Method for identifying adulteration of tea oil based on C-4 dimethyl sterol composition

Abstract

The invention provides a method for identifying adulteration of tea oil based on composition of C-4 dimethyl sterol, which comprises the following steps: carrying out least square linear regression analysis on the proportion of C-4 dimethyl sterol peak area in the total peak area in collected tea oil adulteration samples with different adulteration proportions and different adulteration proportions, and determining the adulteration ratio of the tea oil adulteration samples according to the Filipin III fluorescence signal intensity. An adulteration detection model is established, so that whether other grease adulteration exists in the tea oil sample or not is judged, and the adulteration proportion can be judged according to the ratio of the Filipin III fluorescence signal intensity to the tea oil standard threshold value. The method not only can identify adulteration of the tea oil, but also can detect the adulteration proportion, has the characteristics of simplicity, rapidness, good repeatability, high accuracy and the like, and can be practically applied to adulteration detection work of the tea oil.

Description

Technical Field

[0001] The invention belongs to the field of oil chemical analysis and detection, relates to the application of fluorescent probe technology in oil component analysis, and specifically relates to a method for identifying tea oil adulteration based on the composition of C-4 dimethyl sterol. Background Art

[0002] Plant sterols are abundant in oils. The key step of sterol biosynthesis is to control the carbon flow into the isoprene pathway to generate C4-dimethyl sterols (cycloartenol and 24-methylenecycloartenol), which are then converted into common methyl-free sterols (sitosterol and campesterol). Sterol methyltransferase (SMT) and sterol methyloxidase (SMO) play a key role in the conversion of C4-dimethyl sterols into methyl-free sterols. Studies have shown that the differential expression of sterol-related metabolic genes produces unique sterol fingerprints for oils. For example, camellia oleifera has lower SMT1 gene expression, and its sterol fingerprint has a higher content of C4-dimethyl sterols; soybean has a higher transcription level of SMO, so the soybean oil sterol fingerprint contains a smaller proportion of C4-dimethyl sterols. While oil crops form differentiated sterol fingerprints during evolution, sterol fingerprints between the same species are still highly conserved, such as camellia oil, tea seed oil and shea butter. Therefore, the structural composition of plant sterols can be analyzed and the identification of edible oil adulteration can be achieved based on the plant sterol fingerprint information. The molecular polarity generated by the 3β-hydroxyl group (3β-OH) in phytosterols is the main reason for the affinity with Filipin III. Compared with methyl-free sterols, C4-dimethyl sterols have weaker polarity due to the shielding effect of the two methyl groups (CH3) next to 3β-OH, and cannot be compactly arranged in the interface layer of the measurement medium. The affinity with Filipin decreases, and the fluorescence intensity produced is weaker. The difference in the proportion of different C4-dimethyl sterols and methyl-free sterols in edible oils will affect the affinity efficiency of Filipin III. When the total amount of sterol content in the measurement medium is the same, the fluorescence signal formed after binding with Filipin III is also specific. This fluorescence signal feature can be used for adulteration detection of oils and fats. CN106950241B discloses a method for predicting the types and contents of other adulterated oils in tea oil, and CN112305108A discloses a method for detecting adulteration of tea seed oil based on the ratio of oleic acid / behenic acid and β-amyrinol / campesterol. Both of them have carried out adulteration detection on tea oil, but the technical schemes of both involve not only complicated extraction processes and sample detection processes, such as nuclear magnetic resonance 1H-NMR detection and OPLS-DA analysis; but also the analysis and processing process of the final data of the samples is complicated, which is difficult to meet the requirements of efficient identification of edible oil adulteration. CN103217393B discloses a method for detecting adulterated tea oil. On the basis of detecting refractive index, iodine value, saponification value and oleic acid content, the invention adds full-wave scanning of ultraviolet spectrum. However, the invention needs to be fitted and identified under the model of known target adulteration components, and cannot achieve accurate qualitative and quantitative analysis of unknown components. At present, the products in my country's edible oil market are diversified, segmented and high-end. Improving the ability to identify edible oil adulteration is of great significance to combating edible oil adulteration and ensuring the quality and safety of edible oil products. Phytosterols in oils and fats have high inertness, stability, and specific distribution in oils, can produce specific fingerprint information, and can be quickly identified by Filipin III fluorescent probes. Identification of edible oil adulteration based on phytosterol fingerprint information has important application value. Summary of the invention

[0003] Technical problem to be solved: In view of the defects of the existing related technologies, the purpose of the present invention is to provide a method for identifying tea oil adulteration based on the composition of C-4 dimethyl sterols, and the specific steps are: preparation of tea oil adulteration samples, fluorescence signal collection, phytosterol composition analysis, establishment of adulteration detection model and adulteration discrimination. The method can identify whether tea oil is adulterated with other oils and determine its adulteration ratio, so as to solve the technical problems of cumbersome and inefficient detection methods of edible oil adulteration with other oils in the prior art, and low accuracy of qualitative and quantitative detection of adulteration.

[0004] Technical solution: A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition, comprising the following steps: S1. Extraction of phytosterols: Mix tea oil with other oils to obtain tea oil adulterated samples with different adulteration ratios, and quickly extract the unsaponifiable matter of tea oil and tea oil adulterated samples to obtain phytosterols; S2. Analysis of phytosterol composition: After the phytosterols in the tea oil adulterated sample were derivatized, GC-MS was performed to determine the peak area of ​​C-4 dimethyl sterol and the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area a were obtained based on the gas chromatography-mass spectrometry and fingerprint data; S3. Fluorescence signal acquisition: Filipin III fluorescence probe was used to detect and measure the fluorescence signal intensity of phytosterols in tea oil. The average value was taken as the reference threshold value f for identifying tea oil adulteration. At the same time, the fluorescence signal intensity x of phytosterols in tea oil adulterated samples was collected. S4. Adulteration detection model establishment: The ratio a of the peak area of ​​C-4 dimethyl sterols in the adulterated tea oil samples with different adulteration ratios collected was subjected to least squares linear regression analysis with different adulteration ratios y, and the regression equation y=-42.083a+99.281 was obtained. Combined with the Filipin III fluorescence signal intensity x, an adulteration detection model was established. S5. Adulteration determination: Rapidly extract the unsaponifiable matter of the unknown oil sample to be tested, match the measured fluorescence signal intensity x and the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area a with the adulteration detection model, determine whether the tea oil is adulterated with other oils, and calculate the adulteration ratio by the ratio difference method, adulteration ratio = |xf| / f×100%. Furthermore, the tea oil in step S1 is camellia oil; the other oils include one or more of peanut oil, soybean oil, cottonseed oil, and rapeseed oil. Furthermore, the different adulteration ratios in step S1 are 5%, 10%, 15%, 20%, 30% and 50% respectively. Furthermore, the preparation method of phytosterols in step S1 is specifically as follows: 8% NaOH solution is added to tea oil and tea oil adulterated sample respectively, vortexed for 0.5-1.0 min, and saponified under ultrasonic heating conditions for 15 min to obtain a treated liquid; an equal volume of deionized water is first added to the treated liquid, shaken and diluted, and then an equal volume of n-hexane is added to shake and stand until the aqueous phase and the organic phase are separated, then the unsaponifiable matter is extracted, and evaporated with N2 to obtain phytosterols. Furthermore, the specific steps of the derivatization treatment in step S2 are: adding 200 μL of silanization reagent to the plant sterols in the tea oil adulterated sample, placing it in a 75°C water bath for 30 minutes for derivatization treatment, and waiting for the sample to cool to room temperature, blowing it dry with N2 again, redissolving it in 100 μL of n-hexane, and filtering it through a 0.22 μm organic microporous filter membrane for testing. Furthermore, the silanization reagent is a mixed solution of BSTFA and TMCS in a volume ratio of 99:1. Furthermore, the GC-MS measurement conditions in step S2 are: Chromatographic conditions: Chromatographic column: DB-5 column, 30m×0.25mm, 0.25μm; Temperature program: the initial column temperature was set at 200°C and maintained for 0.5 min; the temperature was increased to 250°C at a rate of 15°C / min and maintained for 8 min; a "low-rate gradient temperature program" was used with a temperature increase rate of 1°C / min, and the column temperature was maintained for 1.5 min after it reached 270°C; Detector and injection port temperature: 290°C; Injection volume: 1.0 μL; Split ratio: 1:100; Carrier gas: 99.99% high purity helium, flow rate 1.2mL / min; Total program running time: 40min; Mass spectrometry conditions: Inlet temperature: 300°C; Solvent delay: 10min; Ion source: EI; Electron energy: 70eV; Scan time: 0.5s; Mass scan range: 50-550 m / z. Furthermore, the specific steps of Filipin III fluorescent probe detection in step S3 are: adding 100 μL of Filipin III working solution to the phytosterols in the tea oil and tea oil adulterated samples, incubating at room temperature for 30 minutes in the dark, removing the staining solution, and performing fluorescence analysis. Furthermore, the fluorescence signal intensity in step S3 refers to the fluorescence absorption collected by the Filipin III probe under 300-400nm excitation light and 400-500nm emission light. Beneficial effects: 1. The present invention utilizes Filipin III fluorescent probe to have specific affinity with phytosterol 3β-OH, and determines the adulteration amount by means of characteristic absorption of Filipin III fluorescent probe under 300-400nm excitation light and 400-500nm emission light; the proportion of C4-dimethyl sterol in tea oil is high, and the polarity of C4-dimethyl sterol is weaker due to the shielding effect of two methyl groups (CH3) next to 3β-OH, so the affinity with Filipin III is reduced; compared with other oils and fats, which are richer in methyl-free sterols, its weaker Filipin III fluorescence intensity value can be used to indicate the adulteration ratio of tea oil; compared with the traditional technology that only relies on GC-MS, infrared spectroscopy or nuclear magnetic resonance, the Filipin III fluorescence characteristic can not only identify the adulteration amount, but also has the characteristics of simplicity, rapidity, good repeatability and high accuracy. 2. The present invention utilizes a high concentration of 8% NaOH solution to quickly extract phytosterols. Under the action of the high concentration NaOH solution, a large amount of hydroxide ions (OH-) can be effectively provided in a short time. This alkaline condition helps to separate the phytosterol molecules from the oil. Since the hydroxide ions can form water-soluble complexes with the hydroxyl groups in the sterol molecules, the sterols are more soluble under alkaline conditions. In addition, the NaOH solution at a concentration of 8% can usually selectively and fully dissolve the sterols, while the dissolving effect on other plant components such as tocopherol and polyphenols is relatively small. This selectivity can improve the purity and yield of sterol extraction, which is beneficial to Filipin III fluorescence detection and GC-MS composition analysis of phytosterols. 3. The present invention uses a regression analysis method to establish an adulteration detection model for adulterated tea oil, which has a good fitting constant and high accuracy. The use of Filipin III fluorescent probe detection alone has many influencing factors and a relatively large error. Therefore, the present invention uses GC-MS composition analysis and Filipin III fluorescent probe detection to construct an adulteration detection model, which makes the model more accurate. At the same time, the Filipin III fluorescence intensity threshold and adulteration detection model provided by the present invention can be repeatedly used to facilitate rapid detection of different batches. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 This is a flow chart for identifying tea oil adulteration based on C-4 dimethyl sterol composition; Figure 2 is the GC-MS chromatogram of phytosterols in tea oil samples; Figure 3 The linear regression equations obtained for the tea oils and tea oil adulterated samples prepared in Examples 1-6. DETAILED DESCRIPTION The present invention is further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments: Example 1 A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition, comprising the following steps: S1. Extraction of phytosterols: Add high alkalinity saponification solution (8% NaOH) to tea oil sample 1, vortex for 0.5-1.0 min, and saponify under ultrasonic heating for 15 min to obtain a treated solution. Add an equal volume of deionized water to the treated solution, shake well and dilute, then add an equal volume of n-hexane, shake and stand until the aqueous phase and the organic phase are separated, extract the unsaponifiable matter, evaporate with N2 to obtain phytosterols; S2. Analysis of plant sterol composition: 200 μL of silanization reagent BSTFA+TMCS (99:1, v / v) was added to the plant sterol, and the mixture was placed in a 75°C water bath for 30 min for derivatization. The sample was cooled to room temperature, dried with N2 again, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter membrane, and subjected to GC-MS determination. According to the gas chromatography-mass spectrometry and fingerprint data, the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area was obtained. S3. Fluorescence signal collection: add 100 μL of Filipin III working solution to the phytosterols, incubate at room temperature for 30 min in the dark, remove the staining solution, perform Filipin III fluorescent probe detection, and collect the fluorescence signal intensity x of the phytosterols in tea oil. Example 2 A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition, comprising the following steps: S1. Extraction of phytosterols: Add high alkalinity saponification solution (8% NaOH) to tea oil sample 2, vortex for 0.5-1.0 min, and saponify under ultrasonic heating for 15 min to obtain a treated solution. Add an equal volume of deionized water to the treated solution, shake well and dilute, then add an equal volume of n-hexane, shake and stand until the aqueous phase and the organic phase are separated, extract the unsaponifiable matter, evaporate with N2 to obtain phytosterols; S2. Analysis of plant sterol composition: 200 μL of silanization reagent BSTFA+TMCS (99:1, v / v) was added to the plant sterol, and the mixture was placed in a 75°C water bath for 30 min for derivatization. The sample was cooled to room temperature, dried with N2 again, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter membrane, and subjected to GC-MS determination. According to the gas chromatography-mass spectrometry and fingerprint data, the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area was obtained. S3. Fluorescence signal collection: add 100 μL of Filipin III working solution to the phytosterols, incubate at room temperature for 30 min in the dark, remove the staining solution, perform Filipin III fluorescent probe detection, and collect the fluorescence signal intensity x of the phytosterols in tea oil. Example 3 A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition, comprising the following steps: S1. Extraction of phytosterols: Add high alkalinity saponification liquid (8% NaOH) to the adulterated tea oil sample (tea oil sample 95% + 5% soybean oil), vortex for 0.5-1.0 min, and saponify under ultrasonic heating for 15 min to obtain a treated liquid. First, add an equal volume of deionized water to the treated liquid and shake to dilute it. Then, add an equal volume of n-hexane and shake it until the aqueous phase and the organic phase are separated. Then extract the unsaponifiable matter and evaporate it with N2 to obtain phytosterols. S2. Analysis of plant sterol composition: 200 μL of silanization reagent BSTFA+TMCS (99:1, v / v) was added to the plant sterol, and the mixture was placed in a 75°C water bath for 30 min for derivatization. The sample was cooled to room temperature, dried with N2 again, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter membrane, and subjected to GC-MS determination. According to the gas chromatography-mass spectrometry and fingerprint data, the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area was obtained. S3. Fluorescence signal collection: add 100 μL of Filipin III working solution to the plant sterols, incubate at room temperature for 30 min in the dark, remove the staining solution, perform Filipin III fluorescent probe detection, and collect the fluorescence signal intensity x of the plant sterols in the tea oil adulterated sample. Example 4 A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition, comprising the following steps: S1. Extraction of phytosterols: Add high alkalinity saponification solution (8% NaOH) to the adulterated tea oil sample (tea oil sample 50% + 50% peanut oil), vortex for 0.5-1.0 min, and saponify under ultrasonic heating for 15 min to obtain a treated solution. First, add an equal volume of deionized water to the treated solution and shake to dilute it. Then, add an equal volume of n-hexane and shake it until the aqueous phase and the organic phase are separated. Then extract the unsaponifiable matter and evaporate it with N2 to obtain phytosterols. S2. Analysis of plant sterol composition: 200 μL of silanization reagent BSTFA+TMCS (99:1, v / v) was added to the plant sterol, and the mixture was placed in a 75°C water bath for 30 min for derivatization. The sample was cooled to room temperature, dried with N2 again, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter membrane, and subjected to GC-MS determination. According to the gas chromatography-mass spectrometry and fingerprint data, the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area was obtained. S3. Fluorescence signal collection: add 100 μL of Filipin III working solution to the plant sterols, incubate at room temperature for 30 min in the dark, remove the staining solution, perform Filipin III fluorescent probe detection, and collect the fluorescence signal intensity x of the plant sterols in the tea oil adulterated sample. Example 5 A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition, comprising the following steps: S1. Extraction of phytosterols: Add high alkalinity saponification liquid (8% NaOH) to the adulterated tea oil sample (tea oil sample 80% + 10% soybean oil + 10% rapeseed oil), vortex for 0.5-1.0 min, and saponify under ultrasonic heating for 15 min to obtain a treated liquid. First, add an equal volume of deionized water to the treated liquid and shake to dilute it. Then, add an equal volume of n-hexane and shake it until the aqueous phase and the organic phase are separated. Then extract the unsaponifiable matter and evaporate it with N2 to obtain phytosterols. S2. Analysis of plant sterol composition: 200 μL of silanization reagent BSTFA+TMCS (99:1, v / v) was added to the plant sterol, and the mixture was placed in a 75°C water bath for 30 min for derivatization. The sample was cooled to room temperature, dried with N2 again, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter membrane, and subjected to GC-MS determination. According to the gas chromatography-mass spectrometry and fingerprint data, the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area was obtained. S3. Fluorescence signal collection: add 100 μL of Filipin III working solution to the plant sterols, incubate at room temperature for 30 min in the dark, remove the staining solution, perform Filipin III fluorescent probe detection, and collect the fluorescence signal intensity x of the plant sterols in the tea oil adulterated sample. Example 6 A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition, comprising the following steps: S1. Extraction of phytosterols: Add high alkalinity saponification liquid (8% NaOH) to the adulterated tea oil sample (tea oil sample 70% + 10% soybean oil + 10% rapeseed oil + 10% cottonseed oil), vortex for 0.5-1.0 min, and saponify under ultrasonic heating for 15 min to obtain a treated liquid. First, add an equal volume of deionized water to the treated liquid and shake to dilute it. Then, add an equal volume of n-hexane and shake it until the aqueous phase and the organic phase are separated. Then extract the unsaponifiable matter and evaporate it with N2 to obtain phytosterols. S2. Analysis of plant sterol composition: 200 μL of silanization reagent BSTFA+TMCS (99:1, v / v) was added to the plant sterol, and the mixture was placed in a 75°C water bath for 30 min for derivatization. The sample was cooled to room temperature, dried with N2 again, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter membrane, and subjected to GC-MS determination. According to the gas chromatography-mass spectrometry and fingerprint data, the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area was obtained. S3. Fluorescence signal collection: add 100 μL of Filipin III working solution to the plant sterols, incubate at room temperature for 30 min in the dark, remove the staining solution, perform Filipin III fluorescent probe detection, and collect the fluorescence signal intensity x of the plant sterols in the tea oil adulterated sample. The results of the determination of the C-4 dimethyl sterol ratio and Filipin III fluorescence intensity of the tea oil and tea oil adulterated samples prepared in Examples 1-6 are shown in Table 1 below: Table 1 Test results of tea oil and tea oil adulterated samples prepared in Examples 1-6 As shown in Table 1, the fluorescence intensities of Filipin III in Example 1 and Example 2 are 4.11 and 4.32, respectively, and their average values ​​are taken as the reference threshold value f=4.215. The ratio a of the peak area of ​​C-4 dimethyl sterol in the tea oil prepared in Examples 1-6 and the tea oil adulterated samples with different adulteration ratios to the total peak area and the different actual adulteration ratios y were subjected to least squares linear regression analysis, and the linear regression equation y=-42.083a+99.281(R 2 =0.9878), combined with the Filipin III fluorescence signal intensity x, an adulteration detection model was established, such as Figure 1 shown. Example 7 A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition, comprising the following steps: S1. Extraction of phytosterols: Add high alkalinity saponification liquid (8% NaOH) to the adulterated tea oil sample (tea oil sample 80% + 20% rapeseed oil), vortex for 0.5-1.0 min, and saponify under ultrasonic heating for 15 min to obtain a treated liquid. First, add an equal volume of deionized water to the treated liquid, shake well and dilute, then add an equal volume of n-hexane, shake and stand until the aqueous phase and the organic phase are separated, extract the unsaponifiable matter, evaporate with N2 to obtain phytosterols; S2. Analysis of plant sterol composition: 200 μL of silanization reagent BSTFA+TMCS (99:1, v / v) was added to the plant sterol, and the mixture was placed in a 75°C water bath for 30 min for derivatization. The sample was cooled to room temperature, dried with N2 again, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter membrane, and subjected to GC-MS determination. According to the gas chromatography-mass spectrometry and fingerprint data, the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area was obtained. S3. Fluorescence signal collection: add 100 μL of Filipin III working solution to the plant sterols, incubate at room temperature for 30 min away from light, remove the staining solution, perform Filipin III fluorescent probe detection, and collect the fluorescence signal intensity x of the plant sterols in the tea oil adulterated sample; S4. Adulteration determination: Rapidly extract the unsaponifiable matter of the oil sample to be tested, match the measured fluorescence signal intensity x and the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area a with the adulteration detection model, determine whether the tea oil is adulterated with other oils, and calculate the adulteration ratio by the ratio difference method, adulteration ratio = |xf| / f×100%. Example 8 A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition, comprising the following steps: S1. Extraction of phytosterols: Add high alkalinity saponification liquid (8% NaOH) to the adulterated tea oil sample (tea oil sample 90% + 10% cottonseed oil), vortex for 0.5-1.0 min, and saponify under ultrasonic heating for 15 min to obtain a treated liquid. First, add an equal volume of deionized water to the treated liquid and shake to dilute it. Then, add an equal volume of n-hexane and shake it until the aqueous phase and the organic phase are separated. Then extract the unsaponifiable matter and evaporate it with N2 to obtain phytosterols. S2. Analysis of plant sterol composition: 200 μL of silanization reagent BSTFA+TMCS (99:1, v / v) was added to the plant sterol, and the mixture was placed in a 75°C water bath for 30 min for derivatization. The sample was cooled to room temperature, dried with N2 again, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter membrane, and subjected to GC-MS determination. According to the gas chromatography-mass spectrometry and fingerprint data, the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area was obtained. S3. Fluorescence signal collection: add 100 μL of Filipin III working solution to the plant sterols, incubate at room temperature for 30 min away from light, remove the staining solution, perform Filipin III fluorescent probe detection, and collect the fluorescence signal intensity x of the plant sterols in the tea oil adulterated sample; S4. Adulteration determination: Rapidly extract the unsaponifiable matter of the unknown oil sample to be tested, match the measured fluorescence signal intensity x and the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area a with the adulteration detection model, determine whether the tea oil is adulterated with other oils, and calculate the adulteration ratio by the ratio difference method, adulteration ratio = |xf| / f×100%. Verify the accuracy of the equation: Substitute the ratio a of the peak area of ​​C-4 dimethyl sterol to the total peak area in the tea oil prepared in Examples 1-8 and the tea oil adulterated samples with different adulteration ratios into the linear regression equation to obtain the preliminary adulteration ratio, and then confirm the adulteration ratio by the fluorescence intensity x, as shown in Table 2 below: Table 2 Predicted adulteration identification results of tea oil and tea oil adulterated samples prepared in Examples 1-8 It can be seen from Examples 1-8 in Table 2 that the relative deviations of the tea oil adulteration ratio identified by using the C-4 dimethyl sterol ratio (a) and the Filipin III fluorescence intensity (x) are all less than 3.42%, and the average relative deviation is 2.05%, which shows that the adulteration identification result is accurate and the effect is stable. From the comparison between Example 3 and Example 4, it can be seen that the smaller the proportion of other oils in the adulterated tea oil sample, the greater the relative deviation obtained by the detection, which shows that the adulteration identification model is more accurate in the adulteration identification detection of high-proportion adulterated tea oil samples. Comparative Example 1 A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition, comprising the following steps: S1. Extraction of phytosterols: Add high alkalinity saponification solution (8% NaOH) to peanut oil, vortex for 0.5-1.0 min, and saponify under ultrasonic heating for 15 min to obtain a treatment solution. First, add an equal volume of deionized water to the treatment solution and shake to dilute it. Then, add an equal volume of n-hexane and shake it until the aqueous phase and the organic phase separate. Then extract the unsaponifiable matter and evaporate it with N2 to obtain phytosterols. S2. Analysis of plant sterol composition: 200 μL of silanization reagent BSTFA+TMCS (99:1, v / v) was added to the plant sterol, and the mixture was placed in a 75°C water bath for 30 min for derivatization. The sample was cooled to room temperature, dried with N2 again, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter membrane, and subjected to GC-MS determination. According to the gas chromatography-mass spectrometry and fingerprint data, the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area was obtained. S3. Fluorescence signal collection: add 100 μL of Filipin III working solution to the phytosterols, incubate at room temperature for 30 min away from light, remove the staining solution, perform Filipin III fluorescent probe detection, and collect the fluorescence signal intensity x of the phytosterols in peanut oil; S4. Adulteration determination: The obtained fluorescence signal intensity x and the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area a are matched with the adulteration detection model to determine whether the peanut oil is adulterated with other oils, and the adulteration ratio is calculated based on the difference method between the fluorescence signal intensity and the reference threshold of tea oil. Comparative Example 2 A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition, comprising the following steps: S1. Extraction of phytosterols: Add high alkalinity saponification solution (8% NaOH) to soybean oil, vortex for 0.5-1.0 min, and saponify under ultrasonic heating for 15 min to obtain a treatment solution. Add an equal volume of deionized water to the treatment solution and shake to dilute it. Then add an equal volume of n-hexane and shake it until the aqueous phase and the organic phase separate. Extract the unsaponifiable matter and evaporate it with N2 to obtain phytosterols. S2. Analysis of plant sterol composition: 200 μL of silanization reagent BSTFA+TMCS (99:1, v / v) was added to the plant sterol, and the mixture was placed in a 75°C water bath for 30 min for derivatization. The sample was cooled to room temperature, dried with N2 again, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter membrane, and subjected to GC-MS determination. According to the gas chromatography-mass spectrometry and fingerprint data, the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area was obtained. S3. Fluorescence signal collection: add 100 μL of Filipin III working solution to the phytosterols, incubate at room temperature for 30 min away from light, remove the staining solution, perform Filipin III fluorescent probe detection, and collect the fluorescence signal intensity x of the phytosterols in soybean oil; S4. Adulteration determination: The obtained fluorescence signal intensity x and the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area a are matched with the adulteration detection model to determine whether the soybean oil is adulterated with other oils, and the adulteration ratio is calculated based on the difference method between the fluorescence signal intensity and the reference threshold of tea oil. Comparative Example 3 A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition, comprising the following steps: S1. Extraction of phytosterols: Add high alkalinity saponification solution (8% NaOH) to cottonseed oil, vortex for 0.5-1.0 min, and saponify under ultrasonic heating for 15 min to obtain a treated solution. Add an equal volume of deionized water to the treated solution, shake well to dilute, then add an equal volume of n-hexane, shake and stand until the aqueous phase and the organic phase are separated, extract the unsaponifiable matter, evaporate with N2 to obtain phytosterols; S2. Analysis of plant sterol composition: 200 μL of silanization reagent BSTFA+TMCS (99:1, v / v) was added to the plant sterol, and the mixture was placed in a 75°C water bath for 30 min for derivatization. The sample was cooled to room temperature, dried with N2 again, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter membrane, and subjected to GC-MS determination. According to the gas chromatography-mass spectrometry and fingerprint data, the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area was obtained. S3. Fluorescence signal collection: add 100 μL of Filipin III working solution to the phytosterols, incubate at room temperature for 30 min away from light, remove the staining solution, perform Filipin III fluorescent probe detection, and collect the fluorescence signal intensity x of the phytosterols in cottonseed oil; S4. Adulteration determination: The obtained fluorescence signal intensity x and the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area a are matched with the adulteration detection model to determine whether the cottonseed oil is adulterated with other oils, and the adulteration ratio is calculated based on the difference method between the fluorescence signal intensity and the reference threshold of tea oil. Comparative Example 4 A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition, comprising the following steps: S1. Extraction of phytosterols: A high alkalinity saponification solution (8% NaOH) was added to rapeseed oil, vortexed for 0.5-1.0 min, and saponified under ultrasonic heating for 15 min to obtain a treated solution. An equal volume of deionized water was first added to the treated solution, and then an equal volume of n-hexane was added and shaken to stand until the aqueous phase and the organic phase were separated, and the unsaponifiable matter was extracted and evaporated with N2 to obtain phytosterols; S2. Analysis of plant sterol composition: 200 μL of silanization reagent BSTFA+TMCS (99:1, v / v) was added to the plant sterol, and the mixture was placed in a 75°C water bath for 30 min for derivatization. The sample was cooled to room temperature, dried with N2 again, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter membrane, and subjected to GC-MS determination. According to the gas chromatography-mass spectrometry and fingerprint data, the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area was obtained. S3. Fluorescence signal collection: add 100 μL of Filipin III working solution to the phytosterols, incubate at room temperature for 30 min away from light, remove the staining solution, perform Filipin III fluorescent probe detection, and collect the fluorescence signal intensity x of the phytosterols in rapeseed oil; S4. Adulteration determination: The obtained fluorescence signal intensity x and the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area a are matched with the adulteration detection model to determine whether the rapeseed oil is adulterated with other oils, and the adulteration ratio is calculated based on the difference method between the fluorescence signal intensity and the reference threshold of tea oil. The fingerprint information of phytosterol composition measured by GC-MS is shown in Table 3. Table 3 Fingerprint information of phytosterol composition measured by GC-MS Time (min) Chinese name Molecular formula Molecular weight 26.39 Ergo-7,22-dien-3β-ol <![CDATA[C 28 H 46 O]]> 398.66 27.08 Cholesterol <![CDATA[C 27 H 46 O]]> 386.62 28.53 Ergo-3,25-diene-3β-ol <![CDATA[C 28 H 46 O]]> 398.35 28.82 Campesterol <![CDATA[C 28 H 48 O]]> 400.69 29.16 Lupanol <![CDATA[C 30 H 52 O]]> 428.73 29.86 Stigmasterol <![CDATA[C 29 H 48 O]]> 412.70 30.39 Ergo-8(14)-en-3β-ol <![CDATA[C 28 H 48 O]]> 400.68 30.58 Ergo-5,22-dien-3β-ol <![CDATA[C 28 H 46 O]]> 398.66 30.91 Borasterol <![CDATA[C 29 H 48 O]]> 412.69 31.29 Ergo-7-en-3β-ol <![CDATA[C 28 H 48 O]]> 400.68 31.70 Bletilla striata <![CDATA[C 30 H 50 O]]> 426.72 32.36 β-Sitosterol <![CDATA[C 29 H 50 O]]> 414.72 32.68 β-Amyl alcohol <![CDATA[C 30 H 50 O]]> 426.73 32.88 Sitostanol <![CDATA[C 29 H 48 O]]> 416.73 33.02 Mannitrophenol <![CDATA[C 30 H 50 O]]> 426.73 33.04 δ5-Avenasterol <![CDATA[C 29 H 48 O]]> 412.70 33.33 Myclobutanol <![CDATA[C 30 H 50 O]]> 426.73 33.53 Stigmasterol <![CDATA[C 29 H 50 O]]> 414.41 33.66 Parker <![CDATA[C 30 H 50 O]]> 426.72 34.01 Isofuccasterol <![CDATA[C 29 H 48 O]]> 412.70 34.36 α-Amyl alcohol <![CDATA[C 30 H 50 O]]> 426.73 34.47 Stigmasterol <![CDATA[C 30 H 50 O]]> 426.72 34.75 Lupeol <![CDATA[C 30 H 50 O]]> 426.73 34.97 Stigmasterol <![CDATA[C 29 H 50 O]]> 414.41 35.03 Cycloartenol <![CDATA[C 30 H 50 O]]> 426.72 35.21 Lanosterol <![CDATA[C 30 H 50 O]]> 426.72 35.64 δ7-Avenasterol <![CDATA[C 29 H 48 O]]> 412.70 36.02 Taraxasterol <![CDATA[C 30 H 50 O]]> 426.72 37.44 24-Methylenecycloartanol <![CDATA[C 31 H 52 O]]> 440.74 37.85 Gorgonsterol <![CDATA[C 30 H 50 O]]> 426.72 38.52 Limonostanol <![CDATA[C 30 H 52 O]]> 428.73 39.26 Limonostadienol <![CDATA[C 30 H 50 O]]> 426.73 The adulteration determination results of Comparative Examples 1-4 are shown in Table 4. Table 4 Identification results of tea oil adulteration in different embodiments and comparative examples C-4 dimethyl sterol ratio (a) FilipinIII fluorescence intensity (x) Comparative Example 1 Peanut oil sample 0.08 1.93 Comparative Example 2 Soybean oil sample 0.07 2.01 Comparative Example 3 Cottonseed oil sample 0.04 2.06 Comparative Example 4 Rapeseed oil sample 0.08 2.76 By comparing Table 4 with Table 1, it can be seen that the C-4 dimethyl sterol ratio (a) and Filipin III fluorescence intensity (x) in the tea oil samples of the comparative example show significant differences from those of Examples 1-6. The combination of the two indicators of C-4 dimethyl sterol ratio (a) and Filipin III fluorescence intensity (x) can be used to determine the adulteration of tea oil. The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for identifying adulteration of tea oil based on C-4 dimethyl sterol composition, characterized in that: The following steps are involved: S1. Extraction of phytosterols: Mix tea oil with other oils to obtain tea oil adulterated samples with different adulteration ratios, and quickly extract the unsaponifiable matter of tea oil and tea oil adulterated samples to obtain phytosterols; S2. Analysis of phytosterol composition: After the phytosterols in the tea oil adulterated sample were derivatized, GC-MS was performed to determine the peak area of ​​C-4 dimethyl sterol and the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area a were obtained based on the gas chromatography-mass spectrometry and fingerprint data; S3. Fluorescence signal acquisition: Filipin III fluorescence probe was used to detect the fluorescence signal intensity of phytosterols in tea oil, and the average value was taken as the reference threshold f for identifying adulterated tea oil. At the same time, the fluorescence signal intensity x of phytosterols in adulterated tea oil samples was collected; S4. Establishment of adulteration detection model: The ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area in the collected tea oil adulterated samples with different adulteration ratios a and the different adulteration ratios y were subjected to least squares linear regression analysis to obtain the regression equation y=-42.083a+99.

281. Combined with the Filipin III fluorescence signal intensity x, an adulteration detection model was established; S5. Adulteration determination: Rapidly extract the unsaponifiable matter of the unknown oil sample to be tested, match the measured fluorescence signal intensity x and the ratio of the peak area of ​​C-4 dimethyl sterol to the total peak area a with the adulteration detection model, determine whether the tea oil is adulterated with other oils, and calculate the adulteration ratio by the ratio difference method, adulteration ratio = |xf| / f×100%.

2. A method for identifying adulteration of tea oil based on C-4 dimethyl sterol composition according to claim 1, characterized in that: The tea oil in step S1 is camellia oil; the other oils include one or more of peanut oil, soybean oil, cottonseed oil and rapeseed oil.

3. A method for identifying adulteration of tea oil based on C-4 dimethyl sterol composition according to claim 1, characterized in that: The different adulteration ratios in step S1 are 5%, 10%, 15%, 20%, 30% and 50% respectively.

4. The method for identifying tea oil adulteration based on C-4 dimethyl sterol composition according to claim 1, characterized in that: The preparation method of phytosterols in step S1 is specifically as follows: 8% NaOH solution is added to tea oil and tea oil adulterated sample respectively, vortexed for 0.5-1.0 min, and saponified under ultrasonic heating conditions for 15 min to obtain a treated solution; an equal volume of deionized water is first added to the treated solution and shaken to dilute, then an equal volume of n-hexane is added to shake and stand until the aqueous phase and the organic phase are separated, then the unsaponifiable matter is extracted, and evaporated with N2 to obtain phytosterols.

5. The method for identifying adulteration of tea oil based on C-4 dimethyl sterol composition according to claim 1, characterized in that: The specific steps of the derivatization treatment in step S2 are: adding 200 μL of silanization reagent to the phytosterols in the tea oil adulterated sample, placing it in a 75°C water bath for 30 minutes for derivatization treatment, cooling the sample to room temperature, blowing it dry with N2 again, redissolving it in 100 μL of n-hexane, passing it through a 0.22 μm organic microporous filter membrane, and testing it.

6. A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition according to claim 5, characterized in that: The silanization reagent is a mixed solution of BSTFA and TMCS, with a volume ratio of 99:

1.

7. The method for identifying adulteration of tea oil based on C-4 dimethyl sterol composition according to claim 1, characterized in that: The GC-MS measurement conditions in step S2 are: Chromatographic conditions: Chromatographic column: DB-5 column, 30m×0.25mm, 0.25μm; Temperature program: The initial column temperature was set at 200°C and maintained for 0.5 min; the temperature was increased to 250°C at a rate of 15°C / min and maintained for 8 min; a "low rate gradient temperature program" was used with a temperature increase rate of 1°C / min, and the column temperature was maintained for 1.5 min after it reached 270°C; Detector and injection port temperature: 290°C; Injection volume: 1.0 μL; Split ratio: 1:100; Carrier gas: 99.99% high purity helium, flow rate 1.2mL / min; Total program running time: 40min; Mass spectrometry conditions: Inlet temperature: 300°C; Solvent delay: 10min; Ion source: EI; Electron energy: 70eV; Scan time: 0.5s; Mass scan range: 50-550 m / z.

8. The method for identifying adulteration of tea oil based on C-4 dimethyl sterol composition according to claim 1, characterized in that: The specific steps of Filipin III fluorescent probe detection in step S3 are: adding 100 μL of Filipin III working solution to the phytosterols in tea oil and tea oil adulterated samples, incubating at room temperature for 30 minutes in the dark, removing the staining solution, and performing fluorescence analysis.

9. The method for identifying adulteration of tea oil based on the C-4 dimethyl sterol composition according to claim 1, characterized in that: The fluorescence signal intensity in step S3 refers to the fluorescence absorption collected by the Filipin III probe under 300-400nm excitation light and 400-500nm emission light.

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