Rapid detection kit and rapid detection method for phytosterol

Abstract

The invention provides a rapid detection kit and a rapid detection method for phytosterol, and belongs to the field of food detection.The rapid detection kit for phytosterol comprises a phytosterol standard substance, a standard substance diluent, a substrate solution, an enzyme mixed solution, a stop solution and an elisa plate; the standard substance diluent comprises isopropanol or dimethyl sulfoxide; the substrate solution is prepared from TMB (Tetramethylbenzidine) and a substrate buffer solution; the enzyme mixed solution is a mixed solution of a cholesterol oxidase solution and a horse radish peroxidase solution. By optimizing the composition of each working reagent in the kit, the sensitivity and accuracy of phytosterol detection are remarkably improved. Meanwhile, the rapid detection method provided by the invention is simple and convenient to operate, short in detection time and suitable for on-site rapid detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food detection, in particular to a rapid detection kit and method for phytosterol. BACKGROUND

[0002] The incidence and mortality of cardiovascular diseases are increasing year by year with the improvement of people's living standards, changes in dietary structure and population aging, and have become the "first killer" that endangers health. Phytosterols can effectively reduce the levels of total cholesterol and low-density lipoprotein cholesterol, thereby reducing the risk of cardiovascular disease. In the phytosterol intake of residents' diet, the contribution of edible vegetable oil accounts for 46%, so increasing the phytosterol content of oil plants and creating high-phytosterol edible oil are of great significance for the prevention and treatment of cardiovascular diseases. Its content has become a key indicator for measuring the nutritional value of food. Accurate and efficient detection of phytosterols is of great significance in food quality control, nutritional component evaluation and other fields.

[0003] Detection is the basis for high-phytosterol oil variety breeding and high-phytosterol edible oil creation. At present, there are various detection methods for phytosterols. Infrared spectroscopy in spectral method can realize material structure analysis by judging the difference of molecular group vibration frequency and combination frequency vibration information, and has the advantages of rapidity, efficiency and environmental friendliness, and is widely used in oil detection, especially near-infrared spectroscopy, which can obtain characteristic information of organic molecules by measuring the frequency and combination frequency absorption of hydrogen-containing group vibration. However, infrared spectroscopy needs to rely on a reliable mathematical model to process data, and the process is complicated, and pure oil samples are easily disturbed by factors such as variety, climate and processing, resulting in insufficient stability of the detection results. Raman spectroscopy analyzes molecular information based on scattering light effect, but its application in phytosterol detection is limited by signal intensity and selectivity. In chromatographic methods, high-performance liquid chromatography (HPLC) is widely used, but the pretreatment needs complex steps such as saponification and extraction, and the detection period is as long as 2-4 hours. Although gas chromatography (GC) has high separation efficiency, it needs to perform derivatization treatment on phytosterols, which may cause oxidation loss of phytosterols. Supercritical fluid chromatography (SFC) uses supercritical fluid as mobile phase, combining the characteristics of gas chromatography and liquid chromatography, and can analyze high-boiling and low-volatility samples with fast analysis speed and high column efficiency, but its equipment cost is high, and the technical requirements are high, which limits its large-scale promotion. The enzyme-linked immunosorbent assay (ELISA) in immunoassay has complex pretreatment operation and is difficult to realize high-throughput detection. In addition, solid-phase extraction combined with ultraviolet detection improves the sensitivity to a certain extent, but still has obvious defects, such as the need for centrifugal separation steps in the operation process, the process is complicated, the reagent kit has a storage period of less than 1 month at 4°C, and the stability is poor, which is difficult to meet the needs of on-site rapid detection.

[0004] In summary, the existing phytosterol detection technology generally has the problems of complex pretreatment, long detection time, poor on-site applicability, poor stability of the kit, and large detection error. These drawbacks greatly restrict the rapid screening application of phytosterol detection in food processing process monitoring, market quality supervision and other scenes. Therefore, it is urgent to develop a phytosterol detection technology and a corresponding kit which are simple in operation, rapid and efficient, good in stability and strong in specificity. SUMMARY

[0005] In view of the technical problems in the background art, the present application provides a rapid detection kit and a rapid detection method for phytosterols, aiming to solve the technical problems of complex pretreatment, long detection time, poor on-site applicability, poor stability of the kit and large detection error in the existing phytosterol detection technology.

[0006] In a first aspect, the present application provides a rapid detection kit for phytosterols, comprising a phytosterol standard, a standard diluent, a substrate solution, an enzyme mixture, a termination solution and an enzyme-labeled plate. The standard diluent comprises isopropyl alcohol or dimethyl sulfoxide. The substrate solution is obtained by configuring TMB and a substrate buffer solution, and has a mass concentration of 0.1% to 0.2%. The enzyme mixture is a mixture of a cholesterol oxidase solution and a horseradish peroxidase solution. The enzyme activity concentration of cholesterol oxidase in the cholesterol oxidase solution is 1 to 25 U / mL. The enzyme activity concentration of horseradish peroxidase in the horseradish peroxidase solution is 1 to 10 U / mL.

[0007] In a second aspect, the present application provides a rapid detection method for phytosterols, comprising the following steps: S1, sample pretreatment: mixing the plant oil sample to be detected with an extraction agent, oscillating and mixing uniformly, centrifuging, removing the upper organic phase, and diluting with isopropyl alcohol to obtain a test solution; wherein the extraction agent is a mixed solution of methanol and isopropyl alcohol. S2, rapid detection using the rapid detection kit provided in the first aspect: Dilute the phytosterol standard to different concentrations with the standard diluent, add the substrate solution and the enzyme mixture to the microwells of the enzyme-labeled plate, then add the standard solution or the test solution of different concentrations to the microwells of the enzyme-labeled plate, mix uniformly, react at room temperature, finally add the termination solution, and measure the absorbance of each well at 450 / 630 nm. According to the concentration and absorbance value of the phytosterol standard solution, a standard curve is drawn, so as to determine the content of phytosterols in the test solution. Compared with the prior art, the present application has the following advantages: The kit of the present application adopts enzyme catalysis method, cooperates with effective phytosterol extraction process, and significantly improves the sensitivity and specificity of detection by optimizing the reagent components and reaction conditions in the kit. The method does not need complex pretreatment steps, is simple to operate, can complete the content test of phytosterol in a short time, and is suitable for on-site rapid screening demand. In addition, the enzyme protectant formula in the kit is carefully designed, which effectively improves the stability of the enzyme, prolongs the shelf life of the kit, and ensures the reliability and repeatability of the detection results. Compared with the traditional detection method, the present application has obvious advantages in detection efficiency, stability and applicability. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 The figure is an average value graph of the influence of each level of the orthogonal test factor of the enzyme protectant component on P / N value; Figure 2 The figure is an average value graph of the influence of each level of the orthogonal test factor of the enzyme protectant component on the absorbance of negative samples; Figure 3 The figure is the standard sample absorbance fluctuation under different reaction times; Figure 4 The figure is a schematic diagram of the components of the rapid detection kit for phytosterol; The figure is a schematic diagram of the components of the rapid detection kit for phytosterol; DETAILED DESCRIPTION

[0009] The embodiments of the technical solutions of the present application will be described in detail below with reference to the drawings. The following examples are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, but cannot limit the protection scope of the present application.

[0010] In order to solve the technical problems of complex pretreatment, long detection time, poor on-site applicability, poor stability of the kit and large detection error in the existing phytosterol detection technology, the present application provides a rapid detection kit and rapid detection method for phytosterol, wherein through enzyme catalysis method, cooperating with effective phytosterol extraction process, and optimizing the reaction system, the detection sensitivity is higher, the specificity is stronger, and the accuracy is higher.

[0011] In a first aspect, the present application provides a rapid detection kit for phytosterol, which comprises phytosterol standard, standard diluent, substrate solution, enzyme mixture, termination solution and enzyme-labeled plate; The standard diluent comprises isopropyl alcohol or dimethyl sulfoxide; The substrate solution is obtained by configuring TMB and substrate buffer, and the mass concentration is 0.1%~0.2%; The enzyme mixture is a mixture of cholesterol oxidase solution and horseradish peroxidase solution; the enzyme activity concentration of cholesterol oxidase in the cholesterol oxidase solution is 1~25 U / mL; the enzyme activity concentration of horseradish peroxidase in the horseradish peroxidase solution is 1~10 U / mL.

[0012] In the technical solution of this invention embodiment, the detection principle of this invention is as follows: when TMB solution and a mixed solution of cholesterol oxidase and horseradish peroxidase are added to the ELISA plate, and then a standard or sample solution is added, the phytosterols in the standard or sample solution generate hydrogen peroxide under the action of cholesterol oxidase. This product reacts with TMB to generate a blue product under the action of horseradish peroxidase. After termination, the absorbance value is detected. The absorbance value of the sample is positively correlated with its sterol content.

[0013] Furthermore, in some embodiments, the substrate solution includes: 0.02M disodium hydrogen phosphate-citric acid buffer at pH 5 and 0.15% TMB.

[0014] Furthermore, in some embodiments, the cholesterol oxidase solution is prepared by cholesterol oxidase and an enzyme protectant; the horseradish peroxidase solution is prepared by horseradish peroxidase and an enzyme protectant. The enzyme protectants include: 0.01-0.05M disodium hydrogen phosphate-citric acid buffer at pH 5-7, sodium chloride at a concentration of 0.5%-2%, sucrose at a concentration of 1%-3%, glycine at a concentration of 0.1%-1%, aspartic acid at a concentration of 0.1%-1%, bovine serum albumin at a concentration of 0.1%-1%, PEG20000 at a concentration of 0.01%-0.1%, and proclin950 at a volume fraction of 0.05%-0.2%.

[0015] Further, in some embodiments, the cholesterol oxidase solution comprises: cholesterol oxidase with an enzyme activity concentration of 1 U / mL, 0.02 M disodium hydrogen phosphate-citrate buffer at pH 5, sodium chloride at a mass concentration of 1%, sucrose at a mass concentration of 2%, glycine at a mass concentration of 0.5%, aspartic acid at a mass concentration of 0.5%, bovine serum albumin at a mass concentration of 0.5%, PEG 20000 at a mass concentration of 0.05%, and proclin950 at a volume fraction of 0.1%. The horseradish peroxidase solution comprises: horseradish peroxidase with an enzyme activity concentration of 1 U / mL, 0.02 M disodium hydrogen phosphate-citric acid buffer at pH 5, 1% sodium chloride, 2% sucrose, 0.5% glycine, 0.5% aspartic acid, 0.5% bovine serum albumin, 0.05% PEG 20000, and 0.1% proclin950. The enzyme mixture was prepared by mixing cholesterol oxidase solution and horseradish peroxidase solution at a volume ratio of 1:1.

[0016] In the technical solution of this invention, the enzyme mixture consists of cholesterol oxidase, horseradish peroxidase, and an enzyme protectant. The inventors further discovered that the enzyme activity concentration and the composition of the enzyme protectant have a significant impact on the stability of enzyme activity and the detection sensitivity. Through orthogonal experimental design, the optimal ratio range of each component was further determined, enabling the enzyme protectant to exhibit excellent protective effects at different temperatures. The enzyme protectant uses a 0.02M disodium hydrogen phosphate-citric acid buffer system at pH 5, placing cholesterol oxidase and horseradish peroxidase in a high-activity plateau region. 2% sucrose, 0.5% glycine, 0.5% aspartic acid, and 0.5% bovine serum albumin form a ternary protective network of sugar, amino acids, and protein, and this multi-component composite stabilizer enables long-term liquid preservation. 1% sodium chloride provides adequate ionic strength to maintain osmotic pressure. 0.05% PEG 20000 reduces surface tension, minimizing microbubbles and "wall adhesion." 0.1% proclin950 effectively inhibits microbial growth and extends the shelf life of the reagent after opening.

[0017] Furthermore, in some embodiments, the ELISA plate is an uncoated or unused high-adsorption ELISA plate.

[0018] Furthermore, in some embodiments, the stop solution is a 0.5 mol / L hydrochloric acid buffer solution.

[0019] In the technical solution of this invention embodiment, 0.5 mol / L hydrochloric acid buffer can effectively terminate the enzymatic reaction in a short time, avoiding detection errors caused by over- or incomplete reaction.

[0020] Secondly, embodiments of the present invention provide a rapid detection method for phytosterols, comprising the following steps: S1. Sample pretreatment: Take the vegetable oil sample to be tested and mix it with the extractant, shake to mix well and centrifuge, transfer the upper organic phase, and dilute it with isopropanol to obtain the test solution; wherein, the extractant is a mixed solution of methanol and isopropanol; S2. Perform rapid testing using the rapid test kit provided in the first aspect: Phytosterol standards were diluted to different concentrations with standard diluent. Substrate solution and enzyme mixture were added to the microwells of the microplate. Then, standard solutions or test solutions of different concentrations were added to the microwells of the microplate. After mixing, the reaction was allowed to stand at room temperature. Finally, stop solution was added, and the absorbance of each well was measured at dual wavelengths of 450 / 630 nm. A standard curve is plotted based on the concentration and absorbance values ​​of the phytosterol standard solution to determine the content of phytosterol in the test solution.

[0021] In the technical solution of this invention, a mixed solution of methanol and isopropanol is used as the extraction agent, which can efficiently extract phytosterol components from plant oils while reducing interference from impurities. Detection at a main wavelength of 450 nm and a secondary wavelength of 630 nm can effectively eliminate background interference and improve the accuracy of detection.

[0022] Furthermore, in some embodiments, in step S1, the mass-to-volume ratio of the vegetable oil sample to be tested to the extractant is 1:6.

[0023] Furthermore, in some embodiments, in step S1, the extractant is prepared by mixing methanol and isopropanol in a volume ratio of 1:1; or, the extractant is prepared by mixing methanol and dimethyl sulfoxide in a volume ratio of 1:1.

[0024] Furthermore, in some embodiments, in step S1, the vegetable oil sample to be tested includes vegetable edible oil or oilseed crop seed powder.

[0025] In the technical solutions of this invention, vegetable edible oils include, but are not limited to, peanut oil, rapeseed oil, and sesame oil; oilseed crops include, but are not limited to, rapeseed and peanut kernels.

[0026] Furthermore, in some embodiments, step S2 specifically includes: The phytosterol standards were diluted with standard diluent to different concentrations, specifically: 0 mg / L, 6.25 mg / L, 12.5 mg / L, 25 mg / L, 50 mg / L, and 100 mg / L. Add 50 μL of substrate solution and 100 μL of enzyme mixture to the microwells of the microplate. Then add 50 μL of standard solution or test solution of different concentrations to the microwells of the microplate respectively. After mixing, let it stand at room temperature for 15 min. Finally, add 100 μL of stop solution and measure the absorbance of each well at 450 / 630 nm using a microplate reader. A standard curve is plotted based on the concentration and absorbance values ​​of the phytosterol standard solution to determine the content of phytosterol in the test solution.

[0027] In the technical solution of this invention embodiment, the reaction time is 15 minutes, which can obtain the optimal absorbance value and also ensure the stability of absorbance.

[0028] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0029] Example 1: Optimization of the reagent formulation for the rapid phytosterol test kit 1. Optimization of standard dilution and enzyme activity concentration 1) The standard diluent is prepared by mixing isopropanol and methanol in different proportions. Preparation of the standard diluent: Standard dilution 1: Mix isopropanol and methanol at a ratio of 1:1 (v / v). Standard dilution 2: Mix isopropanol and methanol at a ratio of 4:1 (v / v). Standard diluent 3: isopropanol.

[0030] 2) Preparation of high-concentration standard: Accurately weigh 100 mg of β-sitosterol standard powder, add 5 mL of isopropanol, and dissolve by sonication to achieve a concentration of 20 mg / mL.

[0031] 3) Take the above three standard dilutions and add 20 mg / mL of β-sitosterol high-concentration standard to make the concentration 100 mg / L. Use this as a positive control (P), and the standard dilution is the negative control (N).

[0032] 4) Preparation of reagent A: Prepare a 0.02M disodium hydrogen phosphate-citric acid buffer solution with a pH of 5.0, add TMB (3,3',5,5'-tetramethylbenzidine) to make the final mass concentration (m / v) 0.15%, and store at 4°C protected from light for later use.

[0033] 5) Preparation of enzyme dilution buffer: Prepare a 0.02M disodium hydrogen phosphate-citrate buffer with a pH of 7.0, add ovalbumin to make its final mass concentration (m / v) 0.5%.

[0034] 6) Weigh cholesterol oxidase powder (1 KU / mg), dissolve it in 0.02 M disodium hydrogen phosphate-citric acid buffer at pH 6.0 to make its concentration 100 U / mL, and continue to dilute to 25 U / mL, 15 U / mL, 5 U / mL and 1 U / mL for use.

[0035] 7) Weigh horseradish peroxidase powder (1 KU / mg), dissolve it in 0.02 M disodium hydrogen phosphate-citric acid buffer at pH 6.0 to make its concentration 100 U / mL, and continue to dilute to 10 U / mL, 5 U / mL and 1 U / mL for use.

[0036] 8) Preparation of test C: Prepare a hydrochloric acid solution with a molar concentration of 0.5 mol / L and store it at 4℃ for later use.

[0037] 9) Take an appropriate amount of microplate wells and determine the absorbance using a square matrix titration test. Use a blank control absorbance value close to 0 as the reference standard. The higher the P / N value, the more sensitive the reagent and the better the effect. Add 50 μL of reagent A, 50 μL of the above-diluted horseradish peroxidase, and 50 μL of the above-diluted cholesterol oxidase to each well. Finally, add 50 μL of the above-concentration phytosterol negative control and positive control to each well. Mix well, cover with a light-proof plate, and incubate at room temperature (25℃) for 30 minutes. Add 100 μL of reagent C and measure the absorbance value using an automated microplate reader at wavelengths of 450 nm / 630 nm. The results are shown in Table 1.

[0038] Table 1 Cross-test of enzyme activity concentration and standard dilution in sterol kits

[0039] According to the data in Table 1, when the standard diluent is isopropanol, the cholesterol oxidase is diluted to an enzyme activity concentration of 1 U / mL and the horseradish peroxidase is diluted to an enzyme activity concentration of 1 U / mL using enzyme dilution buffer. The negative control has the lowest absorbance value and the highest P / N value. This condition can be selected as the current optimal group for continuing the experiment.

[0040] In addition, experiments showed that when the standard diluent was dimethyl sulfoxide, its effect was very similar to that of isopropanol, and it could be used as an alternative.

[0041] 2. Optimization of component B and standard curve 1) Preparation of enzyme protectant: Control reagent: Prepare a 0.02M disodium hydrogen phosphate-citrate buffer solution with a pH of 7.0, add ovalbumin to make its final mass concentration (m / v) 0.5%.

[0042] Orthogonal experimental design for the protective agent: Prepare 0.02M disodium hydrogen phosphate-citric acid buffer solution with pH levels of 5.0, 6.0, and 7.0, add glycine at mass concentrations (m / v) of 1%, 0.5%, and 0.1%, and add PEG 20000 at mass concentrations (m / v) of 0.1%, 0.05%, and 0.01%.

[0043] Common components of the protective agent: In the above orthogonal experimental design group, add sodium chloride to make its final mass concentration (m / v) 1%, add sucrose to make its final mass concentration (m / v) 2%, add aspartic acid to make its final mass concentration (m / v) 0.5%, add ovalbumin to make its final mass concentration (m / v) 0.5%, and add proclin950 to make its final volume fraction (v / v) 0.1%.

[0044] 2) Test B: Add cholesterol oxidase to the above 3 enzyme protectants to make the enzyme activity concentration 1U / mL, add horseradish peroxidase to make the enzyme activity concentration 1U / mL, mix the two enzyme dilutions at a volume ratio of 1:1 to obtain the test B reaction solution.

[0045] 3) Standards: The high concentration of 20 mg / mL β-sitosterol standard was diluted to 100 mg / L with isopropanol as a positive control, and isopropanol was used as a blank control (i.e., 0 mg / L).

[0046] 4) Preparation of reagent A: Prepare a 0.02M disodium hydrogen phosphate-citric acid buffer solution with a pH of 5.0, add TMB (3,3',5,5'-tetramethylbenzidine) to make the final mass concentration (m / v) 0.15%, and store at 4°C protected from light for later use.

[0047] 5) Preparation of test C: Prepare a hydrochloric acid solution with a molar concentration of 0.5 mol / L and store it at 4℃ for later use.

[0048] 6) Take an appropriate amount of microplate wells, add 50 μL of test A to each well, then add 100 μL of test B to the corresponding well, and finally add 50 μL of phytosterol negative control and positive control standards to each well. After mixing, cover with a light-proof plate and incubate at room temperature (25℃) for 30 minutes. Add 100 μL of test C and measure the absorbance value at 450 nm / 630 nm wavelength using an automated microplate reader.

[0049] Table 2. Raw data from the orthogonal experiment of enzyme protectant components

[0050] Table 3. Range analysis data of orthogonal experiment for enzyme protectant components

[0051] Figure 1 This is a plot showing the mean influence of each factor level on the P / N value in an orthogonal experiment. Figure 2 This is a graph showing the average effect of each level of the orthogonal experimental factor on the absorbance of the negative sample.

[0052] The orthogonal experiment results show that the optimal levels are pH 5.0, glycine concentration of 0.5%, and PEG 20000 concentration of 0.05%. Therefore, this concentration can be selected as the current optimal group for further experiments.

[0053] 3. Standard curve linearity level 1) Test B: First, prepare a 0.02M disodium hydrogen phosphate-citrate buffer solution with a pH of 5.0. Add sodium chloride to achieve a final concentration (m / v) of 1%, add sucrose to achieve a final concentration (m / v) of 2%, add glycine to achieve a final concentration (m / v) of 0.5%, add aspartic acid to achieve a final concentration (m / v) of 0.5%, add bovine serum albumin to achieve a final concentration (m / v) of 0.5%, add PEG 20000 to achieve a final concentration (m / v) of 0.05%, and add proclin 950 to achieve a final volume fraction (v / v) of 0.1% as an enzyme protectant. Add cholesterol oxidase to achieve an enzyme activity concentration of 1 U / mL, add horseradish peroxidase to achieve an enzyme activity concentration of 1 U / mL, and mix the two enzyme solutions at a volume ratio of 1:1 to obtain the enzyme mixture solution for Test B.

[0054] 2) Standard curve: Dilute the 20 mg / mL β-sitosterol high concentration standard to 6.25 mg / L, 12.5 mg / L, 25 mg / L, 50 mg / L and 100 mg / L with isopropanol, and use isopropanol as a blank control (i.e. 0 mg / L).

[0055] 3) Preparation of reagent A: Prepare a 0.02M disodium hydrogen phosphate-citric acid buffer solution with a pH of 5.0, add TMB (3,3',5,5'-tetramethylbenzidine) to make the final mass concentration (m / v) 0.15%, and store at 4°C protected from light for later use.

[0056] 4) Preparation of test C: Prepare a hydrochloric acid solution with a molar concentration of 0.5 mol / L and store it at 4℃ for later use.

[0057] 5) Take an appropriate amount of microplate wells, add 50 μL of test A to each well, then add 100 μL of test B as described above, and finally add 50 μL of phytosterol negative control and positive control standards to the corresponding wells. Mix well, cover with a light-proof plate, and incubate at room temperature (25℃) for 30 minutes. Add 100 μL of test C and measure the absorbance value at a wavelength of 450 nm / 630 nm using an automated microplate reader.

[0058] Table 4. Data table of phytosterol standard curve

[0059] 4. Stability of reaction reagents The optimal protective agent was determined through matrix titration experiments. Using the absorbance ratio of the negative control to the positive control (P / N value fluctuation less than 10%) as the reference standard, the above-mentioned optimal protective agent was used as the dilution buffer for the enzyme reaction solution. Cholesterol oxidase was diluted to an enzyme activity concentration of 1 U / mL, and horseradish peroxidase was diluted to an enzyme activity concentration of 1 U / mL. The two enzyme dilutions were mixed at a 1:1 (v / v) ratio to obtain test B. 50 μL of test A and 100 μL of the above test B were added to each well of the ELISA plate. Then, 50 μL of phytosterol negative and positive control standards were added to the corresponding wells. After mixing, the plate was covered with a light-proof label and incubated at room temperature (25℃) for 30 minutes. 100 μL of test C was then added, and the absorbance was measured using an automated ELISA reader at wavelengths of 450 nm / 630 nm. After determining the reagents involved in the reaction, the plate was divided into two portions: one portion was refrigerated at 4℃, and the other portion was placed in a 37℃ incubator. Accelerated comparison experiments were conducted at different intervals. The experimental data are as follows.

[0060] Table 5. Accelerated Comparison Experiments with Enzyme Protectants

[0061] The results showed that this enzyme protectant can be used to dilute and preserve reaction reagents.

[0062] 5. Optimization of reaction time 1) Take 20 mg / mL of high-concentration β-sitosterol standard and dilute it with isopropanol to a final concentration of 100 mg / L and 25 mg / L as positive controls (P) and isopropanol negative controls (N).

[0063] 2) Take 11 groups of microplate wells. In each group, add 50 μL of reagent A, then 100 μL of reagent B, followed by 50 μL of phytosterol negative and positive controls of the above concentrations. Incubate at room temperature (25℃). After the blue product is formed, add 100 μL of reagent C to the corresponding group's wells every 3 minutes to stop the reaction. Measure the absorbance at a microplate reader at a main wavelength of 450 nm and a secondary wavelength of 630 nm. The results are shown in the table below and appendix. Figure 3 .

[0064] Table 6. Absorbance changes of different concentrations of sterol standards with increasing reaction time.

[0065] Figure 3 The absorbance fluctuations of the standard were observed under different reaction times. The results showed that a reaction time of 15 min yielded the optimal absorbance value while also ensuring absorbance stability.

[0066] Example 2: Composition of the Phytosterol Rapid Detection Kit Develop a rapid detection kit for phytosterols in vegetable oils and oilseed crops, such as... Figure 4 As shown, it includes the following components: ELISA plates are uncoated or unused high-adsorption ELISA plates. Test A is the substrate solution, prepared by mixing TMB (3,3',5,5'-tetramethylbenzidine) and substrate buffer: Prepare a 0.02M disodium hydrogen phosphate-citric acid buffer solution with pH 5.0, and add TMB (3,3',5,5'-tetramethylbenzidine) to achieve a final mass concentration (m / v) of 0.15%; Test B is an enzyme mixture prepared by mixing cholesterol oxidase and horseradish peroxidase with an enzyme protectant in a specific ratio. The specific formula is as follows: Cholesterol oxidase solution: cholesterol oxidase with an enzyme activity concentration of 1 U / mL, 0.02M disodium hydrogen phosphate-citric acid buffer at pH 5, 1% sodium chloride, 2% sucrose, 0.5% glycine, 0.5% aspartic acid, 0.5% bovine serum albumin, and 0.05% PEG. 20000, 0.1% (v / v) proclin950; Horseradish peroxidase solution: horseradish peroxidase with an enzyme activity concentration of 1 U / mL, 0.02M disodium hydrogen phosphate-citric acid buffer at pH 5, 1% (w / w) sodium chloride, 2% (w / w) sucrose, 0.5% (w / w) glycine, 0.5% (w / w) aspartic acid, 0.5% (w / w) bovine serum albumin, 0.05% (w / w) PEG 20000, 0.1% (v / v) proclin950; The enzyme mixture was obtained by mixing cholesterol oxidase solution and horseradish peroxidase solution at a volume ratio of 1:1. Test C is the stop solution for 0.5 mol / L hydrochloric acid solution; The standard was a β-sitosterol gradient standard (6.25 mg / L~100 mg / L) obtained by diluting with isopropanol; Reaction procedure: Add 50 μL of test A to the sterol microplate, then add 100 μL of test B, gently tap to mix, and add 50 μL of standard and sample solution to the microwells in sequence. Let the reaction stand at room temperature for 15 min. After the blue product is formed, add 100 μL of test C and gently tap to mix. Detect the OD value at the main wavelength of 450 nm and the secondary wavelength of 630 nm using a microplate reader. Fit a standard curve according to the standard concentration and absorbance, and calculate the total amount of phytosterols in the sample.

[0067] Example 3: Rapid Detection of Phytosterol Content in Samples 1) Five samples of vegetable edible oil and five samples of oilseeds were selected and tested using rapid detection method and GC-MS method, respectively.

[0068] 2) The comparison of results between the two methods must be based on the GC-MS results to evaluate the rapid detection method.

[0069] 3) Pretreatment method for total sterols in edible oil samples: Take the vegetable oil sample to be tested and shake it thoroughly to ensure sample homogeneity. Treat the sample according to the sample:extractant ratio of 1:6 (m / v), vortex on a shaker for 1 min, and centrifuge at 4000 r / min for 5 min. Transfer the upper organic phase and dilute it with isopropanol to the standard curve range. Record the total dilution factor for later use. The sample extractant is prepared by mixing methanol and isopropanol at a volume ratio of 1:1 and can be stored at room temperature in a sealed container.

[0070] This pretreatment method for total sterols in edible oils is applicable to the determination of total sterols in edible oils such as peanut oil, rapeseed oil, and sesame oil.

[0071] 4) Pretreatment method for total sterols in oilseed crop seed samples: Select fresh, mold-free oilseed crop seed samples (such as rapeseed, peanut kernels, etc.), remove impurities, and pulverize the seeds to 80 mesh using a high-speed grinder (avoid sample heating during pulverization; pulverization can be intermittent to prevent excessive temperature from affecting sterol stability). Treat the samples according to a sample:extractant ratio of 1:6 (m / v), vortex on a shaker for 1 min, and centrifuge at 4000 r / min for 5 min. Transfer the upper organic phase, dilute with isopropanol to the standard curve range, and record the total dilution factor for later use. The sample extractant is prepared by mixing methanol and isopropanol at a volume ratio of 1:1 and can be stored at room temperature in a sealed container.

[0072] This pretreatment method for total sterols in oilseed crops is applicable to the determination of samples such as rapeseed and peanuts.

[0073] 5) Take the appropriate number of microwells, add 50 μL of test A to each well, then add 100 μL of test B, gently tap to mix, and sequentially add 50 μL of standard and each sample solution to the microwells. Incubate at room temperature (25℃) for 15 min. After a blue product is formed, add 100 μL of test C, gently tap to mix, and detect the absorbance value at a microplate reader with a main wavelength of 450 nm and a secondary wavelength of 630 nm. At the same time, establish a standard working curve with the standard concentration as the x-axis and the corresponding absorbance as the y-axis. Substitute the sample absorbance into the working curve to calculate the result, and then multiply it by the total dilution factor during sample pretreatment to obtain the sterol content in the sample.

[0074] Table 7 Comparison of results from the phytosterol rapid detection kit with GC-MS results

[0075] The results obtained by the method provided by this invention are compared with those obtained by GC-MS, and the relative error is about ±10%. The rapid detection method is accurate, fast and simple, and can achieve relatively faster results. It can also perform simultaneous detection on a large scale, and is suitable for the screening and quality control needs of processing and manufacturing enterprises.

[0076] Example 4: Reagent Kit Quality Testing 1. Inter-batch difference experiment Edible oil samples with a phytosterol content of 2925 mg / kg were tested using three different batches of reagent kits. Each batch of the sample was tested five times, and the coefficient of variation was calculated. The results are shown in Table 8.

[0077] Table 8. Inter-batch variation of the phytosterol rapid test kit

[0078] The results showed that the coefficient of variation of the edible oil samples was all within 15%, which met the reference evaluation criteria of the kit.

[0079] 2. Sample repeatability experiment Three edible oil samples with phytosterol contents of 1269 mg / kg, 2925 mg / kg, and 6256 mg / kg were taken and tested according to the method of the kit of the present invention. Four parallel tests were performed for each sample, and the recovery rate and coefficient of variation were calculated. The results are shown in Table 9.

[0080] Table 9. Sample repeatability of the phytosterol rapid test kit

[0081] The results showed that the accuracy of the edible oil samples was 85%–113%, and the coefficient of variation was within 15%, which met the reference evaluation criteria of the kit.

[0082] 3. Reagent kit preservation experiment The kit was stored at 2–8°C. After 12 months of testing, the minimum absorbance (zero standard), maximum P / N ratio, and phytosterol recovery were all within the normal range. Considering that abnormal storage conditions may occur during the transport and use of the kit, the kit was placed at 37°C for 10 days for accelerated aging testing.

[0083] Table 10 Stability of the Phytosterol Rapid Detection Kit

[0084] The results show that all indicators of the kit fully meet the requirements. These results also indicate that the kit of this invention can be stored at 2-8℃ for more than 12 months.

[0085] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A rapid detection kit for phytosterols, characterized in that, Includes phytosterol standards, standard diluents, substrate solutions, enzyme mixtures, stop solutions, and ELISA plates; The standard diluent includes isopropanol or dimethyl sulfoxide; The substrate solution was prepared by mixing TMB and substrate buffer solution; The enzyme mixture is a mixture of cholesterol oxidase solution and horseradish peroxidase solution; the enzyme activity concentration of cholesterol oxidase in the cholesterol oxidase solution is 1~25 U / mL; the enzyme activity concentration of horseradish peroxidase in the horseradish peroxidase solution is 1~10 U / mL.

2. The rapid detection kit for phytosterols according to claim 1, characterized in that, The substrate solution comprises: 0.02M disodium hydrogen phosphate-citric acid buffer solution with a pH of 5 and 0.15% TMB.

3. The rapid detection kit for phytosterols according to claim 1, characterized in that, The cholesterol oxidase solution is prepared by cholesterol oxidase and an enzyme protectant; the horseradish peroxidase solution is prepared by horseradish peroxidase and an enzyme protectant. The enzyme protectant comprises: 0.01-0.05M disodium hydrogen phosphate-citric acid buffer with a pH of 5-7, 0.5%-2% sodium chloride, 1%-3% sucrose, 0.1%-1% glycine, 0.1%-1% aspartic acid, 0.1%-1% bovine serum albumin, 0.01%-0.1% PEG20000, and 0.05%-0.2% proclin950.

4. The rapid detection kit for phytosterols according to claim 3, characterized in that, The cholesterol oxidase solution comprises: cholesterol oxidase with an enzyme activity concentration of 1 U / mL, 0.02 M disodium hydrogen phosphate-citrate buffer with a pH of 5, sodium chloride with a mass concentration of 1%, sucrose with a mass concentration of 2%, glycine with a mass concentration of 0.5%, aspartic acid with a mass concentration of 0.5%, bovine serum albumin with a mass concentration of 0.5%, PEG 20000 with a mass concentration of 0.05%, and proclin950 with a volume fraction of 0.1%. The horseradish peroxidase solution comprises: horseradish peroxidase with an enzyme activity concentration of 1 U / mL, 0.02 M disodium hydrogen phosphate-citric acid buffer at pH 5, sodium chloride at a mass concentration of 1%, sucrose at a mass concentration of 2%, glycine at a mass concentration of 0.5%, aspartic acid at a mass concentration of 0.5%, bovine serum albumin at a mass concentration of 0.5%, PEG 20000 at a mass concentration of 0.05%, and proclin950 at a volume fraction of 0.1%. The enzyme mixture is obtained by mixing the cholesterol oxidase solution and the horseradish peroxidase solution at a volume ratio of 1:

1.

5. The rapid detection kit for phytosterols according to claim 1, characterized in that, The enzyme-labeled plate is an uncoated or unused high-adsorption enzyme-labeled plate.

6. The rapid detection kit for phytosterols according to claim 1, characterized in that, The termination solution is a 0.5 mol / L hydrochloric acid buffer solution.

7. A rapid detection method for phytosterols, characterized in that, Includes the following steps: S1. Sample pretreatment: Take the vegetable oil sample to be tested, mix it with the extractant, shake to mix, centrifuge, transfer the upper organic phase, and dilute with isopropanol to obtain the test solution; wherein, the extractant is a mixed solution of methanol and isopropanol; S2. Rapid detection using the rapid detection kit for phytosterols according to any one of claims 1 to 6: Phytosterol standards were diluted to different concentrations with standard diluent. Substrate solution and enzyme mixture were added to the microwells of the ELISA plate. Then, standard solutions of different concentrations or the test solution were added to the microwells of the ELISA plate. After mixing, the reaction was allowed to stand at room temperature. Finally, stop solution was added, and the absorbance of each well was measured at dual wavelengths of 450 / 630 nm. A standard curve was plotted based on the concentration and absorbance values ​​of the phytosterol standard solution to determine the content of phytosterol in the test solution.

8. The rapid detection method for phytosterols according to claim 7, characterized in that, In step S1, the mass-to-volume ratio of the vegetable oil sample to be tested to the extractant is 1:6; The extractant was prepared by mixing methanol and isopropanol in a volume ratio of 1:

1. Alternatively, the extractant is prepared by mixing methanol and dimethyl sulfoxide in a volume ratio of 1:

1.

9. The rapid detection method for phytosterols according to claim 7, characterized in that, In step S1, the vegetable oil sample to be tested includes vegetable edible oil or oilseed crop seed powder.

10. The rapid detection method for phytosterols according to claim 7, characterized in that, Step S2 specifically involves: The phytosterol standards were diluted with standard diluent to different concentrations, specifically: 0 mg / L, 6.25 mg / L, 12.5 mg / L, 25 mg / L, 50 mg / L, and 100 mg / L. Add 50 μL of substrate solution and 100 μL of enzyme mixture to the microwells of the microplate. Then add 50 μL of standard solution or test solution of different concentrations to the microwells of the microplate respectively. After mixing, let it stand at room temperature for 15 min. Finally, add 100 μL of stop solution and measure the absorbance of each well at 450 / 630 nm using a microplate reader. A standard curve was plotted based on the concentration and absorbance values ​​of the phytosterol standard solution to determine the content of phytosterol in the test solution.

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