A quantitative detection kit and method for total phytosterol content in rapeseed

Abstract

The application relates to the technical field of detection methods, in particular to a quantitative detection kit and detection method for the total amount of phytosterols in rapeseed. In the detection of the total amount of phytosterols in rapeseed, the peroxidase mimics of copper-based MOF carbide in the application are used as artificial catalytic enzymes to replace the conventional biological horseradish peroxidase, so that the cost is reduced, the stability of the method is improved, the signal stability of biological enzyme catalysis is inferior to that of artificial enzyme, and the accuracy of quantitative effect is also inferior to that of artificial enzyme.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection methods, and particularly relates to a quantitative detection kit and detection method for total phytosterol content in rapeseed. BACKGROUND

[0002] Phytosterols are an important class of lipids with various biological activities. Studies have shown that appropriate intake of phytosterols can reduce the risk of cardiovascular disease. Edible vegetable oil is the main way for humans to intake phytosterols, and the phytosterol content of rapeseed oil ranks first among edible vegetable oils, only next to corn oil. As a primary agricultural product and raw material for related food, developing a method for rapidly and quantitatively detecting the total phytosterol content of rapeseed is of great significance for raw material procurement and grading processing of oil product processing enterprises. The current detection methods for phytosterols mainly include large instrument methods such as gas chromatography and high-performance liquid chromatography (e.g., CN 202210654018.8 and ZL 201310289363.7), which are time-consuming, complex to operate, and require high-quality instruments and skilled operators. Currently, there are few quantitative methods suitable for high-throughput and rapid detection of the total phytosterol content in rapeseed. CN 202210224430.6 reports a rapid detection method and kit for the sterol content of vegetable oil, which uses horseradish peroxidase as a catalytic enzyme to generate hydrogen peroxide from phytosterols and cholesterol oxidase, and then catalyzes the substrate to produce a color signal for the quantitative detection of phytosterols. However, the horseradish peroxidase used in this method is a biological enzyme with high cost and poor stability. SUMMARY

[0003] The present application provides a quantitative detection kit and detection method for the total phytosterol content in rapeseed, which overcomes the high cost and poor stability of biological enzymes and develops a rapid quantitative detection kit for the total phytosterol content in rapeseed with high sensitivity, high accuracy, high throughput, and simple operation method. The kit can be used for batch and rapid detection of phytosterols in rapeseed procurement and grading.

[0004] The present application provides a copper-based MOF carbide, and a preparation method thereof. The preparation method comprises carbonizing Cu MOF powder at 400-600 DEG C to obtain a copper-based MOF carbide. The copper-based MOF carbide provided by the present application is a peroxidase mimic. By utilizing its catalytic properties, the present application overcomes the high cost and poor stability of biological enzymes. The copper-based MOF carbide has peroxidase properties and can replace biological enzymes (horseradish peroxidase) at a low cost.

[0005] According to the copper-based MOF carbide, the Cu MOF powder is heated to 400-600°C at a temperature increasing rate of 1-10°C / min under inert gas protection, and kept for 2h or more, to obtain the copper-based MOF carbide with a particle size of 400-600nm.

[0006] Preferably, the Cu MOF powder is Cu-BTC.

[0007] Preferably, the inert gas protection is argon or nitrogen.

[0008] Preferably, the carbonization is performed in a tube furnace.

[0009] Preferably, the keeping is for 2-3h.

[0010] Preferably, the copper-based MOF carbide is obtained by grinding after carbonization.

[0011] The present application provides a copper-based MOF carbide solution, wherein the copper-based MOF carbide is mixed with water, and ultrasonic treatment is performed to obtain a copper-based MOF carbide solution with a concentration of 0.25-1.5mg / mL. The copper-based MOF carbide solution is a peroxidase mimic solution.

[0012] According to the copper-based MOF carbide solution, the ultrasonic treatment is performed at an intensity of 50-100KHZ for 10-30min.

[0013] Preferably, the peroxidase mimic solution of the copper-based MOF carbide is a powder of the copper-based MOF carbide, which is dissolved in water, ultrasonic treatment is performed for 10-30min at an intensity of 50-100KHZ, and the concentration of the solution is 0.25-1.5mg / mL. The preparation process of the peroxidase mimic of the copper-based MOF carbide is as follows: Cu MOF (preferably Cu-BTC) powder is placed in a tube furnace, heated to 400-600°C at a temperature increasing rate of 1-10°C / min under argon or nitrogen protection, and kept for 2h, and then ground to obtain the product.

[0014] The present application provides a sterol content detection kit, which comprises a copper-based MOF carbide.

[0015] Preferably, the kit comprises the copper-based MOF carbide or the copper-based MOF carbide solution.

[0016] According to the sterol content detection kit, it further comprises a buffer, a cholesterol oxidase solution, a sterol standard solution, a chromogenic solution, and a reaction termination solution. The kit comprising the peroxidase mimic solution of the copper-based MOF carbide, the buffer, the cholesterol oxidase solution, the series of plant sterol mixed standard solution, the chromogenic solution, and the reaction termination solution can be used to quantitatively detect the total amount of plant sterols in rapeseed.

[0017] Preferably, the reaction termination solution is a sulfuric acid solution.

[0018] Preferably, the 1L buffer solution includes sodium chloride 7.5-8.5g, potassium chloride 0.15-0.25g, disodium hydrogen phosphate dodecahydrate 3-4g and potassium 0.2-0.35g, and the rest is water, and the pH value of the buffer solution is 6.8-7.2.

[0019] Further preferably, the buffer solution is a phosphate buffered saline solution, with a pH of 5.5-7.4 and a concentration of 0.01 mol / L. The formula is to add 8g of sodium chloride, 0.2g of potassium chloride, 3.58g of disodium hydrogen phosphate dodecahydrate and 0.27g of potassium dihydrogen phosphate to 1000mL of water, and adjust the pH with NaOH or HCl.

[0020] Preferably, the color developing solution is prepared by mixing A liquid and B liquid in a volume ratio of 4:2.8-3.2, wherein the A liquid is a sodium acetate buffer solution, and the B liquid is a 3,3',5,5'-tetramethylbenzidine (TMB) solution.

[0021] The present application provides a sterol content detection method, which utilizes the copper-based MOF carbide, the copper-based MOF carbide solution, or the sterol content detection kit.

[0022] Preferably, the detection method is as follows: add sample extraction solution to each well of a 96-well plate, then sequentially add buffer solution and cholesterol oxidase solution, cover the well with a cover, incubate at 50℃ for 3min, sequentially add color developing solution and peroxidase mimic solution of copper-based MOF carbide, perform color developing reaction, add reaction termination solution after 6min, and finally measure absorbance value at 450nm or take a photo to read G value using a mobile phone color picker APP. Substitute into the standard curve to obtain the content of phytosterol in rapeseed.

[0023] According to the sterol content detection method, buffer solution and cholesterol oxidase solution are sequentially added to the sterol extract of the sample to be tested, incubated at 45-55℃ for 2.5-3.5min, color developing solution and copper-based MOF carbide solution are sequentially added, color developing reaction is performed, reaction termination solution is added after 5.5-6.5min, and finally the absorbance value is measured at 450nm or a photo is taken to read G value using a mobile phone color picker APP, and the sterol content is substituted into the standard curve.

[0024] According to the sterol content detection method, the volume ratio of sample sterol extraction solution: buffer solution: cholesterol oxidase solution: color developing solution: copper-based MOF carbide solution: reaction termination solution is 2.8-3.2:3.8-4.2:7.8-8.2:6.8-7.2:2.8-3.2:4.8-5.2.

[0025] Preferably, the total solution volume added in each well of the 96-well plate in the detection method is 300 μL, and the volume ratio of the added solution is sample extraction solution: buffer: cholesterol oxidase solution: color developing solution: copper-based MOF carbide peroxidase mimic solution: reaction termination solution = 3:4:8:7:3:5

[0026] The detection analysis principle of the kit of the present application is: a certain volume of sample extraction solution is added in each well of the 96-well plate, then buffer and cholesterol oxidase solution are sequentially added, and after incubation for a period of time, different contents of hydrogen peroxide solution are generated, then color developing solution and copper-based MOF carbide peroxidase mimic solution are sequentially added, after the color develops stably, reaction termination solution is added dropwise, and the signal value is detected by an enzyme-labeled instrument or a smart phone, the larger the signal value (the darker the color), the higher the total content of phytosterols in the sample. According to the standard curve prepared by detecting the known phytosterol mixed standard product concentration, the concentration of the total amount of the phytosterol to be detected can be calculated.

[0027] Preferably, after saponification by potassium hydroxide-ethanol solution, the phytosterol sample extraction solution is obtained by extraction with n-hexane.

[0028] Preferably, the sample extraction solution is prepared as follows: 4 mL of 2 mol / L potassium hydroxide-90% ethanol aqueous solution is added to 0.5 g of rapeseed powder, and after extraction by magnetic bead crushing for 2 min, 2 mL of water and 3 mL of n-hexane solution are added, vortexed for 3-10 min, centrifuged at 4000 rpm for 3 min, and a certain volume of the upper n-hexane layer is taken, filtered with a 0.22 μm organic membrane, dried in an incubator, and then redissolved with half the volume of anhydrous ethanol to obtain.

[0029] According to the sterol content detection kit or the sterol content detection method, the sterol is a plant sterol.

[0030] Preferably, the sterol is selected from Brassica plants.

[0031] Further preferably, the sterol is selected from rapeseed.

[0032] The present application has the following beneficial effects:

[0033] 1. The copper-based MOF carbide peroxidase mimic of the present application serves as an artificial catalytic enzyme, replacing the conventional biological horseradish peroxidase, thereby reducing the cost and improving the stability of the method. The signal stability of biological enzyme catalysis is not as good as that of artificial enzyme, and the accuracy of quantitative effect is also not as good as that of artificial enzyme.

[0034] 2. The artificial enzyme used in the present application has a more relaxed condition environment for reagents and a low temperature requirement for storage.

[0035] 3、The optimal condition of the detection method of the application is determined after optimization, and a large number of detection can be completed within 30 minutes, without large instruments, meeting the needs of on-site detection.

[0036] 4、The application has important practical significance for solving the on-site detection technology of phytosterol of a large number of rapeseed samples. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the application or prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0038] Figure 1 is a scanning electron microscope image of the copper-based MOF carbide provided by the application.

[0039] Figure 2 is a standard curve of total rapeseed phytosterol drawn by OD value and G value provided by the application.

[0040] Figure 3 is the correlation of the sample detection results of the instrument method and the method of the application. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical scheme and advantages of the application more clear, the technical scheme in the application will be described clearly and completely in combination with the drawings in the application. Obviously, the described embodiments are part of the embodiments of the application, not all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor belong to the protection scope of the application.

[0042] In this embodiment, the Cu-BTC powder is purchased from Jiangsu Xianfeng Nanometer Material Technology Co., Ltd. (https: / / www.xfnano.com / product / search?searchKey=cubtc).

[0043] The cholesterol oxidase solution is purchased from Shanghai Aladdin Reagent Co., Ltd. (China Shanghai) (https: / / www.aladdin-e.com / zh_cn / c139686.html), dissolved in buffer, and the concentration is 6-50 U / mL.

[0044] The phytosterol mixed standard solution is a 4 mg / mL phytosterol mixed standard anhydrous ethanol solution. The phytosterol mixed standard is purchased from Larodan AB (https: / / www.larodan.com / contact / ), and the composition is 13% brassicasterol, 26% campesterol, 7% stigmasterol, and 53% beta-sitosterol.

[0045] The chromogenic solution is prepared by mixing A solution and B solution at a volume ratio of 4:3. The A solution is a sodium acetate buffer solution, and the B solution is a 3,3',5,5'-tetramethylbenzidine (TMB) solution. The formula of the A solution is 32.8 mg of sodium acetate and 120 uL of acetic acid per 100 mL of water; the formula of the B solution is a TMB anhydrous ethanol solution with a concentration of 2-10 mmol / L.

[0046] The reaction termination solution is a sulfuric acid aqueous solution with a concentration of 2 mol / L.

[0047] Example 1: Preparation of peroxidase mimic solution of copper-based MOF carbide

[0048] The Cu-BTC powder purchased from Jiangsu Xianfeng Nanometer Material Technology Co., Ltd. is ground and placed in a tube furnace. Argon gas is used to expel air (about 20 min). Then, under the protection of argon, the temperature is raised to 550°C at a rate of 5°C / min, and the temperature is maintained for 2 h. After the tube furnace cools to room temperature, the calcined black powder material is collected, ground, and copper-based MOF carbide is prepared. Figure 1 The scanning electron microscope image thereof is shown in the figure. As can be seen from the figure, the material size is about 500 nm, the morphology is rod-shaped, and the size is uniform.

[0049] 5 mg of copper-based MOF carbide is added to 10 mL of deionized water solution, vortexed, and then set to ultrasonic intensity of 70 KHZ for 20 min in an ultrasonic cleaning machine to prepare a peroxidase mimic solution of copper-based MOF carbide with a concentration of 0.5 mg / mL.

[0050] Example 2: Assembly of quantitative detection kit for total phytosterol content in rapeseed

[0051] In this example, the kit contains the following parts:

[0052] (1) 96-well plate and its matching well cover

[0053] (2) Peroxidase mimic solution of copper-based MOF carbide 0.5 mg / mL.

[0054] (3) Reaction termination solution. The preparation method is as follows: take 178.3 mL of distilled water, and add 21.7 mL of concentrated sulfuric acid (98%) dropwise, and mix well.

[0055] (3) Buffer. The preparation method is as follows: 8 g of sodium chloride, 0.2 g of potassium chloride, 3.58 g of disodium hydrogen phosphate dodecahydrate, 0.27 g of potassium dihydrogen phosphate are weighed, 800 mL of water is added, after dissolution, 2 mol / L sodium hydroxide aqueous solution is used to adjust the pH to 7, and then water is added to make up to 1 L.

[0056] (4) Cholesterol oxidase solution. Cholesterol oxidase (5KU) commercially available from Aladdin is taken, 400 mL of PBS buffer solution with pH = 7 is added, and vortex mixing is performed to prepare a cholesterol oxidase solution with a concentration of 12.5 U / mL.

[0057] (5) 4 mg / mL phytosterol mixed standard solution in anhydrous ethanol. Commercially available 25 mg / mL phytosterol mixed standard solution is evaporated to dryness by nitrogen blowing, and is redissolved in 6.25 mL of anhydrous ethanol.

[0058] (6) Chromogenic solution A liquid: 32.8 mg of sodium acetate is weighed and added to 80 mL of water, 120 uL of acetic acid solution is further added, and water is added to make up to 100 mL.

[0059] (7) Chromogenic solution B liquid. 48 mg of TMB is weighed, added to 20 mL of anhydrous ethanol, vortex mixed, and filtered through an organic membrane.

[0060] Kit operation and result calculation of Example 3

[0061] Preparation of 2 mol / L potassium hydroxide-90% ethanol aqueous solution: 11.2 g of potassium hydroxide solid is dissolved in 90 mL of ethanol and 10 mL of deionized water, and mixed.

[0062] 0.5 g of rapeseed powder is weighed, 4 mL of 2 mol / L potassium hydroxide-90% ethanol aqueous solution is added, after magnetic bead crushing extraction for 2 min, 2 mL of water is added, 3 mL of n-hexane solution is added, vortexed for 5 min, centrifuged at 4000 rpm for 3 min, the upper n-hexane layer is taken, filtered through a 0.22 mu m organic membrane. 60 mu L of filtered n-hexane is taken, added to the hole plate, dried in an incubator, 30 mu L of anhydrous ethanol is added for redissolution. 40 mu L of buffer solution, 80 mu L of cholesterol oxidase solution are sequentially added in the hole plate, the hole plate is covered and incubated in a 50 DEG C incubator for 3 min, 70 mu L of chromogenic solution, 30 mu L of copper-based MOF carbide peroxidase mimic solution (concentration of 0.5 mg / mL) are sequentially added, after standing at room temperature for 6 min, 50 mu L of stop solution is added, and the OD value of each hole at a wavelength of 450 nm is measured on an enzyme marker instrument, or a mobile phone colorimeter APP is used to take a photo to read G value.

[0063] Dilute 4 mg / mL phytosterol mixed standard anhydrous ethanol solution into a series of phytosterol standard solutions of 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8 and 2 mg / mL with anhydrous ethanol. Take 30 μL of the above standard solution and add it to the wells of a 96-well plate. Add 40 μL of buffer, 80 μL of cholesterol oxidase solution to the wells of the plate in turn, cover the plate and incubate in a 50°C incubator for 3 min. Add 70 μL of chromogenic solution, 30 μL of copper-based MOF carbide peroxidase mimic solution (concentration of 0.5 mg / mL) in turn, and let stand at room temperature for 6 min. Add 50 μL of stop solution, and measure the OD value of each well at a wavelength of 450 nm on an enzyme marker or take a photo to read the G value using a mobile phone color picker APP.

[0064] Take the OD value of the standard sample well as the ordinate and the corresponding standard sample concentration value as the abscissa to draw a standard curve. According to the linear equation of the curve, the concentration of the corresponding sample can be calculated. The linear equation of the curve is Y = 1.299 * X - 0.1473, R 2 = 0.9937; or take the G value read by the mobile phone color picker APP as the ordinate and the corresponding standard sample concentration value as the abscissa to draw a standard curve. The linear equation of the curve is Y = 31.48 * X + 96.07, R 2 = 0.9684. The lowest detection limit is calculated by multiplying the standard deviation of 12 parallel measurements of the lowest signal value by 3 and dividing by the slope of the standard, which is 0.3 mg / g. The total amount of phytosterols in the sample is calculated by substituting the OD value or the G value read by the mobile phone color picker APP into the standard curve (as the standard curve). Figure 2

[0065] Example 4 Recovery experiment

[0066] Take 9 portions of 0.5 g of rapeseed and add a certain amount of 4 mg / mL phytosterol mixed standard anhydrous ethanol solution. Set 3 standard addition levels of 0.8, 2.0 and 5.0 mg / g, and set 3 replicates for each level. Perform the determination according to the steps of Example 3. The determination results are shown in Table 1 below. As can be seen from the table, the standard addition recovery rate of the method is 89.30-102.82%, and the RSD is between 2.61-9.57%, indicating that the method has good accuracy.

[0067] Table 1 Standard addition recovery rate and coefficient of variation of total phytosterol content in rapeseed (n = 3)

[0068]

[0069] Example 5 Actual sample detection

[0070] ​15 local rapeseed samples were randomly collected, and the total content of phytosterols in the rapeseed was determined by the kit method and the NY / T 3945-2021 GC-MS method respectively. The total content of phytosterols in the rapeseed detected by the two methods was compared, and a standard curve was drawn Figure 3 . It can be seen from Figure 3 that the detection results of the actual samples by the GC-MS and the method have good correlation R 2 = 0.98, and the method has high coincidence rate and can be used for actual sample detection.

[0071] Finally, it should be pointed out that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A kit for detecting the content of sterol, characterized by, The copper-based MOF carbide solution, the color developing solution, the buffer solution, the cholesterol oxidase solution, the sterol standard solution, and the reaction termination solution; The preparation method of the copper-based MOF carbide comprises: carbonizing Cu-BTC powder at 400-600 DEG C to obtain the copper-based MOF carbide, and the copper-based MOF carbide is rod-shaped. The color developing solution is prepared by mixing A solution and B solution in a volume ratio of 4:2.8-3.2, wherein the A solution is a sodium acetate buffer solution, and the B solution is a 3,3',5,5'-tetramethylbenzidine solution. The buffer solution 1L comprises 7.5-8.5g of sodium chloride, 0.15-0.25g of potassium chloride, 3-4g of disodium hydrogen phosphate dodecahydrate, and 0.2-0.35g of potassium, and the rest is water, and the pH value of the buffer solution is 6.8-7.

2. The reaction termination solution is a sulfuric acid solution.

2. The kit for detecting the content of sterol according to claim 1, wherein, The Cu-BTC powder is heated to 400-600 DEG C at a heating rate of 1-10 DEG C / min under inert gas protection, and is kept for more than 2h to obtain the copper-based MOF carbide with a particle size of 400-600nm.

3. The kit of claim 2, wherein the sterol content detection reagent is a sterol oxidase. The inert gas protection is argon or nitrogen.

4. The kit for detecting the content of sterol according to claim 2, wherein The carbonization is performed in a tube furnace.

5. The kit of claim 2, wherein the sterol content detection reagent is a sterol oxidase. The keeping time is 2-3h.

6. The kit for detecting the content of sterol according to claim 2, wherein The copper-based MOF carbide is obtained by grinding after carbonization.

7. The kit for detecting the content of sterol according to any one of claims 1 to 6, characterized in that, The preparation method of the copper-based MOF carbide solution comprises: mixing the copper-based MOF carbide and water, and ultrasonicating to obtain the copper-based MOF carbide solution with a concentration of 0.25-1.5mg / mL.

8. The kit of claim 7, wherein the sterol content detection reagent is a sterol oxidase. The ultrasonic intensity is 50-100KHZ, and the ultrasonic time is 10-30min.

9. A method for detecting the content of sterols, characterized by, The sterol content detection kit according to any one of claims 1-8 is used.

10. The method for detecting sterol content according to claim 9, characterized in that, The buffer solution and the cholesterol oxidase solution are sequentially added to the sterol extract of the sample to be tested, and are incubated at 45-55 DEG C for 2.5-3.5min, the color developing solution and the copper-based MOF carbide solution are sequentially added for color developing reaction, the reaction termination solution is added dropwise after 5.5-6.5min, finally, the absorbance value is measured at 450nm of an enzyme label instrument or the G value is read by a mobile phone colorimeter APP, and the sterol content is calculated according to the standard curve.

11. The method for detecting sterol content according to claim 9 or 10, characterized in that, The volume ratio of the sample sterol extract solution, the buffer solution, the cholesterol oxidase solution, the color developing solution, the copper-based MOF carbide solution, and the reaction termination solution is 2.8-3.2:3.8-4.2:7.8-8.2:6.8-7.2:2.8-3.2:4.8-5.

2.

12. The method for detecting sterol content according to claim 9 or 10, characterized in that, The sterol is selected from plant sterols.

13. The method for detecting sterol content according to claim 12, characterized in that, The sterol is selected from Brassica plants.

14. The method for detecting sterol content according to claim 12, characterized in that, The sterol is selected from rapeseed.

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