Method for rapidly detecting content of diglyceride in oil sample

Abstract

The invention provides a method for rapidly detecting the content of diglyceride in an oil sample, which comprises the following steps of: heating an oil sample to be detected and excessive heptafluorobutyryl imidazole in a water bath to react to generate heptafluorobutyryl diglyceride and imidazole; after the reaction is completed, adding water to react with the residual heptafluorobutyryl imidazole to generate imidazole and heptafluorobutyric acid; adding S-benzylisothiourea hydrochloride to react with heptafluorobutyric acid to generate a precipitate which is insoluble in water; and calculating the content of the diglyceride according to the generation amount of the precipitate. The detection method has good linear correlation when the mass fraction range of the diglyceride is 5-70%, the lowest detection limit of the diglyceride in the oil sample is 1.89%, the quantitation limit is 6.31%, the standard deviation of the measured value of the obtained to-be-detected sample is less than 4%, the deviation from the actual value is less than 2%, and the method has good sensitivity, precision and accuracy. According to the invention, the problem that a detection method in the prior art is difficult to give consideration to detection accuracy, precision, rapidness and economy is solved, so that the overall detection capability of the diglyceride detection method is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of food, relates to the detection of edible oil, and particularly relates to a rapid detection method for the content of diglyceride in an oil sample. Background Art

[0002] Edible oil is an important part of daily diet. With the continuous development of society and the remarkable improvement of people's living standards, its safety and nutritional value have attracted increasing attention. The main component of traditional edible oil is triglyceride, and excessive intake may induce a series of chronic diseases such as atherosclerosis, fatty liver, and diabetes. As a new alternative to ordinary edible oil, diglyceride oil exhibits many unique advantages in the human metabolic process and has various positive effects on health. Research shows that the metabolic pathway of diglyceride in the human body is significantly different from that of ordinary oils, and it can effectively reduce the synthesis and accumulation of triglyceride, thus helping to reduce body fat and prevent obesity; in addition, diglyceride can significantly reduce the cholesterol level in the blood, which is of great significance for preventing cardiovascular diseases; diglyceride also has the function of improving insulin sensitivity and helps to improve the insulin resistance condition; at the same time, diglyceride can reduce the accumulation of fat in the liver, relieve the metabolic burden of the liver, and thus play a role in protecting the liver to a certain extent.

[0003] In summary, compared with ordinary edible oil, diglyceride oil shows significant advantages in terms of digestion and absorption characteristics and health benefits. According to the "Announcement on Approving Seven Kinds of Substances such as Diglyceride Oil as New Resource Foods" (No. 18, 2009), it is stipulated that only oils with a diglyceride content of more than 40% can be called diglyceride oil. At present, the price of diglyceride oil products on the market is mainly determined by the diglyceride content therein. Therefore, the development of a rapid detection method for the content of diglyceride in an oil sample has important practical significance for the quality supervision and quality control of diglyceride oil products.

[0004] At present, the detection methods of diglycerides at home and abroad are mostly chromatography, spectroscopy, etc. Thin-layer chromatography is based on the separation of diglycerides and triglycerides on a silica gel plate through the movement of a solvent, so as to quantitatively analyze them. This method is simple to operate, but its sensitivity and accuracy are relatively low. Gas and liquid chromatography are based on the difference in the distribution coefficients of diglycerides and triglycerides between the stationary phase. Through the separation of the chromatographic column, their residence times in the column are different, so as to achieve separation and detection. Although this method has good separation effect and high sensitivity, it requires complex pretreatment of samples, which will produce harmful waste to pollute the environment, and requires large-scale instruments; Spectroscopic methods such as nuclear magnetic resonance and infrared spectroscopy are based on the interaction between diglycerides and light. By analyzing the wavelength and intensity distribution of absorbed, emitted or scattered light, the composition and structure are determined. Although this method does not require sample pretreatment and is more environmentally friendly, it also has the defects of high instrument cost and the need for relatively complex analysis and calculation of spectra. Xu Tongcheng et al. mentioned a rapid detection method based on the reaction of citric acid with diglycerides to form a precipitate in the patent "A Method for Rapidly Detecting the Content of Diglycerides in Oils and Fats". After adding the reaction reagents citric acid and concentrated sulfuric acid to the oil sample, centrifugation is carried out after the reaction is completed under the heating condition of 120 °C. By weighing the mass of the precipitate, the content can be calculated according to the standard curve. The required detection time is 40 - 140 min, and the detection limit is 23.1 g / 100 g. However, this method has problems such as too high reaction temperature, long time consumption, high detection limit, and the use of strongly corrosive reagents. Therefore, there is an urgent need for a rapid detection method with mild reaction conditions, short time consumption, higher sensitivity, and relatively safe reaction reagents. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a rapid detection method for the content of diglycerides in an oil sample, and solve the technical problem that the overall detection ability of the existing detection methods for diglycerides needs to be further improved.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions to achieve:

[0007] A rapid detection method for the content of diglycerides in an oil sample. In this method, the oil sample to be tested is heated in a water bath with excessive heptafluorobutyryl imidazole to react, generating heptafluorobutyryl diglyceride and imidazole; after the reaction is completed, water is added to react with the remaining heptafluorobutyryl imidazole, generating imidazole and heptafluorobutyric acid; then S-benzylisothiourea hydrochloride is added to react with heptafluorobutyric acid to generate a precipitate that is insoluble in water; the content of diglycerides is calculated based on the amount of the precipitate formed.

[0008] The present invention also has the following technical features:

[0009] This method includes the following steps:

[0010] Step 1, Prepare the sample to be tested:

[0011] Weigh the sample to be tested, place the weighed sample to be tested in the first centrifuge tube, add n-hexane, and wash all the sample to be tested to the bottom of the first centrifuge tube.

[0012] Step 2, add heptafluorobutyryl imidazole for water bath reaction:

[0013] After adding heptafluorobutyryl imidazole to the first centrifuge tube in Step 1, vortex it, seal the mouth of the first centrifuge tube with a sealing film, and heat it in a water bath for reaction. After the reaction is completed, add distilled water, and centrifuge to discard the upper organic phase.

[0014] Step 3, prepare S-benzylisothiourea hydrochloride solution:

[0015] Weigh S-benzylisothiourea hydrochloride, transfer it to a volumetric flask for volume fixation, and filter to obtain a clear S-benzylisothiourea hydrochloride solution.

[0016] Step 4, weigh the precipitate mass after adding S-benzylisothiourea hydrochloride solution:

[0017] Weigh the mass of the empty second centrifuge tube in advance, transfer the lower aqueous phase obtained in Step 2 to the empty second centrifuge tube, dilute it with water, add S-benzylisothiourea hydrochloride solution, let it stand for a period of time, then centrifuge to discard the supernatant, weigh the total mass of the second centrifuge tube and the precipitate in the second centrifuge tube, and subtract the mass of the empty second centrifuge tube to obtain the precipitate mass.

[0018] Step 5, calculate and obtain the content of diglyceride in the sample to be tested:

[0019] Substitute the precipitate mass obtained in Step 4 into the linear regression equation with the absolute mass of diglyceride as the independent variable X and the precipitate mass as the dependent variable Y to obtain the absolute mass of diglyceride in the sample to be tested, and then divide the absolute mass by the sample mass to calculate the content of diglyceride in the sample to be tested.

[0020] In Step 1, the mass of the sample to be tested weighed is 0.250 g to 0.350 g.

[0021] In Step 1, the volume of n-hexane added is 50 μL to 300 μL.

[0022] In Step 2, the addition amount of heptafluorobutyryl imidazole is 50 μL to 300 μL.

[0023] In Step 2, the reaction conditions of the water bath reaction are: temperature 45 °C to 95 °C, reaction time 20 min to 60 min.

[0024] In Step 2, the volume of distilled water added is 100 μL to 500 μL.

[0025] In Step 2, the centrifugal force is (500 - 3500)×g, and the centrifugation time is 10 - 60 s.

[0026] In Step 2, when discarding the supernatant, rinse with n - hexane 2 - 4 times.

[0027] In Step 3, the concentration of the S - benzylisothiourea hydrochloride solution is 0.030 - 0.080 g / mL, and the filtration uses a 0.45 - μm aqueous filter membrane.

[0028] In Step 4, the second centrifuge tube is a 50 - mL graduated conical - bottom centrifuge tube. When transferring, rinse with water 2 - 4 times and add water to dilute to the 5 - 15 mL graduation line.

[0029] In Step 4, the addition amount of the S - benzylisothiourea hydrochloride solution is 1.0 mL - 5.0 mL.

[0030] In Step 4, the standing time is 10 min - 50 min, the centrifugal force is (4000 - 10000)×g, and the centrifugation time is 5 - 20 min.

[0031] In Step 5, the method for obtaining the linear regression equation is as follows: Mix the diglyceride standard with triglyceride oil at different mass ratios to obtain multiple standard samples. Measure and obtain the precipitation masses of multiple standard samples according to the above Steps 1 to 4. Use the absolute mass of diglyceride as the independent variable X, and use the precipitation mass obtained from the reaction of the standard sample as the dependent variable Y to obtain the linear regression equation and draw the standard curve.

[0032] Compared with the prior art, the present invention has the following technical effects:

[0033] (Ⅰ) The detection method of the present invention shows a good linear correlation when the mass fraction range of diglyceride is 5% - 70%. The lowest detection limit of diglyceride in the oil sample is 1.89%, the quantification limit is 6.31%. The standard deviation of the measured value of the sample to be detected is less than 4%, and the deviation from the actual value is less than 2%. It has good sensitivity, precision, and accuracy. The present invention solves the problem that the detection methods in the prior art are difficult to balance detection accuracy, precision, speed, and economy, and significantly improves the overall detection ability of the diglyceride detection method.

[0034] (Ⅱ) The detection method of the present invention is easy to operate and can shorten the detection time to within 60 min. The method of the present invention mainly utilizes the structural differences between diglyceride and triglyceride, and calculates the diglyceride content by converting the diglyceride content into the corresponding precipitation defect mass. This method is simple, sensitive, reliable, and has a low detection cost.

[0035] (Ⅲ) The detection method of the present invention, compared with the rapid detection method based on the reaction of citric acid with diglyceride to form a precipitate, does not use toxic and harmful chemical reagents, produces less harmful waste, and is environmentally friendly.

[0036] (Ⅳ) The rapid detection method provided by the present invention can realize the detection of samples only by using common laboratory instruments such as a water bath, a balance, and a centrifuge. The detection cost for each sample is only about 6.8 yuan. The detection personnel do not need to undergo professional training, and it can be realized without expensive instruments, complex processing, and professional detection personnel. Description of the Drawings

[0037] Figure 1 It is the infrared spectrogram of diglyceride oil and its reaction product with heptafluorobutyryl imidazole.

[0038] Figure 2 It is the linear regression equation and standard curve graph for calculating the diglyceride content of the sample to be measured in the present invention.

[0039] Figure 3 It is the photo of the precipitation formed after the reaction of diglyceride with different mass fractions.

[0040] The following further explains the specific content of the present invention in detail in conjunction with examples. Specific Embodiments

[0041] In the present invention, the purity of heptafluorobutyryl imidazole is 97%, and it is purchased from Shanghai Macklin Biochemical Co., Ltd.

[0042] In the present invention, the purity of S-benzylisothiourea hydrochloride is 98.5 wt%, and it is purchased from Shanghai Macklin Biochemical Co., Ltd.

[0043] In the present invention, the infrared spectrometer is purchased from Bruker Corporation of Germany, and the model is vertex70.

[0044] The method of the present invention reacts the oil sample to be measured with excessive heptafluorobutyryl imidazole by heating in a water bath to generate heptafluorobutyryl diglyceride and imidazole; after the reaction is completed, water is added to react with the remaining heptafluorobutyryl imidazole to generate imidazole and heptafluorobutyric acid; then S-benzylisothiourea hydrochloride is added to react with heptafluorobutyric acid to generate a precipitate that is insoluble in water; the content of diglyceride is calculated by the amount of the generated precipitate.

[0045] The specific detection mechanism is as follows:

[0046] The present invention utilizes the special structure of diglyceride, that is, there is a free hydroxyl group on the 2nd carbon atom, which can react with the carbonyl group of heptafluorobutyryl imidazole to generate heptafluorobutyryl diglyceride and imidazole. The specific reaction mechanism is:

[0047]

[0048] An excessive amount of heptafluorobutyryl imidazole is added to the sample. After the reaction in a water bath is completed, distilled water is added to react with the remaining heptafluorobutyryl imidazole to generate imidazole and heptafluorobutyric acid. Then S-benzylisothiourea hydrochloride is added. This reagent reacts with heptafluorobutyric acid to form a precipitate that is insoluble in water. Thus, the higher the content of diglyceride, the less the remaining heptafluorobutyryl imidazole, and further the less the precipitate. Therefore, the content of diglyceride can be calculated by the amount of precipitate formed.

[0049] To prove that diglyceride reacts with heptafluorobutyryl imidazole, the present invention uses an infrared spectrometer to characterize diglyceride oil and the reaction product of it and heptafluorobutyryl imidazole, as Figure 1 shown.

[0050] From Figure 1 the infrared spectrum of the diglyceride sample shown, the characteristic peak at a wave number of 3400 - 3500 cm-1 represents the stretching vibration of O-H, indicating the presence of hydroxyl groups in diglyceride. The weakening of this peak in the reaction product indicates that the hydroxyl groups have reacted. From the infrared spectrum of the reaction product, the peaks at 643 cm-1, 1217 cm-1, and 1189 cm-1 are the bending vibration, antisymmetric stretching, and symmetric stretching vibrations of CF2 respectively, while the peaks at 1269 cm-1 and 1147 cm-1 are the antisymmetric stretching and symmetric stretching vibrations of CF3, indicating that heptafluorobutyryl imidazole has been combined with the diglyceride molecule at this time. In addition, 1742 cm-1 is the characteristic peak of the carbonyl group of diglyceride itself. In the reaction product, the newly added characteristic peak at 1787 cm-1 is the stretching vibration peak of the carbonyl group generated after the heptafluorobutyryl group is incorporated into diglyceride. Since fluorine has a strong electronegativity, the characteristic peak of the carbonyl group of diglyceride at 1742 cm-1 is shifted to the high-frequency band to 1748 cm-1, which also indicates that the heptafluorobutyryl group has been combined with diglyceride at this time.

[0051] In summary, in the infrared spectrum of diglyceride after reaction with heptafluorobutyryl imidazole, multiple characteristic peaks of CF2 and CF3 appear, and the original characteristic peaks also weaken and shift, indicating that diglyceride reacts with heptafluorobutyryl imidazole to generate heptafluorobutyryl diglyceride and imidazole. Therefore, it is feasible to calculate the diglyceride content by converting the diglyceride content into the corresponding precipitate defect mass by adding S-benzylisothiourea hydrochloride.

[0052] It can be known from the above analysis and experiments that if the content of diglyceride in the oil sample is different, it will cause a change in the precipitate mass of the final reaction product. That is to say, measuring the precipitate mass can be used as a means to detect its content.

[0053] In accordance with the above technical solution, specific embodiments of the present invention are given below. It should be noted that the present invention is not limited to the following specific embodiments, and all equivalent transformations made on the basis of the technical solution of this application fall within the protection scope of the present invention.

[0054] Example 1: (Drawing of standard curve)

[0055] In this example, a rapid detection method for the content of diglycerides in an oil sample is given. The method includes the following steps:

[0056] Step 1, prepare the sample to be tested:

[0057] Weigh 0.300 g of the sample to be tested. Among them, for diglycerides, weigh 0.015, 0.030, 0.060, 0.090, 0.105, 0.120, 0.135, 0.150, 0.180, 0.210 g respectively, and make up the remaining mass with triglycerides. Place the weighed samples to be tested in clean 2 mL first centrifuge tubes respectively, and add 200 μL of n-hexane along the tube wall by rotation to wash all the oil samples to the bottom of the tube.

[0058] Step 2, add heptafluorobutyryl imidazole for water bath reaction:

[0059] After preparing the sample to be tested in Step 1, add 100 μL of heptafluorobutyryl imidazole and vortex it. Seal the tube mouth with a sealing film for water bath reaction. The reaction temperature is 70 °C and the time is 30 min. After the reaction is completed, add 300 μL of distilled water, centrifuge at 1500 × g for 30 s, then discard the upper organic phase, and rinse with n-hexane 3 times.

[0060] Step 3, prepare S-benzylisothiuronium hydrochloride solution:

[0061] Weigh S-benzylisothiuronium hydrochloride, transfer it to a volumetric flask and make up the volume to make its concentration 0.057 g / mL, and filter it through a 0.45 μm aqueous filter membrane to obtain a clear solution.

[0062] Step 4, weigh the precipitate mass after adding S-benzylisothiuronium hydrochloride solution:

[0063] Weigh the mass of an empty 50 mL graduated conical bottom second centrifuge tube in advance. Transfer the lower aqueous phase to the empty second centrifuge tube, and rinse it 3 times with water. Dilute it with water to the 10 mL graduation line, add 2.0 mL of S-benzylisothiuronium hydrochloride solution, let it stand for 30 min, centrifuge at 8000 × g for 8 min, then discard the supernatant, and weigh the mass of the second centrifuge tube and the precipitate in it. Subtract the mass of the empty tube to obtain the precipitate mass.

[0064] Step 5, draw the standard curve:

[0065] Taking the absolute mass of diglyceride as the independent variable X and the precipitate mass as the dependent variable Y, the linear regression equation was obtained and the standard curve was plotted. The linear regression equation and the standard curve are as Figure 2 shown.

[0066] Example 2: (Determination of detection limit and quantification limit)

[0067] In this example, a rapid detection method for the content of diglyceride in an oil sample is given. The method includes the following steps:

[0068] Step 1, prepare the sample to be tested:

[0069] Weigh 0.100 g of diglyceride to prepare a sample with a concentration of about 5% and a mass of about 2 g. The remaining mass is made up with triglyceride. Then divide it into 6 portions of about 0.3 g of the sample to be tested, and weigh another 0.280 g. Place the weighed samples to be tested in clean 2 mL first centrifuge tubes respectively, and add 100 μL of n-hexane along the tube wall and rotate to wash all the oil samples to the bottom of the tube.

[0070] Step 2, add heptafluorobutyryl imidazole for water bath reaction:

[0071] After preparing the sample to be tested in Step 1, add 50 μL of heptafluorobutyryl imidazole and vortex. Seal the tube mouth with a sealing film for water bath reaction. The reaction temperature is 85 °C and the time is 20 min. After the reaction is completed, add 400 μL of distilled water, centrifuge at 3000×g for 10 s, discard the upper organic phase, and rinse twice with n-hexane.

[0072] Step 3, prepare S-benzylisothiuronium hydrochloride solution:

[0073] Weigh S-benzylisothiuronium hydrochloride, transfer it to a volumetric flask and make up the volume to make its concentration 0.080 g / mL, and filter it through a 0.45 μm aqueous filter membrane to obtain a clear solution.

[0074] Step 4, weigh the precipitate mass after adding S-benzylisothiuronium hydrochloride solution:

[0075] Weigh the mass of an empty 50 mL graduated conical bottom second centrifuge tube in advance. Transfer the lower aqueous phase to the empty second centrifuge tube, rinse twice with water, dilute with water to the 15 mL scale line, add 3.0 mL of S-benzylisothiuronium hydrochloride solution, let it stand for 50 min, centrifuge at 5000×g for 15 min, discard the supernatant, weigh the mass of the second centrifuge tube and the precipitate in it, and subtract the mass of the empty tube to obtain the precipitate mass.

[0076] Step 5, calculate and obtain the content of diglyceride in the sample to be tested:

[0077] Substitute the precipitate mass of the sample to be measured in Step 4 into the linear regression equation Y = -5.0036X + 1.1141 obtained in Example 1 to calculate the absolute mass of diglyceride. Then, divide the absolute mass by the sample mass to obtain the content of diglyceride in the sample to be measured in this example.

[0078] The measured values of this example are shown in Table 1.

[0079] Table 1 Content of diglyceride in 6 samples to be measured in Example 2

[0080] Sample Serial Number Measured Value (%) 1 4.95 2 4.93 3 5.00 4 5.08 5 5.01 6 5.10

[0081] The calculation formulas for the detection limit and quantification limit in this example are as follows:

[0082] LOD = 3 × S;

[0083] LOQ = 10 × S;

[0084]

[0085] In the formula:

[0086] LOD is the detection limit;

[0087] LOQ is the quantification limit;

[0088] S0 is the standard deviation of the sample;

[0089] n is the number of samples;

[0090] S is the median deviation of the sample.

[0091] From Table 1 and Formulas 1 - 3, it can be obtained that the detection limit of the detection method corresponding to this example is 1.89%, and the quantification limit is 6.31%, with good sensitivity.

[0092] Example 3:

[0093] A rapid detection method for the content of diglyceride in an oil sample is given in this example. The method includes the following steps:

[0094] Step 1, prepare the sample to be measured:

[0095] Weigh 0.320 g of the sample, in which the mass fractions of diglyceride are approximately 13%, 23%, 33%, 38%, 48%, 58%, and 65% respectively. The specific values are shown in Table 2. Place the weighed samples to be measured in clean 2 - mL first centrifuge tubes respectively, and add 50 μL of n - hexane along the tube wall to wash all the oil samples to the bottom of the tube.

[0096] Step 2, add 1 - heptafluorobutyryl imidazole for water - bath reaction:

[0097] Prepare the sample to be tested in Step 1, add 50 μL of heptafluorobutyryl imidazole thereto and vortex, seal the tube mouth with a sealing film and react in a water bath. The reaction temperature is 45 °C and the time is 60 min. After the reaction is completed, add 100 μL of distilled water, centrifuge at 1000×g for 60 s, discard the upper organic phase, and rinse twice with n-hexane.

[0098] Step 3, prepare the S-benzylisothiuronium hydrochloride solution:

[0099] Weigh S-benzylisothiuronium hydrochloride, transfer it to a volumetric flask and make up the volume to make its concentration 0.090 g / mL, and filter through a 0.45 μm aqueous filter membrane to obtain a clear solution.

[0100] Step 4, weigh the precipitate mass after adding the S-benzylisothiuronium hydrochloride solution:

[0101] Weigh the mass of an empty graduated conical bottom second centrifuge tube with a capacity of 50 mL in advance, transfer the lower aqueous phase to the empty second centrifuge tube, rinse twice with water, add water to dilute to the 8 mL scale line, add 2.5 mL of the S-benzylisothiuronium hydrochloride solution, let stand for 10 min, centrifuge at 4000×g for 20 min, discard the supernatant, weigh the mass of the second centrifuge tube and the precipitate therein, and subtract the mass of the empty tube to obtain the precipitate mass.

[0102] Step 5, calculate and obtain the content of diglyceride in the sample to be tested:

[0103] Substitute the precipitate mass of the sample to be tested in Step 4 into the linear regression equation Y = -5.0036X + 1.1141 obtained in Example 1, calculate the absolute mass of diglyceride, and then divide the absolute mass by the sample mass to obtain the content of diglyceride in the sample to be tested in this example.

[0104] Perform 3 parallel tests. The measured values and actual values are shown in Table 2. The measured values are expressed as the mean ± standard deviation. The precipitation conditions during the reaction are shown in Figure 3 , and the second centrifuge tubes correspond to diglyceride samples with contents of 13%, 23%, 33%, 38%, 48%, 58%, and 65% from left to right in sequence.

[0105] Table 2 Contents of diglyceride in 7 samples to be tested in Example 3

[0106] Sample Serial Number Measured Value (%) Actual Value (%) 1 12.94±0.44 12.97±0.07 2 23.64±1.05 22.84±0.15 3 33.43±1.41 33.03±0.22 4 38.50±0.85 38.00±0.14 5 49.12±1.14 47.84±0.40 6 59.85±0.98 57.96±0.16 7 64.03±0.34 64.62±0.15

[0107] As can be seen from Table 2, the error value of the sample to be tested in this example is less than 1.50%, and the average error value is 0.79%, indicating that the detection method corresponding to this example has good accuracy; the standard deviation of the measured values in this example is between 0.34% and 1.41%, indicating that the detection method corresponding to this example has good precision.

[0108] Example 4:

[0109] In this example, a rapid detection method for the content of diglyceride in an oil sample is given. This method includes the following steps:

[0110] Step 1, prepare the sample to be tested:

[0111] Weigh 0.250 g of the sample, where the mass fractions of diglyceride are 6.31%, 15%, 42%, and 62% respectively. For each concentration, prepare about 2 g of the sample and then divide it into 6 portions of about 0.3 g of the sample to be tested, and then weigh 0.280 g. Place the weighed samples to be tested into clean 2 mL first centrifuge tubes respectively, and add 150 μL of n-hexane along the tube wall by rotating, so that all the oil samples are washed to the bottom of the tube.

[0112] Step 2, add heptafluorobutyryl imidazole for water bath reaction:

[0113] After preparing the samples to be tested in Step 1, add 150 μL of heptafluorobutyryl imidazole to them, then vortex, seal the tube mouth with a sealing film for water bath reaction. The reaction temperature is 60 °C and the time is 50 min. After the reaction is completed, add 500 μL of distilled water, centrifuge at 3500×g for 20 s, then discard the upper organic phase, and rinse with n-hexane 3 times.

[0114] Step 3, prepare S-benzylisothiourea hydrochloride solution:

[0115] Weigh S-benzylisothiourea hydrochloride, transfer it to a volumetric flask and make up the volume to a certain value to make its concentration 0.070 g / mL, and filter it through a 0.45 μm aqueous filter membrane to obtain a clear solution.

[0116] Step 4, weigh the precipitate mass after adding S-benzylisothiourea hydrochloride solution:

[0117] Weigh the mass of an empty 50 mL graduated conical-bottom second centrifuge tube in advance. Transfer the lower aqueous phase to the empty second centrifuge tube, and rinse it 3 times with water, add water to dilute it to the 5 mL graduation line, add 1.0 mL of S-benzylisothiourea hydrochloride solution, let it stand for 20 min, centrifuge at 10000×g for 5 min, then discard the supernatant, and weigh the mass of the second centrifuge tube and the precipitate in it. Subtract the mass of the empty tube to obtain the precipitate mass.

[0118] Step 5, calculate and obtain the content of diglyceride in the sample to be tested:

[0119] Substitute the precipitate mass of the sample to be tested in Step 4 into the linear regression equation Y = -5.0036X + 1.1141 obtained in Example 1 to calculate the absolute mass of diglyceride, and then divide the absolute mass by the sample mass to obtain the content of diglyceride in the sample to be tested in this example.

[0120] In this example, the content of diglyceride in the sample to be measured is shown in Table 3.

[0121] Table 3 Content of diglyceride in the sample to be measured in Example 4

[0122] Sample Serial Number Measured Value (%) Actual Value (%) Average Relative Standard Deviation (%) 1 6.31±0.24 6.31 3.9 2 15.39±0.41 15.00 2.8 3 42.80±1.21 42.00 2.6 4 62.48±1.70 62.00 2.9

[0123] As can be seen from Table 2, the standard deviation of the measured values in this example is between 0.24% and 1.70%, and the relative standard deviation is between 2.6% and 3.9%, which proves that the precision and stability of the detection method corresponding to this example are good.

[0124] Example 5:

[0125] In this example, a rapid detection method for the content of diglyceride in an oil sample is given. The method includes the following steps:

[0126] Step 1, prepare the sample to be measured:

[0127] Weigh 0.270 g of the sample, which is a commercially available oil sample. Place the weighed sample to be measured in a clean 2 mL first centrifuge tube respectively, and add 250 μL of n-hexane along the tube wall and rotate to wash the oil sample to the bottom of the tube. If the first measurement result of the sample exceeds 70%, then add the corresponding proportion of n-hexane to dilute it in the first centrifuge tube to make its concentration within the standard curve range, and finally multiply by the corresponding dilution factor to obtain the content of diglyceride in the sample.

[0128] Step 2, add heptafluorobutyryl imidazole for water bath reaction:

[0129] After preparing the sample to be measured in Step 1, add 250 μL of heptafluorobutyryl imidazole and vortex it, seal the tube mouth with a sealing film for water bath reaction. The reaction temperature is 55 °C and the time is 45 min. After the reaction is completed, add 350 μL of distilled water, centrifuge at 2500×g for 35 s, then discard the upper organic phase, and rinse it 3 times with n-hexane.

[0130] Step 3, prepare S-benzylisothiuronium hydrochloride solution:

[0131] Weigh S-benzylisothiuronium hydrochloride, transfer it to a volumetric flask and make the volume constant to make its concentration 0.040 g / mL, and filter it through a 0.45 μm aqueous filter membrane to obtain a clear solution.

[0132] Step 4, add S-benzylisothiuronium hydrochloride solution and then weigh the precipitate mass:

[0133] Weigh the mass of a 50 mL graduated conical bottom second centrifuge tube in advance. Transfer the lower aqueous phase to the empty second centrifuge tube, rinse it with water 3 times, add water to dilute it to the 12 mL graduation line, add 3.5 mL of S-benzylisothiourea hydrochloride solution, let it stand for 35 min, centrifuge at 7000×g for 15 min, then discard the supernatant. Weigh the mass of the second centrifuge tube and the precipitate in it, and subtract the mass of the empty tube to obtain the mass of the precipitate.

[0134] Step Five, calculate and obtain the content of diglyceride in the sample to be measured:

[0135] Substitute the precipitate mass of the sample to be measured in Step Four into the linear regression equation Y = -5.0036X + 1.1141 obtained in Example 1 to calculate the absolute mass of diglyceride. Then divide the absolute mass by the sample mass to obtain the content of diglyceride in the sample to be measured in this example.

[0136] The contents of diglyceride in the 5 samples to be measured in this example are shown in Table 3.

[0137] Table 3 Contents of diglyceride in the 5 samples to be measured in Example 5

[0138] Sample Serial Number Measured Value (%) Label Value (%) 1 57.26±3.97 60 2 79.73±1.34 80 3 54.45±2.62 50 4 53.83±1.55 61.5 5 56.89±1.91 60.5

[0139] As can be seen from Table 3, the error values of the samples to be measured in this example are less than 4.00%, and the average error value is 3.74%, which proves that the detection method corresponding to this example has good accuracy; the standard deviation of the measured values in this example is between 1.34% and 3.97%, indicating that the detection method corresponding to this example has good precision. From the results of sample No. 2, it can be seen that for more than 70% of the samples, they can be measured after dilution with n-hexane, and the measurement results also have good precision and accuracy.

Claims

1. A method for rapid detection of diglyceride content in an oil sample, characterized in that: The method involves heating the oil sample to be tested with an excess of heptafluorobutyrylimidazole in a water bath to react, thereby generating heptafluorobutyryl diglycerol and imidazole; after the reaction is completed, water is added to react with the remaining heptafluorobutyrylimidazole to generate imidazole and heptafluorobutyric acid; S-benzyl isothiourea hydrochloride is then added to react with heptafluorobutyric acid to generate a precipitate that is insoluble in water; and the content of diglycerol is calculated based on the amount of precipitate generated.

2. A rapid detection method for diglyceride content in an oil sample, characterized in that: The method comprises the following steps: Step 1: Prepare the sample to be tested: Weigh the sample to be tested, place the weighed sample to be tested in a first centrifuge tube, and add n-hexane to flush the sample to be tested to the bottom of the first centrifuge tube; Step 2, add heptafluorobutyrylimidazole to react in a water bath: Add heptafluorobutyrylimidazole to the first centrifuge tube in step 1, vortex, seal the tube mouth of the first centrifuge tube with a sealing film, heat in a water bath to react, add distilled water after the reaction is completed, centrifuge and discard the upper organic phase; Step 3, prepare S-benzyl isothiourea hydrochloride solution: Weigh S-benzyl isothiourea hydrochloride, transfer to a volumetric flask to make up to volume, and filter to obtain a clear S-benzyl isothiourea hydrochloride solution; Step 4: After adding S-benzyl isothiourea hydrochloride solution, weigh the precipitate mass: Weigh the mass of the empty second centrifuge tube in advance, transfer the lower aqueous phase obtained in step 2 to the empty second centrifuge tube, dilute with water, add S-benzylisothiourea hydrochloride solution, centrifuge and discard the supernatant after standing for a period of time, weigh the total mass of the second centrifuge tube and the precipitate in the second centrifuge tube, and subtract the mass of the empty second centrifuge tube to obtain the mass of the precipitate; Step 5: Calculate and obtain the content of diglyceride in the sample to be tested: Substitute the precipitate mass obtained in step 4 into the linear regression equation with the absolute mass of diglyceride as the independent variable X and the precipitate mass as the dependent variable Y to obtain the absolute mass of diglyceride in the sample to be tested. Then divide the absolute mass by the sample mass to calculate the content of diglyceride in the sample to be tested.

3. The rapid detection method for diglyceride content in an oil sample as claimed in claim 2, characterized in that: In step 1, the mass of the sample to be tested is 0.250 g to 0.350 g; In step 1, the volume of n-hexane added is 50 μL to 300 μL.

4. The rapid detection method for diglyceride content in an oil sample as claimed in claim 2, characterized in that: In step 2, the amount of heptafluorobutyrylimidazole added is 50 μL to 300 μL; In step 2, the reaction conditions of the water bath reaction are: temperature 45°C to 95°C, reaction time 20min to 60min; In step 2, the volume of distilled water added is 100 μL to 500 μL; In step 2, the centrifugal force is (500-3500)×g, and the centrifugal time is 10-60s; In step 2, the supernatant is discarded and rinsed with n-hexane 2 to 4 times.

5. The method for rapid detection of diglyceride content in an oil sample as claimed in claim 2, characterized in that: In step 3, the concentration of the S-benzyl isothiourea hydrochloride solution is 0.030-0.080 g / mL, and filtration is performed using a 0.45 μm water filter membrane.

6. The rapid detection method for diglyceride content in an oil sample as claimed in claim 2, characterized in that: In step 4, the second centrifuge tube is a 50 mL conical bottom centrifuge tube with a scale, which is rinsed with water 2 to 4 times during transfer and diluted with water to the 5 to 15 mL scale line; In step 4, the amount of S-benzyl isothiourea hydrochloride solution added is 1.0 mL to 5.0 mL; In step 4, the standing time is 10 min to 50 min, the centrifugal force is (4000 to 10000) × g, and the centrifugal time is 5 to 20 min.

7. The method for rapid detection of diglyceride content in an oil sample as claimed in claim 2, characterized in that: In step 5, the method for obtaining the linear regression equation is: mixing the diglyceride standard and the triglyceride oil in different mass ratios to obtain multiple standard samples, measuring and obtaining the precipitate mass of the multiple standard samples according to the above steps 1 to 4, taking the absolute mass of diglyceride as the independent variable X, taking the precipitate mass obtained by the standard sample reaction as the dependent variable Y, obtaining the linear regression equation and drawing a standard curve.

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