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

Abstract

The application provides a method for identifying adulteration of tea oil based on C-4 dimethyl sterol composition, wherein the proportion of the peak area of C-4 dimethyl sterol in the total peak area in collected tea oil adulteration samples with different adulteration proportions is subjected to least square linear regression analysis with different adulteration proportions, a detection model for adulteration is established by combining with the fluorescence signal intensity of Filipin III, and whether other oil is adulterated in the tea oil sample is determined, and the adulteration proportion can be determined according to the ratio of the fluorescence signal intensity of Filipin III to the standard threshold value of tea oil. The method provided by the application can not only identify the adulteration of tea oil, but also detect the adulteration proportion, has the characteristics of simplicity, rapidness, good repeatability, high accuracy and the like, and can be practically applied to the detection of tea oil adulteration.

Description

TECHNICAL FIELD

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

[0002] Plant sterols are abundant in oilseeds, and the key step of sterol biosynthesis is to control the carbon flow into the isoprene pathway to generate C4-dimethyl sterols (cycloartenol and 24-methylene cycloartenol), which are then converted into common non-methyl sterols (sitosterol and campesterol). Sterol methyltransferase (SMT) and sterol methyl oxidase (SMO) play a key role in the process of converting C4-dimethyl sterols into non-methyl sterols. Studies have shown that the differential expression of sterol-related metabolic genes produces a unique sterol fingerprint of oil. For example, oil tea has lower SMT1 gene expression, and the content of C4-dimethyl sterols in its sterol fingerprint is higher; soybean has a higher transcription level of SMO, so the proportion of C4-dimethyl sterols in the sterol fingerprint of soybean oil is smaller. Oilseed crops form differential sterol fingerprints during evolution, and the sterol fingerprints of the same species are still highly conserved, for example, oil tea seed oil, tea seed oil and shea oil. Therefore, analyzing the structural composition of plant sterols can achieve adulteration identification of edible oils according to the plant sterol fingerprint information.

[0003] The molecular polarity produced by 3β hydroxyl (3β-OH) in plant sterols is the main reason for the occurrence of specific interaction with Filipin III. Compared with non-methyl sterols, C4-dimethyl sterols have weaker polarity due to the shielding effect of two methyl groups (CH3) beside 3β-OH, and cannot be tightly arranged in the interface layer of the determination medium, so the affinity ability with Filipin III is reduced, and the fluorescence intensity produced is weaker. The proportion difference of different C4-dimethyl sterols and non-methyl sterols in edible oils will affect the affinity efficiency of Filipin III. When the total amount of sterols in the determination medium is the same, the fluorescence signal formed after binding with Filipin III also has specificity, and this fluorescence signal feature can be used for adulteration detection of oil.

[0004] CN106950241B discloses a method for predicting the type and content of other doped oils in tea oil, and CN112305108A discloses an oil tea seed oil adulteration detection method based on the ratio of oleic acid / behenic acid and beta-amyrin / linoleic acid. Both of them detect the adulteration of tea oil, but both of them involve complex extraction process and sample detection process, such as nuclear magnetic resonance 1H-NMR detection, OPLS-DA analysis; and the analysis and processing of the final data of the sample are complex, which is difficult to meet the requirements of efficient discrimination of edible oil adulteration. CN103217393B discloses a method for detecting adulterated tea oil. The invention increases the full-wave scanning of ultraviolet spectrum on the basis of detecting refractive index, iodine value, saponification value and oleic acid content. However, the invention needs to be fitted and identified under the model of known target adulteration components, and cannot realize accurate and quantitative analysis of unknown components. At present, the products in the edible oil market in China are diversified, subdivided and high-end. It is of great significance to improve the discrimination ability of edible oil adulteration, crack down on edible oil adulteration and fraud, and ensure the quality and safety of edible oil products. The phytosterol in oil has high inertness, stability and specificity in the distribution of oil, can produce specific fingerprint information, and can be quickly identified by Filipin III fluorescent probe. The edible oil adulteration identification based on the phytosterol fingerprint information has important application value. SUMMARY

[0005] The technical problem to be solved is that in view of the defects of the prior art, the purpose of the present application is to provide a method for identifying tea oil adulteration based on C-4 dimethyl sterol composition, and the specific steps are: tea oil adulteration sample preparation, fluorescence signal acquisition, phytosterol composition analysis, adulteration detection model establishment and adulteration discrimination. The method can identify and discriminate whether tea oil is doped with other oils and determine the adulteration proportion, so as to solve the technical problems of complicated, low-efficiency detection method of edible oil adulteration with other oils, qualitative and quantitative detection precision in the prior art.

[0006] Technical scheme: A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition, comprising the following steps:

[0007] S1. Extraction of phytosterol: mix tea oil with other oils to obtain tea oil adulteration samples with different adulteration proportions, and quickly extract unsaponifiable matter of tea oil and tea oil adulteration samples to obtain phytosterol;

[0008] S2. Phytosterol composition analysis: after derivatization treatment of the phytosterol of the tea oil adulteration sample, GC-MS determination is performed, and according to the gas chromatogram and fingerprint data, the peak area of C-4 dimethyl sterol and the proportion a of the peak area of C-4 dimethyl sterol to the total peak area are obtained;

[0009] S3. Fluorescence signal acquisition: Filipin III fluorescent probe detection 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 tea oil adulteration. The fluorescence signal intensity x of phytosterols in tea oil adulteration samples was also collected;

[0010] S5. Adulteration identification: Unsaponifiable matter of the unknown oil sample to be tested was rapidly extracted, and the fluorescence signal intensity x and the peak area ratio a of C-4 dimethyl sterol to the total peak area were matched with the adulteration detection model to identify whether the tea oil was adulterated with other oils. The adulteration ratio was calculated by the difference method, and the adulteration ratio = |x-f| / f x 100%.

[0011] Further, the tea oil in step S1 is camellia seed oil; and the other oils include one or more of peanut oil, soybean oil, cottonseed oil, and rapeseed oil.

[0012] Further, the different adulteration ratios in step S1 are 5%, 10%, 15%, 20%, 30%, and 50%, respectively.

[0013] Further, the preparation method of phytosterols in step S1 is as follows: 8% NaOH solution is added to tea oil and tea oil adulteration samples, respectively, and vortexed for 0.5-1.0 min. After saponification treatment under ultrasonic heating conditions for 15 min, a treatment liquid is obtained. Equal volume of deionized water is added to the treatment liquid, shaken and diluted, and then equal volume of n-hexane is added and shaken. When the water phase and organic phase are separated, the unsaponifiable matter is extracted, dried with N2, and phytosterols are obtained.

[0014] Further, the derivatization treatment in step S2 is as follows: 200 μL of silylating reagent is added to the phytosterols of the tea oil adulteration sample, and derivatization treatment is performed at 75°C water bath for 30 min. After the sample is cooled to room temperature, it is dried with N2 again, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter, and measured.

[0015] Further, the silylating reagent is a mixed solution of BSTFA and TMCS with a volume ratio of 99:1.

[0016] Further, the GC-MS determination conditions in step S2 are as follows:

[0017] Chromatographic conditions:

[0018] Chromatographic column: DB-5 chromatographic column, 30 m x 0.25 mm, 0.25 μm;

[0019] Temperature program: the initial column temperature was set to 200℃ and maintained for 0.5 min; the temperature was raised to 250℃ at a rate of 15℃ / min and maintained for 8 min; the "low rate gradient temperature program" was used to raise the temperature at a rate of 1℃ / min, and the column temperature was maintained at 270℃ for 1.5 min after being raised to 270℃;

[0020] Detector, injection port temperature: 290℃;

[0021] Injection volume: 1.0 μL;

[0022] Split ratio: 1:100;

[0023] Carrier gas: 99.99% high-purity helium, flow rate 1.2 mL / min;

[0024] Total program run time: 40 min;

[0025] Mass spectrometry conditions:

[0026] Injection port temperature: 300℃;

[0027] Solvent delay: 10 min;

[0028] Ion source: EI;

[0029] Electron energy: 70 eV;

[0030] Scan time: 0.5 s;

[0031] Mass scan range: 50-550 m / z.

[0032] Further, the specific steps of the Filipin III fluorescent probe detection in step S3 are as follows: 100 μL of Filipin III working solution is added to the phytosterols in the tea oil and tea oil adulteration samples, and incubated in the dark at room temperature for 30 min; the staining solution is removed, and fluorescence analysis is performed.

[0033] Further, the fluorescence signal intensity refers to the fluorescence absorption of the Filipin III probe under 300-400 nm excitation light and 400-500 nm emission light.

[0034] Beneficial effects:

[0035] 1.The present application uses Filipin III fluorescent probe to specifically bind with phytosterol 3β-OH, and determines the adulteration amount by the characteristic absorption of Filipin III fluorescent probe under 300-400 nm excitation light and 400-500 nm emission light; the proportion of C4-dimethyl sterol in tea oil is high, and C4-dimethyl sterol has weaker polarity due to the shielding effect of two methyl groups (CH3) beside 3β-OH, so the affinity ability of C4-dimethyl sterol with Filipin III decreases; compared with other oils rich in non-methyl sterol, the weaker Filipin III fluorescence intensity value of tea oil can be used to indicate the proportion of tea oil adulteration; compared with the traditional method relying only on GC-MS, infrared spectrum or nuclear magnetic resonance technology, the Filipin III fluorescence characteristics can not only identify the adulteration amount, but also have the characteristics of simple and rapid, good repeatability and high accuracy.

[0036] 2.The present application uses high-concentration 8% NaOH solution to rapidly extract phytosterol, and under the action of high-concentration NaOH solution, a large amount of hydroxyl ions (OH-) can be effectively provided in a short time, and this alkaline condition helps to separate phytosterol molecules from oil; since hydroxyl ions can form water-soluble complexes with the hydroxyl groups in the sterol molecules, the sterol is more easily dissolved under alkaline conditions; in addition, NaOH solution at a concentration of 8% can selectively and fully dissolve sterol, while the dissolution of other plant ingredients such as tocopherol and polyphenol is relatively small, and 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 phytosterol.

[0037] 3.The present application uses regression analysis method to establish an adulteration detection model for adulterated tea oil, which has good fitting constant and high accuracy; the use of Filipin III fluorescent probe alone has many influencing factors and relatively large error, therefore, the present application uses both GC-MS composition analysis and Filipin III fluorescent probe detection to construct the adulteration detection model, so that the accuracy of the model is higher, and the Filipin III fluorescence intensity threshold and adulteration detection model provided by the present application can be repeatedly used for rapid detection of different batches. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 It is a flow chart for identifying tea oil adulteration based on C-4 dimethyl sterol composition;

[0039] Figure 2 It is a GC-MS chromatogram of phytosterol in tea oil sample;

[0040] Figure 3 It is a linear regression equation obtained from the tea oil prepared in Examples 1-6 and the tea oil adulteration sample. DETAILED DESCRIPTION

[0041] The application will be further described in conjunction with the accompanying drawings and examples. The following examples are illustrative of specific embodiments of the application, but the application is not to be limited to the embodiments.

[0042] Example 1

[0043] A method for identifying adulteration of tea oil based on C-4 dimethyl sterol composition, comprising the following steps:

[0044] S1. Extraction of phytosterol: Add high-alkalinity saponification solution (8% NaOH) to tea oil sample 1, vortex for 0.5-1.0 min, and perform saponification treatment under ultrasonic heating for 15 min to obtain a treatment liquid. Add an equal volume of deionized water to the treatment liquid, shake to dilute, then add an equal volume of n-hexane and shake to stand. When the water phase and organic phase are separated, extract the unsaponifiable matter, and dry it with N2 to obtain phytosterol.

[0045] S2. Analysis of phytosterol composition: Add 200 μL of silanization reagent BSTFA+TMCS (99:1, v / v) to the phytosterol, and perform derivatization treatment in a 75°C water bath for 30 min. After the sample cools to room temperature, dry it again with N2, redissolve it in 100 μL of n-hexane, pass it through a 0.22 μm organic microporous filter membrane, and perform GC-MS determination. According to the gas chromatogram and fingerprint data, obtain the proportion a of the peak area of C-4 dimethyl sterol to the total peak area.

[0046] S3. Fluorescence signal collection: Add 100 μL of Filipin III working solution to the phytosterol, incubate at room temperature for 30 min in the dark, remove the staining solution, perform Filipin III fluorescence probe detection, and collect the fluorescence signal intensity x of the phytosterol in the tea oil.

[0047] Example 2

[0048] A method for identifying adulteration of tea oil based on C-4 dimethyl sterol composition, comprising the following steps:

[0049] S1. Extraction of phytosterol: Add high-alkalinity saponification solution (8% NaOH) to tea oil sample 1, vortex for 0.5-1.0 min, and perform saponification treatment under ultrasonic heating for 15 min to obtain a treatment liquid. Add an equal volume of deionized water to the treatment liquid, shake to dilute, then add an equal volume of n-hexane and shake to stand. When the water phase and organic phase are separated, extract the unsaponifiable matter, and dry it with N2 to obtain phytosterol.

[0050] S2. Plant sterol composition analysis: 200 μL of silylating reagent BSTFA+TMCS (99:1, v / v) was added to the plant sterols, and derivatization treatment was performed in a 75°C water bath for 30 min. After the sample cooled to room temperature, it was blown dry again with N2, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter, and determined by GC-MS. According to the gas chromatogram and fingerprint data, the proportion a of the peak area of C-4 dimethyl sterol to the total peak area was obtained;

[0051] S3. Fluorescent signal acquisition: 100 μL of Filipin III working solution was added to the plant sterols, and incubated in the dark at room temperature for 30 min. The staining solution was removed, and Filipin III fluorescent probe detection was performed. The fluorescent signal intensity x of the plant sterols in the tea oil was collected.

[0052] Example 3

[0053] A method for identifying adulteration of tea oil based on C-4 dimethyl sterol composition, comprising the following steps:

[0054] S1. Extraction of plant sterols: A high-alkalinity saponification solution (8% NaOH) was added to the tea oil adulteration sample (tea oil sample 95% + 5% soybean oil), and vortexed for 0.5-1.0 min. Saponification treatment was performed under the action of ultrasonic heating for 15 min to obtain a treatment solution. An equal volume of deionized water was first added to the treatment solution, shaken and diluted, and then an equal volume of n-hexane was added and shaken and allowed to stand until the water phase and organic phase were separated. The unsaponifiable matter was extracted, dried with N2, and plant sterols were obtained;

[0055] S2. Plant sterol composition analysis: 200 μL of silylating reagent BSTFA+TMCS (99:1, v / v) was added to the plant sterols, and derivatization treatment was performed in a 75°C water bath for 30 min. After the sample cooled to room temperature, it was blown dry again with N2, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter, and determined by GC-MS. According to the gas chromatogram and fingerprint data, the proportion a of the peak area of C-4 dimethyl sterol to the total peak area was obtained;

[0056] S3. Fluorescent signal acquisition: 100 μL of Filipin III working solution was added to the plant sterols, and incubated in the dark at room temperature for 30 min. The staining solution was removed, and Filipin III fluorescent probe detection was performed. The fluorescent signal intensity x of the plant sterols in the tea oil was collected.

[0057] Example 4

[0058] A method for identifying adulteration of tea oil based on C-4 dimethyl sterol composition, comprising the following steps:

[0059] S1. Extraction of phytosterols: Add high-alkalinity saponification solution (8% NaOH) to the tea oil adulteration sample (tea oil sample 50% + 50% peanut oil), vortex for 0.5-1.0 min, and perform saponification treatment under ultrasonic heating for 15 min to obtain a treatment liquid. Dilute the treatment liquid by first adding an equal volume of deionized water, then adding an equal volume of n-hexane, and oscillating and standing until the water phase and organic phase are separated. Extract the unsaponifiable matter, dry it with N2, and obtain the phytosterols.

[0060] S2. Analysis of phytosterol composition: Add 200 μL of silanization reagent BSTFA + TMCS (99:1, v / v) to the phytosterols, place in a 75°C water bath for 30 min for derivatization treatment, cool the sample to room temperature, dry it again with N2, redissolve in 100 μL of n-hexane, pass through a 0.22 μm organic microporous filter, and perform GC-MS determination. According to the gas chromatogram and fingerprint data, the proportion a of the peak area of C-4 dimethyl sterol to the total peak area is obtained.

[0061] S3. Fluorescence signal acquisition: 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 fluorescence probe detection, and acquire the fluorescence signal intensity x of the phytosterols in the tea oil adulteration sample.

[0062] Example 5

[0063] A method for identifying tea oil adulteration based on C-4 dimethyl sterol composition, comprising the following steps:

[0064] S1. Extraction of phytosterols: Add high-alkalinity saponification solution (8% NaOH) to the tea oil adulteration sample (tea oil sample 50% + 50% peanut oil), vortex for 0.5-1.0 min, and perform saponification treatment under ultrasonic heating for 15 min to obtain a treatment liquid. Dilute the treatment liquid by first adding an equal volume of deionized water, then adding an equal volume of n-hexane, and oscillating and standing until the water phase and organic phase are separated. Extract the unsaponifiable matter, dry it with N2, and obtain the phytosterols;

[0065] S2. Analysis of phytosterol composition: Add 200 μL of silanization reagent BSTFA + TMCS (99:1, v / v) to the phytosterols, place in a 75°C water bath for 30 min for derivatization treatment, cool the sample to room temperature, dry it again with N2, redissolve in 100 μL of n-hexane, pass through a 0.22 μm organic microporous filter, and perform GC-MS determination. According to the gas chromatogram and fingerprint data, the proportion a of the peak area of C-4 dimethyl sterol to the total peak area is obtained.

[0066] S3. Fluorescence signal acquisition: 100 μL of Filipin III working solution was added to the phytosterol, and incubated at room temperature for 30 min in the dark. The staining solution was removed, and Filipin III fluorescence probe detection was performed. The fluorescence signal intensity x of the phytosterol in the tea oil adulteration sample was collected.

[0067] Example 6

[0068] A method for identifying tea oil adulteration based on the composition of C-4 dimethyl sterol, comprising the following steps:

[0069] S1. Extraction of phytosterol: high-alkalinity saponification solution (8% NaOH) was added to the tea oil adulteration sample (tea oil sample 70% + 10% soybean oil + 10% rapeseed oil + 10% cottonseed oil), vortexed for 0.5-1.0 min, and subjected to saponification treatment under ultrasonic heating for 15 min to obtain a treatment solution. An equal volume of deionized water was added to the treatment solution, shaken and diluted, and an equal volume of n-hexane was added and shaken and allowed to stand until the water phase and organic phase were separated. Unsaponifiable matter was extracted, dried with N2, and phytosterol was obtained.

[0070] S2. Phytosterol composition analysis: 200 μL of silanization reagent BSTFA + TMCS (99:1, v / v) was added to the phytosterol, and derivatization treatment was performed at 75°C water bath for 30 min. After the sample was cooled to room temperature, it was blown dry with N2 again, redissolved in 100 μL of n-hexane, passed through a 0.22 μm organic microporous filter, and subjected to GC-MS determination. According to the gas chromatogram and fingerprint data, the peak area ratio of C-4 dimethyl sterol to the total peak area a was obtained.

[0071] S3. Fluorescence signal acquisition: 100 μL of Filipin III working solution was added to the phytosterol, and incubated at room temperature for 30 min in the dark. The staining solution was removed, and Filipin III fluorescence probe detection was performed. The fluorescence signal intensity x of the phytosterol in the tea oil adulteration sample was collected.

[0072] The C-4 dimethyl sterol ratio and Filipin III fluorescence intensity determination results of the tea oil and tea oil adulteration samples prepared in Examples 1-6 are shown in Table 1 below:

[0073] Table 1 Determination results of tea oil and tea oil adulteration samples prepared in Examples 1-6

[0074]

[0075] From Table 1, the Filipin III fluorescence intensity of Example 1 and Example 2 is 4.11 and 4.32 respectively, and the average value is taken as the reference threshold f = 4.215. The proportion a of the peak area of C-4 dimethyl sterol in the total peak area in the tea oil prepared in Example 1-6 and the tea oil adulteration sample with different adulteration proportions is subjected to linear regression analysis by least square method with the different actual adulteration proportions y, to obtain the linear regression equation y = -42.083a + 99.281 (R 2 = 0.9878), combined with the Filipin III fluorescence signal intensity x, to establish an adulteration detection model as shown in Figure 1 .

[0076] Example 7

[0077] A method for identifying tea oil adulteration based on the composition of C-4 dimethyl sterol, comprising the following steps:

[0078] S1. Extraction of phytosterol: high alkalinity saponification solution (8% NaOH) is added to the tea oil adulteration sample (tea oil sample 80% + 20% rapeseed oil), vortexed for 0.5-1.0 min, and subjected to saponification treatment under ultrasonic heating for 15 min to obtain a treatment liquid. Equal volume of deionized water is first added to the treatment liquid and shaken to dilute, and then equal volume of n-hexane is added and shaken to stand until the water phase and organic phase are separated. Unsaponifiable matter is extracted, dried with N2, and phytosterol is obtained;

[0079] S2. Phytosterol composition analysis: 200 μL of silanization reagent BSTFA + TMCS (99:1, v / v) is added to the phytosterol, which is subjected to derivatization treatment in a 75°C water bath for 30 min. After the sample is cooled to room temperature, it is dried again with N2, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter, and subjected to GC-MS determination. According to the gas chromatogram and fingerprint data, the proportion a of the peak area of C-4 dimethyl sterol in the total peak area is obtained;

[0080] S3. Fluorescence signal acquisition: 100 μL of Filipin III working solution is added to the phytosterol, which is incubated at room temperature for 30 min in the dark. The staining solution is removed, and Filipin III fluorescence probe detection is performed to acquire the fluorescence signal intensity x of the phytosterol in the tea oil adulteration sample;

[0081] S4. Adulteration identification: unsaponifiable matter is quickly extracted from the oil sample to be tested. The measured fluorescence signal intensity x and the proportion a of the peak area of C-4 dimethyl sterol in the total peak area are matched with the adulteration detection model to identify whether the tea oil is adulterated with other oils. The adulteration proportion is calculated by the difference method, and the adulteration proportion = |x-f| / f x 100%.

[0082] Example 8

[0083] A method for identifying adulteration of tea oil based on C-4 dimethyl sterol composition, comprising the following steps:

[0084] S1. Extraction of phytosterol: Add high-alkalinity saponification solution (8% NaOH) to the tea oil adulteration sample (tea oil sample 90% + 10% cottonseed oil), vortex for 0.5-1.0 min, and perform saponification treatment under ultrasonic heating for 15 min to obtain a treatment liquid. Dilute the treatment liquid by first adding an equal volume of deionized water, shaking, and then adding an equal volume of n-hexane and shaking. When the water phase and organic phase are separated, extract the unsaponifiable matter, dry it with N2, and obtain the phytosterol;

[0085] S2. Analysis of phytosterol composition: Add 200 μL of silanization reagent BSTFA+TMCS (99:1, v / v) to the phytosterol and place it in a 75°C water bath for 30 min for derivatization treatment. After the sample cools to room temperature, dry it again with N2, redissolve it in 100 μL of n-hexane, pass it through a 0.22 μm organic microporous filter membrane, and perform GC-MS determination. According to the gas chromatogram and fingerprint data, obtain the proportion a of the peak area of C-4 dimethyl sterol to the total peak area;

[0086] S3. Fluorescence signal acquisition: Add 100 μL of Filipin III working solution to the phytosterol, incubate it in the dark at room temperature for 30 min, remove the staining solution, perform Filipin III fluorescence probe detection, and collect the fluorescence signal intensity x of the phytosterol in the tea oil adulteration sample;

[0087] S4. Adulteration identification: Quickly extract the unsaponifiable matter of the unknown oil sample to be tested, match the measured fluorescence signal intensity x and the proportion a of the peak area of C-4 dimethyl sterol to the total peak area with the adulteration detection model, identify whether the tea oil is adulterated with other oils, and calculate the adulteration proportion by the difference method: adulteration proportion = |x-f| / f x 100%.

[0088] Verification of equation accuracy:

[0089] Substitute the proportion 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 adulteration samples with different adulteration proportions into the linear regression equation to obtain the preliminary judgment of the adulteration proportion, and then confirm the adulteration proportion by the fluorescence intensity x. The results are shown in Table 2 below:

[0090] Table 2 Predicted adulteration identification results of tea oil prepared in Examples 1-8 and tea oil adulteration samples

[0091]

[0092]

[0093] It can be seen from Examples 1-8 in Table 2 that the relative deviations of the proportion of C-4 dimethyl sterol (a) and the fluorescence intensity of Filipin III (x) for identifying the adulteration proportion of tea oil are all less than 3.42%, and the average relative deviation is 2.05%. Therefore, the adulteration identification result is accurate and stable. As can be seen from the comparison of Example 3 and Example 4, the smaller the proportion of other oils in the adulterated tea oil sample, the greater the relative deviation obtained by detection. Therefore, the adulteration identification model is more accurate in the adulteration identification of high proportion of adulterated tea oil samples.

[0094] Comparative Example 1

[0095] A method for identifying the adulteration of tea oil based on the composition of C-4 dimethyl sterol, comprising the following steps:

[0096] S1. Extraction of phytosterol: Add a high-alkalinity saponification solution (8% NaOH) to peanut oil, vortex for 0.5-1.0 min, and perform saponification treatment under ultrasonic heating for 15 min to obtain a treatment liquid. Add an equal volume of deionized water to the treatment liquid, shake and dilute, then add an equal volume of n-hexane and shake and stand until the water phase and organic phase are separated. Extract the unsaponifiable matter, dry it with N2, and obtain the phytosterol.

[0097] S2. Analysis of phytosterol composition: Add 200 μL of silanization reagent BSTFA+TMCS (99:1, v / v) to the phytosterol, and perform derivatization treatment at 75°C water bath for 30 min. After the sample is cooled to room temperature, dry it again with N2, redissolve it in 100 μL of n-hexane, pass it through a 0.22 μm organic microporous filter membrane, and perform GC-MS determination. According to the gas chromatogram and fingerprint data, the proportion a of the peak area of C-4 dimethyl sterol to the total peak area is obtained.

[0098] S3. Fluorescence signal collection: Add 100 μL of Filipin III working solution to the phytosterol, incubate at room temperature for 30 min in the dark, remove the dyeing liquid, perform Filipin III fluorescence probe detection, and collect the fluorescence signal intensity x of the phytosterol in the peanut oil.

[0099] S4. Adulteration identification: Match the obtained fluorescence signal intensity x and the proportion a of the peak area of C-4 dimethyl sterol to the total peak area with the adulteration detection model to identify whether the peanut oil is adulterated with other oils, and calculate the adulteration proportion according to the ratio difference method of the fluorescence signal intensity and the tea oil reference threshold value.

[0100] Comparative Example 2

[0101] A method for identifying the adulteration of tea oil based on the composition of C-4 dimethyl sterol, comprising the following steps:

[0102] S1. Extraction of phytosterol: Add high-alkalinity saponification solution (8% NaOH) to soybean oil, vortex for 0.5-1.0 min, and perform saponification treatment under the action of ultrasonic heating for 15 min to obtain a treatment liquid. Add an equal volume of deionized water to the treatment liquid, shake to dilute, then add an equal volume of n-hexane and shake and stand until the water phase and organic phase are separated. Extract the unsaponifiable matter, dry with N2, and obtain phytosterol;

[0103] S2. Analysis of phytosterol composition: Add 200 μL of silanization reagent BSTFA+TMCS (99:1, v / v) to the phytosterol and place in a 75°C water bath for 30 min to perform derivatization treatment. After the sample cools to room temperature, dry it again with N2, redissolve in 100 μL of n-hexane, pass through a 0.22 μm organic microporous filter membrane, and perform GC-MS determination. According to the gas chromatogram and fingerprint data, obtain the proportion a of the peak area of C-4 dimethyl sterol to the total peak area;

[0104] S3. Fluorescent signal acquisition: Add 100 μL of Filipin III working solution to the phytosterol, incubate at room temperature for 30 min in the dark, remove the staining solution, perform Filipin III fluorescent probe detection, and acquire the fluorescent signal intensity x of phytosterol in soybean oil;

[0105] S4. Discrimination of adulteration: Match the obtained fluorescent signal intensity x and the proportion a of the peak area of C-4 dimethyl sterol to the total peak area with the adulteration detection model to discriminate whether soybean oil is adulterated with other oils and fats, and calculate the adulteration proportion according to the ratio difference method of the fluorescent signal intensity and the tea oil reference threshold value.

[0106] Comparative Example 3

[0107] A method for identifying the adulteration of tea oil based on the composition of C-4 dimethyl sterol, comprising the following steps:

[0108] S1. Extraction of phytosterol: Add high-alkalinity saponification solution (8% NaOH) to soybean oil, vortex for 0.5-1.0 min, and perform saponification treatment under the action of ultrasonic heating for 15 min to obtain a treatment liquid. Add an equal volume of deionized water to the treatment liquid, shake to dilute, then add an equal volume of n-hexane and shake and stand until the water phase and organic phase are separated. Extract the unsaponifiable matter, dry with N2, and obtain phytosterol;

[0109] S2. Phytosterol composition analysis: 200 μL of silylating reagent BSTFA+TMCS (99:1, v / v) was added to the phytosterol, and derivatization treatment was performed in a 75°C water bath for 30 min. After the sample cooled to room temperature, it was blown dry again with N2, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter, and subjected to GC-MS determination. According to the gas chromatogram and fingerprint data, the proportion a of the peak area of C-4 dimethyl sterol to the total peak area was obtained;

[0110] S3. Fluorescent signal acquisition: 100 μL of Filipin III working solution was added to the phytosterol, and incubated at room temperature for 30 min in the dark. The staining solution was removed, and Filipin III fluorescent probe detection was performed to collect the fluorescent signal intensity x of the phytosterol in the cottonseed oil;

[0111] S4. Adulteration discrimination: The obtained fluorescent signal intensity x and the proportion a of the peak area of C-4 dimethyl sterol to the total peak area were matched with the adulteration detection model to discriminate whether the cottonseed oil was adulterated with other oils, and the adulteration proportion was calculated according to the ratio difference method of the fluorescent signal intensity and the tea oil reference threshold value.

[0112] Comparative Example 4

[0113] A method for identifying tea oil adulteration based on the composition of C-4 dimethyl sterol, comprising the following steps:

[0114] S1. Extraction of phytosterol: High-alkali saponification solution (8% NaOH) was added to the rapeseed oil, vortexed for 0.5-1.0 min, and subjected to saponification treatment under the action of ultrasonic heating for 15 min to obtain a treatment liquid. Equal volume of deionized water was first added to the treatment liquid, shaken and diluted, and then equal volume of n-hexane was added, shaken and allowed to stand until the water phase and organic phase were separated. The unsaponifiable matter was extracted, dried with N2, and phytosterol was obtained;

[0115] S2. Phytosterol composition analysis: 200 μL of silylating reagent BSTFA+TMCS (99:1, v / v) was added to the phytosterol, and derivatization treatment was performed in a 75°C water bath for 30 min. After the sample cooled to room temperature, it was blown dry again with N2, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter, and subjected to GC-MS determination. According to the gas chromatogram and fingerprint data, the proportion a of the peak area of C-4 dimethyl sterol to the total peak area was obtained;

[0116] S3. Fluorescent signal acquisition: 100 μL of Filipin III working solution was added to the phytosterol, and incubated at room temperature for 30 min in the dark. The staining solution was removed, and Filipin III fluorescent probe detection was performed to collect the fluorescent signal intensity x of the phytosterol in the cottonseed oil;

[0117] S4. Adulteration discrimination: match the obtained fluorescence signal intensity x and the proportion a of the peak area of C-4 dimethyl sterol to the total peak area with the adulteration detection model, discriminate whether the rapeseed oil is adulterated with other oils, and calculate the adulteration proportion according to the ratio difference method of fluorescence signal intensity and tea oil reference threshold value.

[0118] The fingerprint information of the plant sterol composition measured by GC-MS is shown in Table 3.

[0119] Table 3 Fingerprint information of plant sterol composition measured by GC-MS

[0120] Time (min) Chinese Name Molecular Formula Molecular Weight 26.39 Ergosta-7,22-dien-3β-ol C 28 H 46 O]]> 398.66 27.08 Cholesterol C 27 H 46 O]]> 386.62 28.53 Ergosta-3,25-dien-3β-ol C 28 H 46 O]]> 398.35 28.82 Campesterol C 28 H 48 O]]> 400.69 29.16 Friedelinol C 30 H 52 O]]> 428.73 29.86 Stigmasterol C 29 H 48 O]]> 412.70 30.39 Ergosta-8(14)-en-3β-ol C 28 H 48 O]]> 400.68 30.58 Ergosta-5,22-dien-3β-ol C 28 H 46 O]]> 398.66 30.91 Bassisterol C 29 H 48 O]]> 412.69 31.29 Ergosta-7-en-3β-ol C 28 H 48 O]]> 400.68 31.70 Clerosterol C 30 H 50 O]]> 426.72 32.36 β-Sitosterol C 29 H 50 O]]> 414.72 32.68 β-Amyrin C 30 H 50 O]]> 426.73 32.88 Stigmastanol C 29 H 48 O]]> 416.73 33.02 Clemastanol C 30 H 50 O]]> 426.73 33.04 δ5-Stigmastanol C 29 H 48 O]]> 412.70 33.33 Mucokinol C 30 H 50 O]]> 426.73 33.53 Stigmadienol C 29 H 50 O]]> 414.41 33.66 Pachymic acid C 30 H 50 O]]> 426.72 34.01 Isodocosteroi C 29 H 48 O]]> 412.70 34.36 α-Amyrin C 30 H 50 O]]> 426.73 34.47 Stigmadienol C 30 H 50 O]]> 426.72 34.75 Lupeol C 30 H 50 O]]> 426.73 34.97 Stigmastenol C 29 H 50 O]]> 414.41 35.03 Cycloartanol C 30 H 50 O]]> 426.72 35.21 Lanosterol C 30 H 50 O]]> 426.72 35.64 δ7-Stigmastanol C 29 H 48 O]]> 412.70 36.02 Taraxasterol C 30 H 50 O]]> 426.72 37.44 24-Methylene cycloartanol C 31 H 52 O]]> 440.74 37.85 Gorgostanol C 30 H 50 O]]> 426.72 38.52 Limonisterol C 30 H 52 O]]> 428.73 39.26 Limonisterol C 30 H 50 O]]> 426.73

[0121] The adulteration determination results of Comparative Examples 1-4 are shown in Table 4.

[0122] Table 4 Tea oil adulteration identification results of different examples and comparative examples

[0123] C-4 Dimethyl sterol ratio (a) Filipin III 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

[0124] By comparing Table 4 and Table 1, it can be seen that: the proportion (a) of C-4 dimethyl sterol and the fluorescence intensity (x) of Filipin III in the tea oil samples of the comparative examples all show significant differences from those of Examples 1-6, and the two indicators of the proportion (a) of C-4 dimethyl sterol and the fluorescence intensity (x) of Filipin III can be used to judge the tea oil adulteration.

[0125] The above is only the preferred embodiment of the present application, and does not limit the present application in any form. Any person skilled in the art can make many possible changes and modifications to the technical solutions of the present application, or modify equivalent embodiments, without departing from the spirit and technical solutions of the present application, by using the disclosed methods and technical contents. Therefore, any simple modification, equivalent replacement, equivalent change and modification of the above embodiments according to the technical essence of the present application, which does not deviate from the technical solutions of the present application, still belongs to the protection scope of the technical solutions of the present application.

Claims

1. A method for identifying adulteration of tea oil based on the composition of C-4 dimethyl sterols, characterized by, The method comprises the following steps: S1. Extraction of phytosterols: mixing tea oil with other oils to obtain tea oil adulteration samples with different adulteration proportions, rapidly extracting unsaponifiable matter of tea oil and tea oil adulteration samples to obtain phytosterols; S2. Analysis of phytosterol composition: after derivatization treatment of phytosterols of tea oil adulteration samples, GC-MS determination is performed, and according to the gas chromatogram and fingerprint data, the peak area of C-4 dimethyl sterol and the proportion a of the peak area of C-4 dimethyl sterol in the total peak area are obtained; S3. Fluorescence signal acquisition: Filipin III fluorescence probe detection is used to determine the fluorescence signal intensity of phytosterols in tea oil, and the average value thereof is taken as a reference threshold f for identifying tea oil adulteration, and the fluorescence signal intensity x of phytosterols of tea oil adulteration samples is also acquired; S4. Establishment of adulteration detection model: the proportion a of the peak area of C-4 dimethyl sterol in the total peak area of tea oil adulteration samples with different adulteration proportions y is subjected to linear regression analysis by the least square method to obtain a regression equation y=-42.083a+99.281, and the adulteration detection model is established in combination with the Filipin III fluorescence signal intensity x; S5. Adulteration identification: unsaponifiable matter of an unknown oil sample to be tested is rapidly extracted, the fluorescence signal intensity x and the proportion a of the peak area of C-4 dimethyl sterol in the total peak area are matched with the adulteration detection model, and whether tea oil is adulterated with other oils is identified, and the adulteration proportion is calculated by the difference method, that is, adulteration proportion=|x-f| / 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: In the step S1, the tea oil is camellia seed oil; and 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: In the step S1, the different adulteration proportions are 5%, 10%, 15%, 20%, 30% and 50% respectively.

4. A method for identifying adulteration of tea oil based on C-4 dimethyl sterol composition according to claim 1, characterized in that, In the step S1, the preparation method of phytosterols is specifically as follows: 8% NaOH solution is added to tea oil and tea oil adulteration samples respectively, vortexed and shaken for 0.5-1.0 min, and then subjected to saponification treatment under ultrasonic heating for 15 min to obtain a treatment liquid; equal volume of deionized water is added to the treatment liquid, shaken and diluted, and then equal volume of n-hexane is added, shaken and allowed to stand until the water phase and the organic phase are separated, and then unsaponifiable matter is extracted and dried with N2 to obtain phytosterols.

5. A method for identifying adulteration of tea oil based on C-4 dimethyl sterol composition according to claim 1, characterized in that, In the step S2, the specific steps of derivatization treatment are as follows: 200 μL of silylating reagent is added to phytosterols of tea oil adulteration samples, and then the samples are subjected to derivatization treatment in a 75°C water bath for 30 min, and then cooled to room temperature, dried with N2 again, redissolved in 100 μL of n-hexane, filtered through a 0.22 μm organic microporous filter and then measured.

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

1.

7. A method for identifying adulteration of tea oil based on C-4 dimethyl sterol composition according to claim 1, characterized in that, In the step S2, the GC-MS determination conditions are as follows: Chromatographic conditions: Chromatographic column: DB-5 chromatographic column, 30 m×0.25 mm, 0.25 μm; Temperature program: initial column temperature setting at 200℃ for 0.5 min, then ramped to 250℃ at 15℃ / min and hold for 8 min, then ramped to 270℃ at 1℃ / min and hold for 1.5 min using "low rate gradient temperature program"; Detector, inlet temperature: 290℃; Injection volume: 1.0 μL; Split ratio: 1:100; Carrier gas: 99.99% high purity helium at a flow rate of 1.2 mL / min; Total run time of the program: 40 min; Mass spectrometry conditions: Inlet temperature: 300℃; Solvent delay: 10 min; Ion source: EI; Electron energy: 70 eV; Scan time: 0.5 s; Mass scan range: 50-550 m / z.

8. A 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 Filipin III fluorescent probe detection in the step S3 are as follows: 100 μL of Filipin III working solution is added to the phytosterols in the tea oil and tea oil adulteration samples, and incubated at room temperature in the dark for 30 min, then the staining solution is removed, and fluorescence analysis is performed.

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

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