ~ 10.5:0.6 ~ 3.0:5 ~ 50; the lyophilization protective agent is composed of sucrose and bovine serum albumin and Tween 20 or sodium dodecylbenzene sulfonate, and is obtained by mixing and lyophilizing a horseradish peroxidase aqueous solution, a citric acid aqueous solution, a 3,3',5,5'-tetramethylbenzidine anhydrous ethanol solution and a lyophilization protective solution containing sucrose, bovine serum albumin, Tween 20 or sodium dodecylbenzene sulfonate. The preparation method is as follows: prepare a horseradish peroxidase aqueous solution, a citric acid aqueous solution, a lyophilization protective solution, and a 3,3',5,5'-tetramethylbenzidine ethanol solution, mix them, and lyophilize them. In the mixed system, the concentration range of each substance is: 8 ~ 250 mg / mL, 3.5 ~ 10.5 mg / ml, 0.6 ~ 3.0 mg / mL, and 15 ~ 50 mg / mL; the lyophilization protective solution is composed of 0.3 ~ 1.5 mg / mL sucrose, 0.15 ~ 0.75 mg / mL bovine serum albumin, 0.15 ~ 0.75 It is composed of 0.15 ~ 0.75 mg / mL Tween 20 or 0.15 ~ 0.75 mg / mL sodium dodecylbenzene sulfonate aqueous solution.

6. The rapid testing method according to claim 5, characterized in that: The shaking reaction time is 30 to 60 minutes, and the shaking reaction temperature is 37 to 42°C; The dosage of the sterol oxidase is 10 to 50 µL, and the concentration is 15 to 25 U / mL.

7. The rapid testing method according to claim 5, characterized in that: The vegetable oil and fat samples are vegetable oil samples and edible vegetable oil samples; The amount of the sample solution is 100 ~ 150 μL; The sample extraction method is selected according to the detection needs. Specifically, in the detection of sterol content in vegetable oils and fats: Extract total sterols from vegetable oil samples and detect the total amount of sterols in the samples. Or extract free sterols from vegetable oil and fat samples and detect the free sterol content in the samples; The sterol oxidase is cholesterol oxidase, and the enzyme activity is ≧25 U / mg.

8. The rapid testing method according to claim 5, characterized in that: In the rapid test method, the concentration of the standard substance β-sitosterol is used to establish a standard curve, and the detection signal of the sample to be tested is substituted into the standard curve to obtain the sterol content in the sample.

9. The rapid testing method according to claim 5 or 8, characterized in that: In the rapid test method: an enzyme-labeled instrument is used for detection, a standard curve is drawn based on the OD value detected by the enzyme-labeled instrument and the sterol concentration of the standard substance, and the sterol content is obtained by quantitative detection based on the standard curve.

10. The rapid testing method according to claim 9, characterized in that: The characteristic wavelength of the microplate reader detection is 450nm.

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