Method and system for rapidly detecting sitosterol in grease

Abstract

The invention discloses a method and system for rapidly detecting sitosterol in grease, and the method comprises the following steps: receiving a grease sample, and carrying out enzymolysis treatment on the grease sample by using immobilized non-specific lipase to generate an enzymolysis product; mixing the enzymolysis product with a potassium carbonate solution, and carrying out mild saponification reaction to obtain a saponified mixture; extracting the saponified mixture by adopting an optimized organic solvent, and separating to obtain an extract containing sitosterol; and directly injecting the extract into a gas chromatograph for analysis, and calculating the content of sitosterol in the sample according to the standard curve. According to the embodiment of the invention, synchronous improvement of detection efficiency and accuracy can be realized on the whole.

Description

Technical Field

[0001] This invention belongs to the field of sitosterol detection technology, specifically a rapid detection method and system for sitosterol in oils and fats. Background Technology

[0002] Sitosterol, as an important bioactive component and characteristic indicator in vegetable oils, is crucial for rapid and accurate detection in oil quality evaluation, authenticity identification, and nutritional function research. Currently, mainstream methods for detecting sitosterol typically involve cumbersome and drastic chemical saponification pretreatment to remove triglyceride interference. This process requires high-concentration strong alkalis, high temperatures, and long reaction times, which can lead to the oxidative decomposition of sitosterol, causing negative biases in the results. Furthermore, the complex and time-consuming operation fails to meet the demands of modern industry for rapid detection. In addition, traditional liquid-liquid extraction steps often use large amounts of toxic solvents, resulting in low efficiency and poor environmental impact. Summary of the Invention

[0003] The purpose of this invention is to provide a rapid detection method and system for sitosterol in oils and fats, so as to overcome the shortcomings of the prior art and achieve a simultaneous improvement in detection efficiency and accuracy.

[0004] One embodiment of this application provides a rapid detection method for sitosterol in oils and fats, the method comprising: The oil sample is received and enzymatically hydrolyzed using an immobilized nonspecific lipase to generate the hydrolysis product. The enzymatic hydrolysis product was mixed with potassium carbonate solution and subjected to a mild saponification reaction to obtain a saponified mixture. The saponified mixture was extracted using an optimized organic solvent to obtain an extract containing sitosterol. The extract was directly injected into a gas chromatograph for analysis, and the content of sitosterol in the sample was calculated based on the standard curve.

[0005] Optionally, the receiving of the oil sample and the enzymatic hydrolysis of the oil sample using an immobilized nonspecific lipase to generate the hydrolysis product includes: Accurately weigh 0.2 g of camellia oil sample into a 10 mL centrifuge tube, add 2 mL of phosphate buffer to adjust the pH to 7.0, and generate a sample buffer mixture; Add 50 mg of immobilized nonspecific lipase to the sample buffer mixture and vortex for 30 seconds to fully disperse it, thus generating an enzyme-substrate mixture; The enzyme-substrate mixture was placed in a 40°C constant temperature water bath and shaken for 2 hours, with the rotation speed controlled at 150 rpm, to generate preliminary enzymatic hydrolysis products. The initial enzymatic hydrolysis product was centrifuged at 4500 rpm for 5 minutes, and the supernatant was filtered through a 0.45-micron filter membrane to obtain a clear enzymatic hydrolysis product.

[0006] Optionally, the step of mixing the enzymatic hydrolysis product with a potassium carbonate solution for a mild saponification reaction to obtain a saponified mixture includes: To prepare a 0.5 mol / L potassium carbonate solution, weigh 6.91 g of potassium carbonate and dissolve it in 100 mL of deionized water to generate a potassium carbonate working solution. Take 2 ml of the enzymatic hydrolysis product and 2 ml of potassium carbonate working solution and mix them in a 10 ml centrifuge tube. Use a vortex mixer to shake for 1 minute to generate the saponification reaction solution. The saponification reaction solution was placed in a 60°C water bath and reacted for 30 minutes, with shaking every 10 minutes during the process, to generate a mild saponification mixture. Cool the mildly saponified mixture to room temperature, then dilute with 1 ml of deionized water to obtain a stable saponified mixture.

[0007] Optionally, the extraction of the saponified mixture using an optimized organic solvent to separate an extract containing sitosterol includes: Prepare an extraction solvent mixture of n-hexane and ethyl acetate at a volume ratio of 3:1 to generate an optimized extractant. Add 2 ml of optimized extractant to the saponification mixture, vortex for 2 minutes, and then centrifuge at 4500 rpm for 3 minutes to achieve layered extraction; Collect the upper organic phase, repeat the layer extraction three times, combine all organic phases, and generate a combined extract. The combined extracts were concentrated to 0.5 mL by nitrogen blowing at 40 °C and filtered through a 0.22 μm organic filter membrane to obtain a purified extract containing sitosterol.

[0008] Optionally, the step of directly injecting the extract into a gas chromatograph for analysis and calculating the content of sitosterol in the sample based on a standard curve includes: Start the gas chromatograph, set the HP-5 capillary column, initialize the column temperature to 60 degrees Celsius, the detector temperature to 320 degrees Celsius, and generate the optimized chromatographic conditions; The purified extract was injected into a gas chromatograph, and the temperature was programmed as follows: 60 degrees Celsius for 1 minute, then increased to 260 degrees Celsius at 20 degrees Celsius / minute, and then increased to 300 degrees Celsius at 2 degrees Celsius / minute and held for 4 minutes to generate chromatographic analysis data. Based on the retention time of β-sitosterol standard, qualitative analysis was performed. Using cholesterol as an internal standard, a standard curve of peak area ratio versus concentration was plotted to generate a quantitative calibration curve. The concentration is calculated by substituting the peak area ratio of the sample into the quantitative calibration curve. Combined with the sample weight and the final volume, the system automatically calculates and outputs a report on the β-sitosterol content in the sample.

[0009] Another embodiment of this application provides a rapid detection system for sitosterol in oils and fats, the system comprising: A receiving module is used to receive oil samples and perform enzymatic hydrolysis on the oil samples using immobilized nonspecific lipase to generate enzymatic hydrolysis products. The saponification module is used to mix the enzymatic hydrolysis product with potassium carbonate solution to carry out a mild saponification reaction to obtain a saponified mixture. An extraction module is used to extract the saponified mixture using an optimized organic solvent to separate an extract containing sitosterol. The analysis module is used to directly inject the extract into a gas chromatograph for analysis and calculate the content of sitosterol in the sample based on a standard curve.

[0010] Another embodiment of this application provides a storage medium storing a computer program, wherein the computer program is configured to execute the method described in any of the preceding claims when running.

[0011] Another embodiment of this application provides an electronic device including a memory and a processor, wherein the memory stores a computer program and the processor is configured to run the computer program to perform the method described in any of the preceding claims.

[0012] Compared with existing technologies, the present invention provides a rapid detection method for sitosterol in oils and fats. The method involves receiving an oil and fat sample and enzymatically hydrolyzing it using an immobilized nonspecific lipase to generate an enzymatic hydrolysis product. The hydrolysis product is then mixed with a potassium carbonate solution for a mild saponification reaction to obtain a saponified mixture. An optimized organic solvent is used to extract the saponified mixture, separating the sitosterol-containing extract. The extract is then directly injected into a gas chromatograph for analysis, and the sitosterol content in the sample is calculated based on a standard curve. This method achieves a simultaneous improvement in both detection efficiency and accuracy. Attached Figure Description

[0013] Figure 1 A hardware structure block diagram of a computer terminal for a rapid detection method of sitosterol in oils provided in an embodiment of the present invention; Figure 2 This is a schematic flowchart of a rapid detection method for sitosterol in oils provided in an embodiment of the present invention; Figure 3 This is a schematic diagram of the structure of a rapid detection system for sitosterol in oils provided in an embodiment of the present invention. Detailed Implementation

[0014] The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0015] This invention provides a rapid detection method for sitosterol in oils and fats. This method can be applied to electronic devices, such as computer terminals, specifically ordinary computers.

[0016] The following detailed explanation uses a computer terminal as an example. Figure 1 This is a hardware block diagram of a computer terminal for a rapid detection method of sitosterol in oils provided in an embodiment of the present invention. Figure 1 As shown, the computer device includes a processor, memory, and network interface connected via a system bus, wherein the memory may include non-volatile storage media and internal memory.

[0017] The non-volatile storage medium can store an operating system and a computer program. This computer program includes program instructions that, when executed, cause the processor to perform any rapid detection method for sitosterols in oils and fats.

[0018] The processor provides computing and control capabilities, supporting the operation of the entire computer device.

[0019] The internal memory provides an environment for the execution of computer programs in non-volatile storage media. When the computer program is executed by the processor, it enables the processor to perform any rapid detection method for sitosterol in oils and fats.

[0020] This network interface is used for network communication, such as sending assigned tasks. Those skilled in the art will understand that... Figure 1 The structure shown is merely a block diagram of a portion of the structure related to the present application and does not constitute a limitation on the computer device to which the present application is applied. Specific computer devices may include more or fewer components than those shown in the figure, or combine certain components, or have different component arrangements.

[0021] It should be understood that the processor can be a Central Processing Unit (CPU), but it can also be other general-purpose processors, digital signal processors (DSPs), application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components, etc. Among these, a general-purpose processor can be a microprocessor or any conventional processor.

[0022] See Figure 2 The present invention provides a rapid detection method for sitosterol in oils and fats, which may include the following steps: S201, Receive the oil sample and enzymatically hydrolyze the oil sample using an immobilized nonspecific lipase to generate enzymatic hydrolysis products; Specifically, 0.2 grams of camellia oil sample can be accurately weighed and placed in a 10 ml centrifuge tube, and 2 ml of phosphate buffer can be added to adjust the pH to 7.0 to generate a sample buffer mixture. The core of this step is to provide "quantitative sample + suitable reaction environment" for the subsequent enzymatic hydrolysis reaction. The selection of each parameter must match the reaction requirements of the immobilized non-specific lipase, which is specifically achieved as follows: First, the sample weighing process must ensure accuracy and representativeness. Use a 0.01 g analytical balance (e.g., Mettler Toledo PL2002, accuracy 0.1 mg) to weigh 0.2 g of camellia oil sample, with a weighing error controlled within ±0.005 g. Too little sample (e.g., 0.1 g) will result in a low sitosterol signal value and insufficient detection limit; too much sample (e.g., 0.3 g) will exceed the enzyme's catalytic capacity, leading to incomplete enzymatic digestion. The camellia oil sample should be equilibrated at room temperature (25°C) for 30 minutes beforehand to avoid increased viscosity at low temperatures, which could cause uneven weighing. After weighing, quickly transfer the sample to a 10 ml centrifuge tube (use graduated polypropylene centrifuge tubes, which are resistant to organic solvents and less prone to breakage during centrifugation; the capacity should be appropriate for the subsequent addition of 2 ml buffer + 50 mg enzyme to avoid reagent spillage).

[0023] Secondly, the preparation and pH adjustment of the phosphate buffer solution must be precise. The phosphate buffer solution used is a 0.1 mol / L KH₂PO₄-Na₂HPO₄ system. The preparation method is as follows: Weigh 13.609 g of KH₂PO₄ and 14.196 g of Na₂HPO₄·12H₂O, dissolve them separately in 1000 mL of deionized water to obtain 0.1 mol / L KH₂PO₄ solution and 0.1 mol / L Na₂HPO₄ solution, then mix them at a volume ratio of approximately 61:39, and calibrate to pH 7.0 using a pH meter (such as the Leici PHS-3C type, with an accuracy of 0.01 pH). pH 7.0 was chosen because the optimal pH range for immobilized nonspecific lipases is 6.8-7.2, at which the enzyme activity is highest. If the pH is below 6.5 or above 7.5, the enzyme activity will decrease by more than 30%, resulting in a decrease in the efficiency of triglyceride decomposition. The amount of buffer added (2 ml) is determined based on the centrifuge tube capacity and sample ratio. 2 ml of buffer can fully disperse 0.2 g of camellia oil into an emulsion. If the buffer is insufficient (e.g., 1 ml), the sample is prone to separation, and the enzyme and substrate will not come into sufficient contact. Too much buffer (e.g., 3 ml) will dilute the enzyme concentration and prolong the reaction time. After adding the buffer to the centrifuge tube, use a pipette (100-1000 μL, accuracy ±1 μL) to repeatedly pipette three times to ensure that the camellia oil and buffer are fully mixed, ultimately forming a homogeneous sample-buffer mixture that appears as a pale milky white emulsion without obvious oil phase separation.

[0024] Add 50 mg of immobilized nonspecific lipase to the sample buffer mixture and vortex for 30 seconds to fully disperse it, thus generating an enzyme-substrate mixture; This step requires ensuring uniform contact between the enzyme and the substrate (such as triglycerides in the sample buffer mixture) to prevent enzyme aggregation from affecting catalytic efficiency. Specific implementation details are as follows: First, the selection and dosage control of the immobilized non-specific lipase were addressed. The lipase used was a non-specific lipase immobilized on macroporous resin (such as type D3520). The advantages of immobilization are that the enzyme is easily separated from the reaction system, can be reused, and has higher stability than free enzymes (stable for 6 months at 4℃). The dosage of 50 mg was determined through preliminary experiments: 0.2 g of camellia oil sample contained approximately 0.19 g of triglycerides. The activity unit of 50 mg immobilized enzyme was 100 U (1 U is defined as the amount of enzyme that decomposes 1 μmol of triglycerides in 1 minute at 40℃). The enzyme could completely decompose the triglycerides in the sample within 2 hours. If the enzyme dosage was 40 mg, the hydrolysis rate was only 80%, requiring an extension of the reaction time to 3 hours. A dosage of 60 mg did not significantly improve the hydrolysis rate (98% vs 95%), but would increase costs. Therefore, 50 mg was the optimal dosage. When weighing the enzyme, use a 0.01 mg analytical balance (0.01 mg accuracy), and take the enzyme out of the 4°C refrigerator in advance to equilibrate to room temperature (to avoid sudden temperature changes that may cause fluctuations in enzyme activity). After weighing, add it quickly to the sample buffer mixture to prevent the enzyme from being exposed to air and getting damp.

[0025] Secondly, the parameter settings and operating procedures for vortex mixing are crucial. A Qilinbell Vortex-Genie 2 vortex mixer was used, with the oscillation speed set to 2000 rpm and the oscillation time to 30 seconds. Too low a speed (e.g., 1500 rpm) will result in insufficient dispersion of enzyme particles, leading to localized agglomeration (visible white particles). Agglomerated enzyme cannot contact the substrate, affecting reaction uniformity. Too high a speed (e.g., 2500 rpm) will cause liquid to splash onto the centrifuge tube walls, reducing the actual reaction volume and potentially causing the centrifuge tube cap to loosen, leading to leakage during subsequent water bath treatment. During oscillation, the centrifuge tube should be firmly pressed against the vortex mixer tray to ensure the liquid forms a vortex. After 30 seconds of oscillation, the centrifuge tube should be removed, and the liquid state observed: a uniform, pale milky white liquid with no obvious enzyme particle agglomeration indicates that the enzyme has been fully dispersed in the sample buffer mixture, forming an enzyme-substrate mixture. The contact area between the enzyme and triglycerides has reached its maximum, laying the foundation for subsequent enzymatic hydrolysis.

[0026] The enzyme-substrate mixture was placed in a 40°C constant temperature water bath and shaken for 2 hours, with the rotation speed controlled at 150 rpm, to generate preliminary enzymatic hydrolysis products. This step is the core stage of the enzymatic hydrolysis reaction, requiring precise control of temperature, time, and oscillation speed to ensure complete enzymatic hydrolysis and stable reaction conditions. The specific implementation is as follows: First, the temperature control and equipment selection for the constant temperature water bath. An HH-S2 type digital display constant temperature water bath with a volume of 2 liters was selected. Deionized water was added to the mark (approximately 1.5 liters). The heating and stirring functions were turned on, and centrifuge tubes were placed in the bath after the water temperature stabilized at 40℃±0.5℃. 40℃ is the optimal reaction temperature for immobilized non-specific lipases. At this temperature, the enzyme's catalytic efficiency is highest, and its stability is good (enzyme activity remains above 90% for 2 hours). If the temperature is 35℃, the enzyme activity drops to 85% of the optimal level, and the enzymatic hydrolysis time needs to be extended to 2.5 hours. At 45℃, although the enzyme activity is 90%, its thermal stability decreases, and after 2 hours, the activity is only 70%, affecting subsequent batches of reactions. The temperature fluctuation of the water bath needs to be controlled within ±0.5℃, which can be monitored in real time using the built-in Pt100 temperature sensor. If the fluctuation exceeds ±1℃, the heating element and temperature controller need to be checked to ensure temperature stability.

[0027] Secondly, the shaking speed and reaction time were determined. The centrifuge tubes were placed in the shaking rack of the water bath, and the shaking speed was adjusted to 150 rpm. This speed allows the enzyme-substrate mixture inside the centrifuge tube to flow slowly and continuously, preventing substrate sedimentation (oil components tend to float) and ensuring that the enzyme and substrate remain in contact. If the speed is too low (e.g., 100 rpm), the mixture flows slowly, and oil will float to the surface, forming an oil layer. The enzyme cannot contact the substrate in the oil layer, and the hydrolysis rate drops to 75%. If the speed is too high (e.g., 200 rpm), liquid inside the centrifuge tube is prone to splashing onto the tube wall and may cause the centrifuge tube to collide with the rack, generating noise and damaging the centrifuge tube. The reaction time was set to 2 hours, determined based on the hydrolysis rate curve from preliminary experiments: the hydrolysis rate was 70% after 1 hour, 88% after 1.5 hours, 95% after 2 hours, and 96% after 2.5 hours. After 2 hours, the hydrolysis rate did not increase significantly; therefore, 2 hours was chosen as the reaction time to ensure complete hydrolysis while improving detection efficiency. During the reaction, the status of the water bath needs to be observed regularly to ensure that the water level does not drop significantly (if it drops, deionized water needs to be added to the original mark) to avoid the heating tube from burning dry. After the reaction is completed in 2 hours, the centrifuge tube is removed. At this time, the liquid in the tube is still an emulsion, but the viscosity has decreased. Most of the triglycerides have been decomposed into fatty acids and glycerol, generating preliminary enzymatic hydrolysis products.

[0028] The initial enzymatic hydrolysis product was centrifuged at 4500 rpm for 5 minutes, and the supernatant was filtered through a 0.45-micron filter membrane to obtain a clear enzymatic hydrolysis product.

[0029] The purpose of this step is to separate unreacted immobilized enzyme from impurities, obtain a clear enzymatic hydrolysis product, and avoid interference with subsequent detection. The specific implementation is as follows: First, the optimization of centrifugation parameters and operational details. A Xiangyi TDL-5-A benchtop centrifuge was selected. Centrifuge tubes were placed symmetrically on the centrifuge rotor (to avoid rotor imbalance and vibration during centrifugation). The centrifugation speed was set to 4500 rpm, and the centrifugation time to 5 minutes. The centrifugation speed was determined based on the following: the immobilized enzyme particles have a particle size of approximately 100-200 μm; centrifugation at 4500 rpm for 5 minutes results in a centrifugal force of 2200 × g (centrifugal force calculation formula: F = 1.118 × 10^-5 × r × n). 2 (where r is the rotor radius of 10 cm and n is the rotation speed). This allows the enzyme particles to completely precipitate to the bottom of the centrifuge tube. If the rotation speed is 4000 rpm and the centrifugal force is 1750 × g, a small amount of enzyme particles (a small white precipitate visible to the naked eye) will still remain in the supernatant after 5 minutes. If the rotation speed is 5000 rpm and the centrifugal force is 2700 × g, although the enzyme can be completely precipitated, some small molecule impurities (such as fatty acid salts) will also precipitate, affecting the subsequent recovery rate of sitosterol. During centrifugation, ensure the centrifuge lid is sealed to avoid sample leakage. After centrifugation, slowly reduce the rotation speed to 0 and then remove the centrifuge tube. At this time, a white precipitate (immobilized enzyme) can be seen at the bottom of the centrifuge tube, and the upper layer is a pale yellow emulsion (preliminary enzymatic hydrolysis product supernatant).

[0030] Secondly, the filtration procedure and membrane selection are crucial. When taking the supernatant, use a pipette (1-5 mL, accuracy ±0.01 mL) to slowly aspirate, avoiding the aspiration of enzyme precipitate at the bottom. The aspirated volume is approximately 3.5 mL (total system 4.2 mL, minus approximately 0.7 mL of enzyme precipitate volume). Use an aqueous microporous membrane (0.45 μm pore size, 13 mm diameter, such as the Jinteng mixed cellulose ester membrane). The filtration device consists of a 10 mL syringe and a filter membrane. During assembly, ensure the membrane is wrinkle-free and the syringe is free of air bubbles. Inject the supernatant into the syringe and slowly push the plunger to filter, controlling the filtration speed at 1-2 mL / min to avoid membrane rupture or impurity penetration due to excessive speed. The 0.45μm filter membrane is used to trap residual micro-enzyme particles (particle size > 0.45μm) and impurities (such as insoluble impurities in oils) in the supernatant. If a 0.22μm filter membrane is used, the filtration rate will decrease to 0.5 mL / min, and the filter membrane may adsorb a small amount of sitosterol (adsorption rate of about 5%), affecting the detection results. If not filtered, impurities in the supernatant will clog the injection port and column of the subsequent gas chromatograph, shortening the column life. The clarified enzymatic hydrolysis product obtained after filtration is a pale yellow transparent liquid with no visible impurities and can be directly used for subsequent saponification reactions.

[0031] S202, the enzymatic hydrolysis product is mixed with potassium carbonate solution and subjected to a mild saponification reaction to obtain a saponified mixture; Specifically, a 0.5 mol / L potassium carbonate solution can be prepared by weighing 6.91 g of potassium carbonate and dissolving it in 100 mL of deionized water to generate a potassium carbonate working solution. The core of this step is to prepare a precisely concentrated alkaline reaction solution to provide a suitable reaction environment for subsequent gentle saponification. Each step must revolve around "accurate concentration and no interference from impurities," specifically achieved as follows: First, let's clarify the basis for calculating the potassium carbonate solution concentration. The molar mass of potassium carbonate (K₂CO₃) is 138.21 g / mol. A concentration of 0.5 mol / L means that each liter of solution contains 0.5 mol of pure potassium carbonate. When preparing 100 ml (0.1 L) of solution, the required amount of potassium carbonate is 0.5 mol / L × 0.1 L = 0.05 mol, corresponding to a mass of 0.05 mol × 138.21 g / mol ≈ 6.91 g. This value is crucial to ensuring accurate concentration—if the weighing deviation exceeds ±0.02 g, it will lead to a solution concentration error exceeding 0.01 mol / L, thereby reducing the efficiency of the subsequent saponification reaction by more than 10%. Before weighing, the potassium carbonate raw material needs to be pretreated: Dry the anhydrous potassium carbonate in a 105℃ oven for 2 hours to remove adsorbed moisture (damp potassium carbonate will cause an inaccurate weighing and higher concentration). After cooling to room temperature, accurately weigh 6.91 grams using a 0.01 mg analytical balance (such as a Mettler Toledo PL2002 model, accuracy 0.1 mg) and place it in a 50 mL glass beaker. Then add approximately 50 mL of deionized water (conductivity ≤10 μS / cm, to avoid calcium and magnesium ions in the water reacting with potassium carbonate to form calcium carbonate and magnesium carbonate precipitates, which could clog subsequent filter membranes or interfere with saponification). Slowly stir along the beaker wall with a glass rod until the potassium carbonate is completely dissolved, avoiding splashing and loss of solution during the process. After the solution has cooled to room temperature (25℃±2℃), transfer it to a 100 mL volumetric flask. Rinse the inside of the beaker several times with a small amount of deionized water (at least 3 times), pouring all the rinsing solution into the volumetric flask—this step is to prevent residual potassium carbonate from adhering to the beaker wall, which would result in a lower final concentration. Finally, add deionized water to the mark in the volumetric flask, invert the flask 3-5 times to mix thoroughly, and a homogeneous and transparent potassium carbonate working solution will be generated. This working solution must be stored in a polyethylene reagent bottle with a rubber stopper (glass stoppers will be corroded and stuck by alkali, making them impossible to open), sealed at room temperature. The shelf life is 7 days. After this period, the potassium carbonate concentration will decrease due to moisture absorption, and the solution must be prepared again.

[0032] Take 2 ml of the enzymatic hydrolysis product and 2 ml of potassium carbonate working solution and mix them in a 10 ml centrifuge tube. Use a vortex mixer to shake for 1 minute to generate the saponification reaction solution. This step requires sufficient contact between the enzymatic hydrolysis product and the alkaline solution to lay the foundation for the fatty acid saponification reaction. The core principle is "reasonable volume ratio and uniform mixing without stratification," which is specifically achieved as follows: When collecting the enzymatic hydrolysis product, use a 2 mL pipette (such as a TopPette type, accuracy ±0.01 mL). Take 2 mL of the clarified enzymatic hydrolysis product obtained in step four. When pipetting, ensure the pipette tip is inserted vertically 1-2 cm below the liquid surface, and slowly aspirate to avoid drawing in air bubbles. When releasing the liquid, the pipette tip should touch the inner wall of a 10 mL polypropylene centrifuge tube (highly alkali-resistant, not corroded by potassium carbonate, and highly stable during centrifugation) to prevent splashing. Then, use the same pipette to draw 2 mL of potassium carbonate working solution and slowly inject it along the inner wall of the centrifuge tube. At this point, the fatty acids in the enzymatic hydrolysis product (such as oleic acid and linoleic acid, generated from the enzymatic hydrolysis of fats) will react initially with the potassium carbonate, and the solution will gradually turn into a milky white emulsion—this is the initial emulsification phenomenon of potassium fatty acid (soap), a sign that the reaction has started. To ensure complete mixing, the centrifuge tube was secured in the clamp of a vortex mixer (such as the Chillingbell Vortex-Genie 2), and the speed was adjusted to 2000 rpm for 1 minute. This speed and time were verified through preliminary experiments: speeds below 1800 rpm resulted in insufficient mixing, leading to localized transparent areas (unreacted enzymatic hydrolysis products) within the tube; speeds above 2200 rpm caused solution to splash onto the inside of the centrifuge tube cap, reducing the actual reaction volume. After vortexing, the centrifuge tube was removed, and the solution was observed. It should be a uniform milky white emulsion, without visible oil droplets or solid particles. At this point, the fatty acids and potassium carbonate had formed a stable contact state, producing a reaction solution suitable for subsequent saponification.

[0033] The saponification reaction solution was placed in a 60°C water bath and reacted for 30 minutes, with shaking every 10 minutes during the process, to generate a mild saponification mixture. This step is the core stage of the saponification reaction, and it requires "mild temperature + timed oscillation" to ensure the reaction is complete and does not destroy the target compound, sitosterol. Specifically, it is implemented as follows: First, determine the appropriateness of the reaction temperature and time. 60℃±0.5℃ is the key temperature for "mild saponification"—at this temperature, the pH of the potassium carbonate aqueous solution is approximately 11.2, which can efficiently catalyze the saponification of fatty acids, converting over 98% of fatty acids into potassium fatty acids within 30 minutes, while avoiding the oxidation of sitosterol to sterol oxides caused by high temperatures (such as above 80℃), which would affect the accuracy of subsequent detections. The constant temperature water bath used in the experiment (such as the HH-S2 type) needs to be preheated. Add deionized water to the water level line (approximately 1.5 liters), turn on the stirring function, and wait until the water temperature stabilizes at 60℃±0.5℃. Then, place the centrifuge tubes containing the saponification reaction solution into the shaking rack of the water bath, ensuring that the liquid level in the centrifuge tubes is lower than the water level in the water bath to prevent uneven heating of the liquid inside the tubes. During the reaction, shaking is required every 10 minutes. The specific operation is as follows: remove the centrifuge tubes, place them in a vortex mixer, shake at 1500 rpm for 10 seconds, and then place them back into the water bath. The purpose of this operation is to break the concentration gradient that may form during the reaction—potassium fatty acids will gradually deposit to the bottom of the tube. If the tube is not shaken, the concentration of potassium carbonate at the bottom will decrease, resulting in unreacted fatty acids remaining. After the 30-minute reaction, the centrifuge tube is removed for observation. The solution is still a milky white emulsion, but the viscosity has increased slightly. At this point, the fatty acids have basically completed saponification. The mixture contains potassium fatty acids (soap), glycerol (a byproduct of enzymatic hydrolysis), and sitosterol (which did not participate in saponification and is uniformly dispersed in the solution), thus forming a mildly saponified mixture.

[0034] Cool the mildly saponified mixture to room temperature, then dilute with 1 ml of deionized water to obtain a stable saponified mixture.

[0035] This step requires creating favorable conditions for subsequent organic solvent extraction. The core is "cooling to prevent emulsification and diluting to reduce viscosity," which is specifically achieved as follows: After removing the mildly saponified mixture from the 60℃ water bath, it needs to be allowed to cool naturally to approximately 25℃ at room temperature (25℃±2℃) for about 15 minutes. Rapid cooling with cold water is not recommended, as this could cause the centrifuge tubes to crack due to excessive temperature differences and prevent the potassium fatty acids in the solution from precipitating as solids and clogging the subsequent filter membrane. After cooling, observe the solution's state; it should remain a homogeneous emulsion without any solid precipitate. If precipitation occurs, gently heat it back into a 30℃ water bath until the precipitate dissolves. Adding 1 ml of deionized water dilutes the mixture: the original mixture volume is 4 ml (2 ml of enzymatic hydrolysis product + 2 ml of potassium carbonate), with a high concentration of potassium fatty acids and high viscosity. This makes it prone to forming a stable emulsion layer when organic solvents (such as n-hexane-ethyl acetate) are added, hindering separation. Adding 1 ml of deionized water reduces the total volume to 5 ml, significantly decreasing viscosity and reducing the risk of emulsification by more than 80%. The amount of water added must be strictly controlled to 1 ml. Adding only 0.5 ml will result in insufficient dilution and emulsification; adding 2 ml will over-dilute the sitosterol in the organic phase, leading to a weakened signal in subsequent gas chromatography detection. After adding deionized water, gently pipette 3 times with 1 ml of water, avoiding vigorous stirring to prevent the formation of bubbles, to obtain a stable saponified mixture. This mixture must be used in the next extraction step within 30 minutes to avoid prolonged standing, which could lead to potassium fatty acid hydrolysis and affect the sitosterol extraction recovery rate.

[0036] S203, the saponified mixture is extracted using an optimized organic solvent to obtain an extract containing sitosterol; Specifically, an extraction solvent mixture of n-hexane and ethyl acetate can be prepared at a volume ratio of 3:1 to generate an optimized extractant. The core of this step is to screen an extraction system suitable for the solubility characteristics of sitosterol. By optimizing the solvent polarity and ratio, efficient separation of sitosterol from impurities (such as potassium fatty acids and glycerol) in the saponified mixture is achieved. Specifically, it is implemented as follows: First, clarify the criteria for solvent selection: Sitosterol is a steroid compound with a hydrophobic cyclopentane-phenanthrene skeleton in its molecular structure, exhibiting relatively weak polarity. Therefore, a mixed solvent with low to medium polarity is required. Hexane (polarity parameter 0.1, nonpolar solvent) can efficiently dissolve nonpolar sitosterol, but its solubility for small amounts of polar impurities (such as residual glycerol) is poor. Ethyl acetate (polarity parameter 4.3, weakly polar solvent) can assist in dissolving polar impurities while simultaneously improving the solubility of sitosterol in the organic phase, avoiding incomplete extraction or impurity residues caused by a single solvent. A 3:1 volume ratio of the two is the optimal ratio verified through preliminary experiments. If the ratio is 1:1, the proportion of ethyl acetate is too high, which will cause polar impurities (such as trace amounts of potassium fatty acid dissolved) to enter the organic phase, resulting in impurity peaks in subsequent chromatographic analysis. If the ratio is 5:1, the proportion of n-hexane is too high, which will reduce the solubility of sitosterol and decrease the single extraction recovery rate from 95% to 78%. However, at the 3:1 ratio, the sitosterol recovery rate is stable at over 95%, and the impurity peak interference rate is less than 5%.

[0037] The preparation process must be carried out in a fume hood (both hexane and ethyl acetate are volatile and flammable, with explosion limits of 1.1%-8.7% and 2.0%-11.5% respectively; a fume hood can reduce the concentration of solvent vapors). Specific procedures: Use a 100 mL brown volumetric flask (brown bottles protect against light and prevent solvent photolysis). Accurately measure 75 mL of n-hexane (analytical grade, purity ≥99.5%, water content ≤0.05%, to avoid water affecting extraction layering) using a 50 mL pipette (accuracy ±0.05 mL) and pour it into the volumetric flask. Then, use a 25 mL pipette to measure 25 mL of ethyl acetate (analytical grade, purity ≥99.5%, acidity ≤0.01 mmol / L, to prevent acidic substances from reacting with sitosterol) and slowly pour it into the volumetric flask, avoiding solvent splashing. Tightly stopper the volumetric flask and invert and shake 10 times to ensure thorough mixing of the two phases, generating a homogeneous and transparent optimized extractant. The extractant should be stored in a brown reagent bottle with a polytetrafluoroethylene stopper (the rubber stopper will swell with the solvent, causing the seal to fail), at room temperature (25℃±2℃), and has a shelf life of 7 days. After the expiration date, the solvent is prone to volatilization, which will cause the ratio to change, and it needs to be prepared again.

[0038] Add 2 ml of optimized extractant to the saponification mixture, vortex for 2 minutes, and then centrifuge at 4500 rpm for 3 minutes to achieve layered extraction; This step requires "oscillation to enhance contact + centrifugation to accelerate layering" to ensure that sitosterol is transferred from the aqueous phase (saponified mixture) to the organic phase (extractant). The key is to control the extraction conditions to avoid emulsification and improve efficiency, which is specifically achieved as follows: First, determine the amount of extractant: the volume of the stable saponified mixture generated in the previous steps is 5 ml (2 ml of enzymatic hydrolysis product + 2 ml of potassium carbonate working solution + 1 ml of deionized water). Adding 2 ml of optimized extractant can make the volume ratio of organic phase (2 ml) to aqueous phase (5 ml) 1:2.5. This ratio can ensure sufficient contact between the two phases. If only 1 ml of extractant is added, the volume of organic phase is too small, the solubility of sitosterol is insufficient, and the extraction recovery rate drops to 80%. Adding 3 ml will result in an excess of organic phase, which will increase the time required for subsequent concentration and increase the cost of solvent consumption. 2 ml is the balance point between efficiency and cost.

[0039] Vortex mixing procedure: Place a 10 ml centrifuge tube containing 5 ml of the saponification mixture in the clamp of a vortex mixer (such as a Chillingbell Vortex-Genie 2), adjust the speed to 2000 rpm, and vortex for 2 minutes. This speed and time need precise control—below 1800 rpm, the two phases are not sufficiently mixed, the organic phase only contacts the surface of the aqueous phase, the sitosterol transfer is incomplete, and the organic phase is colorless after separation (sitosterol is not fully extracted); above 2200 rpm, the solution easily forms a stable emulsion layer (potassium fatty acid particles dispersed in the organic phase), and subsequent centrifugation cannot completely separate the phases; a 2-minute vortexing time ensures the formation of a transient "oil-in-water" emulsion, with sitosterol fully dissolved in the organic phase, and the solution is milky white and turbid after vortexing, without obvious separation.

[0040] Centrifugation stratification: Place the shaken centrifuge tubes symmetrically into the rotor of a benchtop centrifuge (such as the Xiangyi TDL-5-A model), set the speed to 4500 rpm and the centrifugation time to 3 minutes. The speed selection is based on the following: 4500 rpm corresponds to a centrifugal force of approximately 2200 × g (centrifugal force calculation formula F = 1.118 × 10^-5 × r × n). 2 (where r is the rotor radius of 10 cm), this centrifugal force can break down the organic phase (density 0.74 g / cm³) within 3 minutes. 3 (Hexane density 0.66, ethyl acetate 0.90, approximately 0.74 after mixing in a 3:1 ratio) and aqueous phase (density 1.05 g / cm³). 3The centrifugal phase (containing potassium fatty acids) completely separates without damaging the sitosterol structure. If the rotation speed is reduced to 3000 rpm, the centrifugal force is only 1100 × g, requiring 6 minutes for separation, resulting in low efficiency. Increasing the speed to 5000 rpm, with a centrifugal force of 2700 × g, allows for rapid separation, but may cause adsorption of the organic phase onto the inner wall of the centrifuge tube, leading to loss. After centrifugation, the centrifuge tube shows clear separation: the upper layer is a pale yellow, transparent organic phase (containing sitosterol), approximately 2 mL in volume; the lower layer is a milky white aqueous phase (containing potassium fatty acids and glycerol), approximately 5 mL in volume. There is no emulsion layer between the two phases, and the interface is clear, indicating preliminary separation extraction has been achieved.

[0041] Collect the upper organic phase, repeat the layer extraction three times, combine all organic phases, and generate a combined extract. This step improves the recovery rate of sitosterol through multiple extractions, avoiding low detection results caused by incomplete extraction in a single extraction. The specific implementation is as follows: First, the necessity of repeated extraction is clarified: During a single extraction, the partition coefficient of sitosterol in the organic phase (K = concentration in the organic phase / concentration in the aqueous phase) is approximately 20. According to the partition law, the recovery rate of a single extraction = K / (K+Vwater / Vorganic)×100%=20 / (20+5 / 2)×100%≈89%, leaving 11% of sitosterol remaining in the aqueous phase. After a second extraction, the recovery rate increases to 98.7%, and after a third extraction, it reaches 99.8%, meeting the requirement of the detection method for a recovery rate (≥95%). Therefore, a three-stage extraction is chosen.

[0042] Organic phase collection procedure: Use a 1 mL pipette (graduated, accuracy ±0.01 mL) to collect the upper organic phase. The pipette tip should be close to the inner wall of the centrifuge tube, and slowly insert it into the organic phase (avoid inserting below the interface to prevent aspiration of the aqueous phase). Control the flow rate during aspiration to avoid generating air bubbles. After the first collection, the volume of organic phase is approximately 1.9 mL (a small amount remains on the tube wall). Pour this into a 50 mL pear-shaped separatory funnel (pre-rinsed with n-hexane to avoid adsorption of sitosterol on the inner wall). Then, add 2 ml of fresh, optimized extraction solvent to the remaining aqueous phase in the centrifuge tube and repeat the "vortexing for 2 minutes (2000 rpm) → centrifugation for 3 minutes (4500 rpm)" operation. Collect approximately 1.8 ml of organic phase for the second time and pour it into the separatory funnel. After the third extraction, collect approximately 1.8 ml of organic phase. The total volume of organic phase in the separatory funnel after combining is approximately 5.5 ml (slightly lower than the theoretical 6 ml due to residue on the tube wall each time). The volume needs to be accurately measured and recorded with a pipette (e.g., 5.52 ml). This volume will be used for subsequent concentration calculations after concentration.

[0043] During the collection process, care must be taken to avoid contamination with the aqueous phase: If the tip of the pipette touches the interface, causing a small amount of aqueous phase (milky white) to enter the separatory funnel, allow it to stand for 5 minutes until the two phases recrystallize. Then, use a dropper to remove and discard the lower aqueous phase to ensure that the combined organic phase contains only the extractant and sitosterol, without any impurities. The combined organic phase should be pale yellow and transparent, without turbidity or precipitation. At this point, the combined extract has been enriched with more than 99.8% of the sitosterol in the sample and can proceed to the subsequent purification steps.

[0044] The combined extracts were concentrated to 0.5 mL by nitrogen blowing at 40 °C and filtered through a 0.22 μm organic filter membrane to obtain a purified extract containing sitosterol.

[0045] This step, through "gentle concentration + filtration purification," increases the concentration of sitosterol to enhance the detection signal and removes trace impurities (such as fibers and tiny enzyme particles) to avoid clogging the gas chromatograph inlet or column. Specifically, it is implemented as follows: Nitrogen blowing concentration: Transfer the combined extract to a 10 mL nitrogen blowing tube (graduated, made of PTFE, resistant to organic solvents), place it in the heating module of a nitrogen blowing instrument (such as the Tianjin Hengao HA-2003 model), and set the temperature to 40℃ and the nitrogen flow rate to 10 mL / min. The reason for choosing 40℃ is that sitosterol has a melting point of 137-140℃ and a boiling point above 360℃. At 40℃, only the solvent (n-hexane boiling point 69℃, ethyl acetate boiling point 77℃) will evaporate, without causing sitosterol decomposition or sublimation. If the temperature is raised to 50℃, the solvent evaporates too quickly, easily causing sitosterol to crystallize on the tube wall, reducing the recovery rate by 5%. Lowering the temperature to 30℃ increases the concentration time from 30 minutes to 60 minutes, resulting in low efficiency. A nitrogen flow rate of 10 mL / min creates a stable gas flow layer that covers the liquid surface, accelerating solvent evaporation while preventing sample splashing due to excessive flow (when the flow rate exceeds 15 mL / min, vortices easily form on the liquid surface, and the sample is easily blown to the upper part of the tube wall, making concentration impossible). During concentration, the liquid volume in the nitrogen blow-off tube must be closely monitored. When the volume approaches 0.5 mL, reduce the nitrogen flow rate to 5 mL / min and slowly concentrate to exactly 0.5 mL. The volume needs to be precisely controlled. If concentrated to 0.4 mL, the concentration will be too high, leading to an overestimated detection result; 0.6 mL will result in a too low concentration and a weakened signal. 0.5 mL is the optimal volume determined in preliminary experiments, which can make the sitosterol concentration reach more than 10 times the gas chromatography detection limit (0.1 μg / mL).

[0046] Filtration and purification procedure: A 0.22-micron organic microporous membrane (made of polytetrafluoroethylene (PTFE), with a pore size of 0.22 μm, capable of retaining trace solid impurities in the organic phase, such as polypropylene particles detached from the inner wall of centrifuge tubes and residual immobilized enzyme fragments) is used, along with a 1 mL syringe and membrane filter. First, the syringe and membrane filter are rinsed with 0.5 mL of optimized extraction solvent to remove trace impurities from the membrane. Then, 0.5 mL of concentrated organic phase is slowly injected into the syringe, and the piston is pushed to allow the liquid to pass through the membrane filter. The filtration rate is controlled at 0.1 mL / s to avoid excessive pressure that could cause the membrane to rupture. The filtered liquid is collected in a 2 mL sample vial (with an inner tube, brown and light-proof), which is the purified extract containing sitosterol—this liquid is colorless and transparent, free of visible impurities, and can be directly used for subsequent gas chromatography analysis. The impurity content is less than 0.01%, and it will not contaminate the chromatographic column or interfere with peak detection.

[0047] S204, the extract is directly injected into a gas chromatograph for analysis, and the content of sitosterol in the sample is calculated according to the standard curve.

[0048] Specifically, you can start the gas chromatograph, set the HP-5 capillary column, initialize the column temperature to 60 degrees Celsius, the detector temperature to 320 degrees Celsius, and generate optimized chromatographic conditions. This step requires setting up a chromatographic system suitable for the separation and detection of sitosterol. The core is to optimize parameters such as the chromatographic column, temperature, and carrier gas to ensure efficient separation of sitosterol from impurities (such as other sterols and residual solvents) and stable detection response. Specifically, the following steps are taken: First, select the gas chromatograph model and core components. The Agilent 7890B gas chromatograph is commonly used, equipped with a flame ionization detector (FID). The FID is sensitive to carbon-containing organic compounds (such as sitosterol, which contains 29 carbon atoms), with a detection limit of up to 0.01 μg / mL. Moreover, its stability is better than other detectors (such as the thermal conductivity detector TCD), making it suitable for the quantification of trace amounts of sitosterol. The chromatographic column used is an HP-5 capillary column with a stationary phase of 5% phenyl-95% dimethylpolysiloxane (medium polarity stationary phase) and dimensions of 30m × 0.32mm × 0.25μm. The 30m column length ensures sufficient resolution and avoids overlap between sitosterol and stigmasterol (which has a similar structure and retention time). The 0.32mm inner diameter is suitable for the usual injection volume (1μL) and avoids column overload. The 0.25μm membrane thickness balances separation efficiency and analysis speed. If the membrane thickness is too thick (e.g., 0.5μm), the sitosterol retention time will be too long (exceeding 20 minutes), while if it is too thin (0.1μm), the resolution will decrease.

[0049] Initialization parameter settings need to be calibrated one by one: High-purity nitrogen (purity ≥99.999%, oxygen content ≤0.001%, to avoid oxidizing sitosterol or contaminating the detector) was selected as the carrier gas. The column flow rate was set to 1.0 mL / min (constant flow mode), and the split ratio was 10:1. The split ratio controls the sample volume at the injection port. A ratio of 10:1 can avoid excessive injection volume leading to peak broadening while ensuring detection sensitivity. The injection port temperature was set to 280 degrees Celsius. This temperature allows sitosterol (boiling point about 360°C) in the purified extract to be completely vaporized without causing its thermal decomposition (sitosterol thermal decomposition temperature >320°C).

[0050] The column temperature was initialized to 60 degrees Celsius and maintained for 30 minutes. This initial low temperature allows the vaporized solvent at the injection port (hexane boiling point 69℃, ethyl acetate boiling point 77℃) to focus at the column tip, preventing solvent peak diffusion from interfering with subsequent component separation. Simultaneously, the detector temperature was started and increased. The FID detector temperature was set to 320 degrees Celsius, which is higher than the boiling point of sitosterol. This ensures that sitosterol is completely combusted after entering the detector (generating CO2 and H2O), producing a stable current signal. The detector hydrogen flow rate was 40 mL / min and the air flow rate was 400 mL / min (the hydrogen to air ratio of 1:10 is the optimal combustion ratio, resulting in the highest response value).

[0051] System stability assessment: After the column temperature, injection port temperature, and detector temperature all reach their set values, observe the baseline fluctuation. When the baseline noise is ≤0.01mV / h and the drift is ≤0.05mV / h, the system is considered stable, optimized chromatographic conditions are generated, and the sample analysis stage can begin.

[0052] The purified extract was injected into a gas chromatograph, and the temperature was programmed as follows: 60 degrees Celsius for 1 minute, then increased to 260 degrees Celsius at 20 degrees Celsius / minute, and then increased to 300 degrees Celsius at 2 degrees Celsius / minute and held for 4 minutes to generate chromatographic analysis data. This step uses programmed temperature rise to achieve the analytical logic of "removing impurities first, then separating, and finally rinsing," ensuring effective separation of sitosterol from other components while obtaining accurate chromatographic peak data. Specifically, the process is as follows: First, prepare the injection procedure: Use a 10μL airtight injection needle (such as a HAMILTON 701N model, accuracy ±0.05μL), and aspirate 1μL of purified extract (1μL is the optimal injection volume determined in pre-experiments—0.5μL results in a weak signal, and 2μL can easily lead to column overload and peak tailing). During injection, quickly insert the needle into the injection port (insertion depth ≥8mm), completing the sample injection within 1 second to avoid sample residue on the injection port wall, which could cause peak distortion.

[0053] The purpose and control details of each stage of the heating process: First stage: Maintain 60 degrees Celsius for 1 minute — This stage is consistent with the initial column temperature and its main function is "solvent focusing": allowing the low-boiling-point solvent (n-hexane, ethyl acetate) vaporized at the injection port to pass rapidly through the chromatographic column, forming a sharp solvent peak on the chromatogram (retention time of about 0.8-1.0 minutes), which is completely separated from the subsequent high-boiling-point sitosterol (retention time of about 15-16 minutes), avoiding the solvent peak from masking the target peak.

[0054] The second stage involves increasing the temperature to 260 degrees Celsius at a rate of 20 degrees Celsius per minute. This rate of increase is called "rapid heating" and is intended to quickly elute impurities in the column whose boiling points are between those of the solvent and sitosterol (such as small amounts of unsaponified fatty acid methyl esters, with a boiling point of approximately 200-250 degrees Celsius), thereby reducing the interference of impurities on sitosterol separation. If the heating rate is too slow (e.g., 10 degrees Celsius per minute), the elution time of impurities will be prolonged, which will lead to an increase in the total analysis time to more than 30 minutes and a decrease in efficiency.

[0055] The third stage: Increase the temperature to 300 degrees Celsius at a rate of 2 degrees Celsius / minute and hold for 4 minutes—this stage, known as "slow heating," is crucial for the separation of sitosterol: sitosterol and stigmasterol (boiling point approximately 358°C), which have similar structures, differ in retention time by only 0.3-0.5 minutes. Slow heating amplifies this retention time difference, ensuring complete separation of the chromatographic peaks (resolution ≥ 1.5, meeting the requirements for quantitative analysis). Finally, hold at 300 degrees Celsius for 4 minutes, known as "column flushing," to remove residual high-boiling-point impurities (such as other sterol polymers) from the column, preventing them from affecting subsequent sample analyses.

[0056] Chromatographic analysis data generation: The detector converts the current signal generated by combustion into a chromatogram, which includes a horizontal axis (retention time, minutes) and a vertical axis (peak area, μV·s). The chromatographic peak corresponding to sitosterol is a symmetrical Gaussian peak (peak shape symmetry factor 0.9-1.1), without tailing or leading edge, with a retention time of about 15.2 minutes. The peak area has a linear relationship with the sitosterol concentration. This chromatogram and the original signal data constitute the generated chromatographic analysis data.

[0057] Based on the retention time of β-sitosterol standard, qualitative analysis was performed. Using cholesterol as an internal standard, a standard curve of peak area ratio versus concentration was plotted to generate a quantitative calibration curve. This step requires "qualitative confirmation of the target peak + quantitative establishment of calibration relationship" to ensure the accuracy and reliability of the detection results. Specifically, it is implemented as follows: First, perform qualitative analysis: Prepare a β-sitosterol standard solution (purity ≥98%, purchased from Sigma-Aldrich) at a concentration of 100 μg / mL (solvent is a 3:1 mixture of n-hexane and ethyl acetate). Inject 1 μL under the chromatographic conditions of step two and record its retention time—the retention time of the standard β-sitosterol is 15.20 ± 0.05 minutes. Identify the chromatographic peaks in the sample chromatogram with retention times between 15.15 and 15.25 minutes as β-sitosterol peaks to achieve qualitative analysis (if the sample peak retention time deviates from the standard by more than 0.1 minutes, check whether the column temperature is stable or whether the injection port is contaminated).

[0058] Internal standard method operation details: Cholesterol was chosen as the internal standard because it is structurally similar to sitosterol (both are steroid compounds), exhibits similar chromatographic behavior, and is present in extremely low amounts (negligible) in camellia oil samples, thus not interfering with detection. Prepare a cholesterol internal standard solution with a concentration of 50 μg / mL (solvent as above). Add an equal volume of internal standard solution to all standard solutions and the purified extract of the sample (e.g., add 10 μL of internal standard solution to every 0.5 mL of purified extract to achieve a final internal standard concentration of 1 μg / mL). This ensures that the internal standard is subjected to the same interferences as the target analyte during injection (such as fluctuations in injection volume and changes in instrument response), thus offsetting systematic errors.

[0059] Standard curve plotting: Five β-sitosterol standard concentrations were set at 10, 20, 50, 100, and 200 μg / mL, with each concentration point containing a 1 μg / mL cholesterol internal standard. The samples were injected three times under chromatographic conditions, and the average peak area was recorded. A linear regression was performed with the ratio of β-sitosterol peak area to cholesterol peak area as the ordinate (y) and β-sitosterol concentration (μg / mL) as the abscissa (x) to obtain the standard curve equation. An example equation is y = 0.0205x + 0.0042, with a correlation coefficient r = 0.9998 (r ≥ 0.999 is considered acceptable, indicating good linearity and a quantitative error ≤ 5%). This standard curve serves as the quantitative calibration curve and needs to be re-plotted daily to avoid calibration deviations caused by instrument drift.

[0060] The concentration is calculated by substituting the peak area ratio of the sample into the quantitative calibration curve. Combined with the sample weight and the final volume, the system automatically calculates and outputs a report on the β-sitosterol content in the sample.

[0061] This step calculates the actual content of sitosterol in the sample using a formula, and generates a test report containing key information to ensure the results are traceable and verifiable. The specific implementation is as follows: First, extract the peak area ratio of the sample: From the chromatographic analysis data of the sample, read the area of ​​the β-sitosterol peak (e.g., sample A = 2050 μV·s) and the area of ​​the cholesterol internal standard peak (e.g., internal A = 100 μV·s), and calculate the peak area ratio ysample = sample A / internal A = 2050 / 100 = 20.5.

[0062] Concentration and content calculation: Substitute y_sample into the quantitative calibration curve equation (e.g., y=0.0205x + 0.0042) to obtain the concentration of β-sitosterol in the purified extract of the sample: x_sample=(y_sample- 0.0042) / 0.0205=(20.5 -0.0042) / 0.0205≈998μg / mL (approximately 1000μg / mL). Based on the previous experimental parameters: sample weight m = 0.2g (0.2×10^-3kg), purified extract volume V = 0.5mL (0.5×10^-3L), the calculation formula is: β-sitosterol content (mg / kg) = (x sample × V) / m, substituting the values: (1000μg / mL × 0.5mL) / 0.2g = (500μg) / 0.2g = 2500μg / g = 2500mg / kg (1μg / g = 1mg / kg).

[0063] Test report generation: The report must include four parts: "sample information, instrument parameters, analysis results, and verification indicators." Example content: Sample information: Sample name (camellia oil), sampling date (2025-10-20), sample weight (0.2005g, actual sample weight, accurate to 0.0001g); Instrument parameters: Chromatograph model (Agilent 7890B), chromatographic column (HP-5, 30m×0.32mm×0.25μm), programmed temperature conditions (same as step two), internal standard (cholesterol, concentration 1μg / mL); Analysis results: β-sitosterol content (2500±50 mg / kg, uncertainty ≤2%), retention time (15.22 minutes); Validation metrics: Relative standard deviation (RSD, n=3) = 1.2% (≤5%, indicating good repeatability), spike recovery rate (95.6%, within the range of 90%-110%, indicating acceptable accuracy).

[0064] The report can be automatically saved as a PDF, including screenshots of chromatograms and standard curves to ensure data traceability, and is finally output to the testing system terminal to complete the entire testing process.

[0065] Another embodiment of the present invention provides a rapid detection system for sitosterol in oils and fats, see [link to relevant documentation]. Figure 3 The system may include: The receiving module 301 is used to receive oil samples and enzymatically hydrolyze the oil samples using immobilized nonspecific lipase to generate enzymatic hydrolysis products. Saponification module 302 is used to mix the enzymatic hydrolysis product with potassium carbonate solution to carry out a mild saponification reaction to obtain a saponified mixture. Extraction module 303 is used to extract the saponified mixture using an optimized organic solvent to separate an extract containing sitosterol; The analysis module 304 is used to directly inject the extract into a gas chromatograph for analysis and calculate the content of sitosterol in the sample according to the standard curve.

[0066] This invention also provides a storage medium storing a computer program, wherein the computer program is configured to execute the steps in any of the above method embodiments when running.

[0067] This invention also provides an electronic device, including a memory and a processor, wherein the memory stores a computer program, and the processor is configured to run the computer program to perform the steps in any of the above method embodiments.

[0068] Specifically, the aforementioned electronic device may further include a transmission device and an input / output device, wherein the transmission device is connected to the aforementioned processor, and the input / output device is connected to the aforementioned processor.

[0069] The above description, based on the embodiments shown in the figures, details the structure, features, and effects of the present invention. The above description is only a preferred embodiment of the present invention, but the present invention is not limited to the scope of implementation shown in the figures. Any changes made in accordance with the concept of the present invention, or equivalent embodiments modified to have equivalent changes, that do not exceed the spirit covered by the specification and figures, should be within the protection scope of the present invention.

Claims

1. A method for rapid detection of sitosterol in oil and fat, characterized by, The method comprises: receiving an oil sample, and performing enzymatic treatment on the oil sample by using immobilized non-specific lipase to generate an enzymatic product; mixing the enzymatic product with a potassium carbonate solution to perform a mild saponification reaction, to obtain a saponification mixture; extracting the saponification mixture by using an optimized organic solvent, and separating to obtain an extract liquid containing stigmasterol; directly injecting the extract liquid into a gas chromatograph for analysis, and calculating the content of stigmasterol in the sample according to a standard curve.

2. The method of claim 1, wherein, The receiving an oil sample, and performing enzymatic treatment on the oil sample by using immobilized non-specific lipase to generate an enzymatic product comprises: accurately weighing 0.2 grams of camellia oil sample into a 10 milliliter centrifuge tube, adding 2 milliliters of phosphate buffer to adjust the pH to 7.0 to generate a sample buffer mixture; adding 50 milligrams of immobilized non-specific lipase to the sample buffer mixture, and oscillating for 30 seconds on a vortex mixer to make it fully dispersed to generate an enzyme-substrate mixture; placing the enzyme-substrate mixture in a 40-degree Celsius constant temperature water bath for oscillation reaction for 2 hours, and controlling the rotation speed to be 150 revolutions per minute to generate a preliminary enzymatic product; centrifuging the preliminary enzymatic product at 4500 revolutions per minute for 5 minutes, taking the supernatant, and filtering through a 0.45-micron filter membrane to obtain a clear enzymatic product.

3. The method of claim 2, wherein, The mixing the enzymatic product with a potassium carbonate solution to perform a mild saponification reaction, to obtain a saponification mixture comprises: preparing a 0.5 mol / L potassium carbonate solution, weighing 6.91 grams of potassium carbonate and dissolving it in 100 milliliters of deionized water to generate a potassium carbonate working solution; taking 2 milliliters of the enzymatic product and mixing it with 2 milliliters of the potassium carbonate working solution in a 10 milliliter centrifuge tube, and oscillating for 1 minute using a vortex mixer to generate a saponification reaction liquid; placing the saponification reaction liquid in a 60-degree Celsius water bath for reaction for 30 minutes, and oscillating every 10 minutes during the reaction to generate a mild saponification mixture; cooling the mild saponification mixture to room temperature, adding 1 milliliter of deionized water for dilution to obtain a stable saponification mixture.

4. The method of claim 3, wherein, The extracting the saponification mixture by using an optimized organic solvent, and separating to obtain an extract liquid containing stigmasterol comprises: preparing an extraction solvent n-hexane-ethyl acetate mixed liquid, and preparing an optimized extractant by a volume ratio of 3:1; adding 2 milliliters of the optimized extractant to the saponification mixture, vortex oscillating for 2 minutes, and then centrifuging at 4500 revolutions per minute for 3 minutes to realize layered extraction; collecting the upper organic phase, repeating layered extraction three times, and combining all the organic phases to generate a combined extract liquid; nitrogen blowing the combined extract liquid at 40 degrees Celsius to concentrate to 0.5 milliliters, filtering through a 0.22-micron organic filter membrane to obtain a purified extract liquid containing stigmasterol.

5. The method of claim 4, wherein, The directly injecting the extract liquid into a gas chromatograph for analysis, and calculating the content of stigmasterol in the sample according to a standard curve comprises: starting the gas chromatograph, setting an HP-5 capillary column, initializing the column temperature to 60 degrees Celsius, and setting the detector temperature to 320 degrees Celsius to generate optimized chromatographic conditions; The purified extraction liquid is injected into a gas chromatograph, and a programmed temperature is used: 60 degrees Celsius for 1 minute, 20 degrees Celsius / minute to 260 degrees Celsius, and then 2 degrees Celsius / minute to 300 degrees Celsius for 4 minutes, to generate chromatographic analysis data; According to the retention time of the β-sitosterol standard, the peak area ratio and the concentration standard curve are drawn by using the internal standard method with cholesterol as the internal standard, to generate a quantitative calibration curve; The sample peak area ratio is substituted into the quantitative calibration curve to calculate the concentration, and the sample mass and the constant volume are combined to automatically calculate and output the β-sitosterol content report in the sample.

6. A rapid detection system for sitosterol in oil and fat, characterized in that, The system comprises: A receiving module configured to receive an oil sample and perform enzymatic treatment on the oil sample by using immobilized non-specific lipase to generate an enzymatic product; A saponification module configured to mix the enzymatic product with a potassium carbonate solution to perform a mild saponification reaction and obtain a saponification mixture; An extraction module configured to extract the saponification mixture by using an optimized organic solvent to separate and obtain an extraction liquid containing sitosterol; An analysis module configured to inject the extraction liquid directly into a gas chromatograph for analysis and calculate the content of sitosterol in the sample according to a standard curve.

7. The system of claim 6, wherein, The receiving module is specifically configured to: Accurately weigh 0.2 grams of camellia oil sample into a 10 milliliter centrifuge tube, add 2 milliliters of phosphate buffer to adjust the pH to 7.0 to generate a sample buffer mixture; Add 50 milligrams of immobilized non-specific lipase to the sample buffer mixture, and shake it on a vortex mixer for 30 seconds to make it fully dispersed to generate an enzyme-substrate mixture; Place the enzyme-substrate mixture in a 40 degrees Celsius constant temperature water bath and shake for 2 hours to generate a preliminary enzymatic product, with the rotation speed controlled at 150 revolutions / minute; Centrifuge the preliminary enzymatic product at 4500 revolutions / minute for 5 minutes, take the supernatant, and filter it through a 0.45 micrometer filter membrane to obtain a clear enzymatic product.

8. The system of claim 7, wherein, The saponification module is specifically configured to: Prepare a 0.5 mole / liter potassium carbonate solution by weighing 6.91 grams of potassium carbonate and dissolving it in 100 milliliters of deionized water to generate a potassium carbonate working solution; Take 2 milliliters of the enzymatic product and mix it with 2 milliliters of the potassium carbonate working solution in a 10 milliliter centrifuge tube, and use a vortex mixer to shake for 1 minute to generate a saponification reaction liquid; Place the saponification reaction liquid in a 60 degrees Celsius water bath for 30 minutes, and shake it every 10 minutes during the reaction to generate a mild saponification mixture; Cool the mild saponification mixture to room temperature, add 1 milliliter of deionized water for dilution, and obtain a stable saponification mixture.

9. A storage medium, characterized by The storage medium stores a computer program, wherein the computer program is configured to execute the method of any one of claims 1-5 when running.

10. An electronic device comprising a memory and a processor, characterized in that, The memory stores a computer program, and the processor is configured to execute the computer program to execute the method of any one of claims 1-5.

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