Characteristic indication substance and identification method of Hainan camellia oil and Liangguang camellia oil

Abstract

The invention discloses a characteristic indicating substance for identifying Hainan camellia oil and Liangguang camellia oil, and belongs to the technical field of camellia oil variety identification, and the characteristic indicating substance is a specific volatile matter 2, 3-Butanediol. The invention also discloses a method for identifying Hainan camellia oil and Shuangguang camellia oil based on the characteristic indication substance, which comprises the following steps: detecting the content of a specific volatile matter 2, 3-Butanediol in a sample to be detected, carrying out integration processing on data, and carrying out normalization processing on the data after integration processing; when the value of the specific volatile matter 2, 3-Butanediol in the sample to be detected after the normalization treatment is greater than 10, judging that the sample to be detected is Hainan camellia oil; and when the value of the specific volatile matter 2, 3-Butanediol in the sample to be detected after the normalization treatment is less than or equal to 10, judging that the sample to be detected is the Shuangguang camellia oil. According to the method, the 2, 3-Butanediol is taken as a characteristic indicating substance, and the Hainan camellia oil and the Shuangguang camellia oil can be quickly identified by detecting the content of the 2, 3-Butanediol in a sample to be detected.

Description

[0001] This application is a divisional application of a patent application with an application date of May 23, 2024, application number 202410645965X, and invention name “A characteristic indicator substance and an identification method for Hainan camellia oil and Guangdong and Guangxi camellia oil”. Technical Field

[0002] The invention belongs to the technical field of camellia oil type identification, and specifically relates to a characteristic indicator substance and an identification method for Hainan camellia oil and Guangdong and Guangxi camellia oil based on the characteristic indicator substance. Background Art

[0003] Camellia oleifera belongs to the genus Camellia of the Theaceae family. It is a natural woody high-end edible oil variety unique to China. It is known as one of the four major woody oil tree species in the world, along with oil palm, olive and coconut. As an important oil crop in the hilly areas of southern China, the oil-tea tree has rich ecological benefits and economic value. It is widely planted in Jiangxi, Fujian, Hainan, Guangxi and Guangdong. The oil-tea tree grows in warm and humid climatic conditions. Its seeds (tea seeds) are rich in oil and oil, and its oil extraction performance is particularly outstanding. It is widely used in the production and processing of camellia oil to produce high-quality vegetable oil. Compared with olive oil, the oil components of camellia oil are mainly composed of monounsaturated fatty acids and oleic acid, and the similarity is extremely high. It is also known as "Oriental Olive Oil" internationally. Tea oil is a unique edible oil rich in functional active ingredients such as polyphenols, squalene and phytosterols. It not only has a mellow smell, but also a pleasant taste.

[0004] Hainan tea oil is called "mountain pomelo oil" by the locals. It is rich in unsaturated fatty acids and has always been considered to be of excellent quality. It is golden in color and has a unique aroma. Further research has found that Hainan mountain pomelo oil contains higher concentrations of tea saponins, tea polyphenols and α-tocopherol, giving it higher medical and health value. This quality advantage is not only due to Hainan's unique ecological environment and soil conditions (such as selenium-rich soil), but also because in the long-term natural adaptation process, Hainan oil tea is believed to have evolved into a geographical subspecies or new species that is different from inland oil tea.

[0005] In recent years, with the increasing attention of consumers to healthy food, camellia oil, as a vegetable oil with multiple health benefits, has been increasingly in demand in the market. However, due to the high market price and widespread popularity of Hainan camellia oil, a large number of counterfeit products have emerged, which has brought difficulties to consumer rights protection and market order. Therefore, a reliable and accurate characteristic indicator substance and identification method are needed to effectively identify the real Hainan camellia oil. Summary of the invention

[0006] In view of one or more of the above defects or improvement needs in the prior art, the present invention provides a characteristic indicator substance and a method for identifying Hainan camellia oil and Guangdong and Guangxi camellia oil. 2,3-Butanediol is used as the characteristic indicator substance. By detecting the 2,3-Butanediol content in the sample to be tested, Hainan camellia oil and Guangdong and Guangxi camellia oil can be quickly identified.

[0007] To achieve the above object, according to a first aspect of the present invention, a characteristic indicator substance for identifying Hainan camellia oil and Guangdong and Guangxi camellia oil is provided, and the characteristic indicator substance is a specific volatile 2,3-Butanediol.

[0008] According to a second aspect of the present invention, there is provided a use of a specific volatile compound 2,3-Butanediol as a characteristic indicator substance for distinguishing Hainan camellia oil and Guangdong and Guangxi camellia oil.

[0009] According to a third aspect of the present invention, a method for identifying Hainan camellia oil and Guangdong and Guangxi camellia oil based on characteristic indicator substances is provided, comprising the following steps:

[0010] Detect the content of the specific volatile 2,3-Butanediol in the sample to be tested and integrate the data, and then normalize the integrated data;

[0011] When the normalized value of the specific volatile 2,3-Butanediol in the sample to be tested is greater than 10, the sample to be tested is judged to be Hainan camellia oil;

[0012] When the normalized value of the specific volatile 2,3-Butanediol in the sample to be tested is less than or equal to 10, it is determined that the sample to be tested is Guangdong and Guangxi camellia oil.

[0013] As a further improvement of the present invention, the same amount of all the samples to be tested is taken and mixed, and the samples are used as quality control samples for detection together with the samples to be tested, the specific volatile 2,3-Butanediol in the samples to be tested and the quality control samples is integrated and quantified, and then the integrated data is normalized to ensure the detection quality.

[0014] As a further improvement of the present invention, the content of the specific volatile 2,3-Butanediol in the test sample and the quality control sample is detected by gas chromatography-mass spectrometry.

[0015] As a further improvement of the present invention, the volatile substance 2,3-Butanediol is first qualitatively analyzed using Qualitative Navigator B.08.00 software, and then quantitatively analyzed using Agilent MassHunter MS quantitative software, and the data are integrated, and finally the integrated data are normalized using LOWESS-Normalization-Tool software.

[0016] As a further improvement of the present invention,

[0017] When using Agilent MassHunter MS quantitative software for quantitative analysis, select ions with high peaks and specificity as quantitative ions, set a 1-min range window before and after a given RT to prevent peak drift, and perform manual integration correction on the data; and / or,

[0018] When using LOWESS-Normalization-Tool software for normalization processing, the specific parameters are set as follows: Span is set to 0.57, the minimum size of the data point is 0.32, and the maximum size is set to 1.

[0019] As a further improvement of the present invention, the conditions of gas chromatography are:

[0020] HP-5MS capillary column, 30m×0.25mm×0.25μm, was used;

[0021] The heating program was as follows: maintain at 40°C for 3 minutes; increase the temperature to 160°C at a rate of 2°C / min, and after reaching 160°C, increase the temperature to 300°C at a rate of 50°C / min as the final temperature, and maintain for 3 minutes;

[0022] The injection mode was splitless mode, the sample volume was 2 ml, the injection temperature was 270 °C, and a 0.75 mm liner was built in; high-purity He was used as the carrier gas and high-purity N2 was used as the collision gas, with flow rates of 1.0 mL / min and 1.5 mL / min respectively; full scan was used to detect volatiles, and then these signals were converted to multiple reaction monitoring mode; HS-SPME fiber was used for adsorption of volatile substances.

[0023] As a further improvement of the present invention, the mass spectrometry conditions are as follows: the ion source is EI; the MS 4-pole temperature is set to 150°C, and the ion source temperature is 230°C; the collision energy is 70eV; the injection port temperature is set to 270°C, the injection volume is 1μL, and the split ratio is set to 10:1; the carrier gas is high-purity He, and the flow rate is 1.0mL / min; the collision gas is high-purity N2, and the flow rate is 1.5mL / min; the mode is full scan, and the scanning range is m / z 50-650.

[0024] In general, the above technical solution conceived by the present invention has the following beneficial effects compared with the prior art:

[0025] The present invention proposes for the first time to use 2,3-Butanediol as a characteristic indicator substance to distinguish Hainan camellia oil from Guangdong and Guangxi camellia oil. By detecting the content of 2,3-Butanediol in the tea oil sample to be tested, Hainan camellia oil and Guangdong and Guangxi camellia oil can be quickly and accurately distinguished, and has the advantages of simple operation, low cost, high detection efficiency, etc. In addition, the content of 2,3-Butanediol can be qualitatively and quantitatively detected by gas chromatography and mass spectrometry in the prior art. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0027] The embodiment of the present invention provides a characteristic indicator substance for identifying Hainan camellia oil and Guangdong and Guangxi camellia oil. The characteristic indicator substance is a specific volatile 2,3-Butanediol, which is used to distinguish Hainan camellia oil from Guangdong and Guangxi camellia oil. The applicant discovered for the first time that 2,3-Butanediol, as a substance generated based on biochemical reaction products, has different content levels in Hainan camellia oil and Guangdong and Guangxi camellia oil. It exists stably and at high concentrations in real Hainan camellia oil, while its content is extremely low or undetectable in Guangdong and Guangxi camellia oil. Therefore, 2,3-Butanediol can be used as an effective characteristic indicator substance. By detecting the content of 2,3-Butanediol in the tea oil sample to be tested, Hainan camellia oil and Guangdong and Guangxi camellia oil can be quickly and accurately distinguished.

[0028] The invention adopts 2,3-Butanediol as a characteristic indicator substance for distinguishing Hainan camellia oil and Guangdong and Guangxi camellia oil, detects the content of the specific volatile 2,3-Butanediol in the sample to be tested, integrates the data, and then normalizes the integrated data.

[0029] Further preferably, all the samples to be tested are mixed with the same amount, and the samples are tested together with the samples to be tested as quality control samples (QC samples), and then the specific volatile 2,3-Butanediol in the samples to be tested and the quality control samples is integrated and quantified, and then the integrated data is normalized. At the same time, the quality control samples (QC samples) are tested to ensure the quality of the test.

[0030] The present invention preferably uses a high performance gas chromatography-mass spectrometry (GC-MS) method to detect the content of the specific volatile 2,3-Butanediol in the test sample and the quality control sample. The gas chromatography conditions are preferably:

[0031] HP-5MS capillary column, 30m×0.25mm×0.25μm, was used;

[0032] The heating program was as follows: maintain at 40°C for 3 minutes; increase the temperature to 160°C at a rate of 2°C / min, and after reaching 160°C, increase the temperature to 300°C at a rate of 50°C / min as the final temperature, and maintain for 3 minutes;

[0033] The injection mode was splitless mode, the sample volume was 2 ml, the injection temperature was 270 °C, and a 0.75 mm liner was built in; high-purity He was used as the carrier gas and high-purity N2 was used as the collision gas, with flow rates of 1.0 mL / min and 1.5 mL / min respectively; full scan was used to detect volatiles, and then these signals were converted to multiple reaction monitoring mode; HS-SPME fiber was used for adsorption of volatile substances.

[0034] The conditions for mass spectrometry are preferably as follows: the ion source is EI; the MS 4-pole temperature is set to 150°C, the ion source temperature is 230°C; the collision energy is 70eV; the injection port temperature is set to 270°C, the injection volume is 1μL, and the split ratio is set to 10:1; the carrier gas is high-purity He (99.999%) with a flow rate of 1.0mL / min; the collision gas is 99.999% high-purity N2 with a flow rate of 1.5mL / min; the mode is full scan (m / z 50-650).

[0035] When processing the data of gas chromatography and mass spectrometry, the area normalization method is used. First, the volatile substance 2,3-Butanediol in the sample to be tested is qualitatively analyzed, and then quantitative software is used to perform quantitative analysis and normalization processing on it and the quality control sample (QC sample). The data processing method is a prior art. For example, the data processing process of the gas chromatography and mass spectrometry of the embodiment of the present invention is as follows:

[0036] The volatile metabolite data were imported into Qualitative Navigator B.08.00 software, and qualitative analysis was performed when the similar retention time (Rt) signal was 5.35. According to the NIST database, the signal was determined to be 2,3-Butanediol, and the CAS number was 24347-58-8.

[0037] Agilent MassHunter quantitative analysis (MS) software was then used to quantify 2,3-Butanediol metabolites: ions with high peaks and specificity were selected as quantitative ions, and a 1-min range window was set before and after a given RT to prevent peak drift, and the data were manually integrated and corrected.

[0038] Finally, the integrated data were normalized using the LOWESS-Normalization-Tool software. The specific parameters for normalization were: Span was set to 0.57, the minimum size of the data point was set to 0.32, and the maximum size was set to 1.

[0039] In order to better understand and illustrate the technical solution of the present invention, the following specific embodiments are provided:

[0040] Unless otherwise specified, the methods involved in the following embodiments are all conventional methods.

[0041] Example 1 Acquisition and Detection Analysis of Camellia Oil Samples

[0042] (1) Selection of experimental materials

[0043] Camellia species of the Camellia genus in tropical and subtropical regions of China were selected, including 11 populations from Hainan Province, 4 populations from Guangdong Province and Guangxi Zhuang Autonomous Region. Three biological replicates were performed for each population, totaling 45 samples (i.e., the Camellia species used in this example were all tropical Camellia species, the difference being that they grew in different regions).

[0044] (2) Obtaining oil products

[0045] Select full and healthy camellia seeds grown in Hainan and Guangdong and Guangxi in the current year, crack the shells, peel and crush, and put them into an oven to dry with hot air until constant weight (75℃). After the camellia seeds are cooled to room temperature, they are pressed with an oil press (LYF-898 screw oil press), and the crude oil obtained by pressing is precipitated and the supernatant oil is taken.

[0046] Take 2 ml of each sample to be tested and mix them, and use them as quality control samples (QC samples) to test together with the samples to be tested.

[0047] (3) GC-MS analysis of oil products

[0048] Camellia oil was analyzed by gas chromatography (7 7890A, Agilent Technologies, USA) and Agilent 7000D mass selective detector. The compounds were separated by gas chromatography using HP-5MS capillary column (30 m×0.25 mm, 0.25 μm; Agilent).

[0049] The heating program was as follows: 40°C for 3 minutes, then the temperature was increased to 160°C at a rate of 2°C / min, and after reaching 160°C, the temperature was increased to 300°C as the final temperature at a rate of 50°C / min, and maintained for 3 minutes.

[0050] The injection mode was splitless mode, the sample volume was 2 ml, the injection temperature was 270 ° C, and a 0.75 mm inlet liner (Agilent Technologies) was built in; high-purity He (99.999%) was used as the carrier gas and high-purity N2 (99.999%) was used as the collision gas, with flow rates of 1.0 mL / min and 1.5 mL / min respectively; full scan was used to detect volatiles, and then these signals were converted to multiple reaction monitoring mode. (At the same time, after the sample test was completed, the QC samples were tested under the above conditions to ensure the quality of the test).

[0051] The volatiles were adsorbed using HS-SPME fibers with approximately 70 passes to ensure the reproducibility of the method.

[0052] The mass spectrometry conditions were as follows: the ion source was EI; the MS 4th pole temperature was set to 150°C, the ion source temperature was 230°C; the collision energy was 70 eV; the injection port temperature was set to 270°C, the injection volume was 1 μL, and the split ratio was set to 10:1; the carrier gas was high-purity He (99.999%) at a flow rate of 1.0 mL / min; the collision gas was 99.999% high-purity N2, at a flow rate of 1.5 mL / min; the mode was full scan (m / z 50-650).

[0053] (4) Qualitative and quantitative analysis of 2,3-Butanediol

[0054] The volatile metabolite data of the sample to be tested and QC were imported into Qualitative Navigator B.08.00 software. When the similar retention time (Rt) signal was 5.35, the sample to be tested had a peak (the peak offset before and after did not exceed 0.2min). According to the NIST database, this signal was qualitatively identified as 2,3-Butanediol when the CAS number was 24347-58-8.

[0055] Based on the qualitative results, the Agilent MassHunter quantitative analysis (MS) software was used to quantitatively analyze 2,3-Butanediol metabolites and QC. Ions with high peaks and specificity were selected as quantitative ions, and a 1-min range window was set before and after a given RT to prevent peak drift, and the data were manually integrated and corrected.

[0056] The integrated data were normalized using the LOWESS-Normalization-Tool software to normalize the quantitative data of the samples to be tested and QC. The specific parameters of normalization were set as follows: Span was set to 0.57, the minimum size of the data point was set to 0.32, and the maximum size was set to 1.

[0057] The experimental results are shown in Table 1 below.

[0058] Table 1 Normalized values ​​of 2,3-Butanediol in camellia oil at different sampling points

[0059]

[0060] The experimental results show that 2,3-Butanediol is a characteristic indicator substance that can effectively distinguish Hainan camellia oil from Guangdong and Guangxi camellia oil. According to the quantitative results, it can be determined whether the sample is Hainan camellia oil. When the normalized value of 2,3-Butanediol in the sample is greater than 10, it is judged to be Hainan camellia oil. On the contrary, if the normalized value of 2,3-Butanediol in the sample does not reach this value (less than or equal to 10), it is judged to be camellia oil from Guangdong and Guangxi.

[0061] The present invention utilizes GC-MS analysis method to detect and identify camellia oil products, and gas chromatography (GC) combined with mass spectrometry detector (MS) is used to analyze the compound components of oil products. In GC analysis, HP-5MS capillary column is used to separate compounds, and the temperature program is set to 40°C for 3 minutes, then heated to 160°C at a rate of 2°C / min, and then heated to 300°C at a rate of 50°C / min and maintained for 3 minutes. The injection mode is splitless mode, the sample load is 2ml, the injection temperature is 270°C, high-purity He and N2 are used as carrier gas and collision gas, and the flow rate is 1.0mL / min and 1.5mL / min. In addition, HS-SPME fiber is used to adsorb volatile substances during the analysis process to ensure the repeatability of the method. At the same time, after the sample test is completed, the quality control sample (QC sample) is inspected to ensure the detection quality.

[0062] Furthermore, the GC-MS data were analyzed and processed using Qualitative Navigator B.08.00 software, and the peak signals were qualitatively identified according to the NIST database. The peak with the Rt signal of 5.35 and the CAS number of 24347-58-8 was qualitatively identified as 2,3-Butanediol, and the Agilent MassHunter MS quantitative software was used for quantitative analysis, and the LOWESS-Normalization-Tool software was used for normalization. According to the results after normalization, when the value of the integral data of 2,3-Butanediol in the sample after normalization is greater than 10, it can be determined to be Hainan camellia oil; on the contrary, if the content in the sample does not reach this value, it is determined to be camellia oil from Guangdong Province and Guangxi Zhuang Autonomous Region.

[0063] The present invention can identify Hainan camellia oil and Guangdong and Guangxi camellia oil by detecting the 2,3-Butanediol content in the sample to be tested and performing several integrals and normalization processing on the data, and has the advantages of simple operation, low cost, high detection efficiency, etc. The application of the characteristic indicator substance of Hainan camellia oil provided by the present invention will not only improve consumers' cognition and selection ability of camellia oil quality, but also provide important technical support for establishing a sound camellia oil standardization management system.

[0064] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for identifying Hainan camellia oil and Guangdong and Guangxi camellia oil based on characteristic indicator substances, characterized in that: The steps include: Take the same amount of all the samples to be tested and mix them, and use them as quality control samples to test together with the samples to be tested. The specific volatile 2,3-Butanediol in the samples to be tested and the quality control samples is integrated and quantified, and then the integrated data is normalized to ensure the detection quality; When the normalized value of the specific volatile 2,3-Butanediol in the sample to be tested is greater than 10, the sample to be tested is judged to be Hainan camellia oil; When the normalized value of the specific volatile 2,3-Butanediol in the sample to be tested is less than or equal to 10, the sample to be tested is judged to be Guangdong and Guangxi camellia oil; The content of the specific volatile 2,3-Butanediol in the test sample and the quality control sample is detected by gas chromatography-mass spectrometry; The conditions for gas chromatography were: HP-5MS capillary column, 30m×0.25mm×0.25μm, was used; The heating program was as follows: maintain at 40°C for 3 minutes; increase the temperature to 160°C at a rate of 2°C / min, and after reaching 160°C, increase the temperature to 300°C at a rate of 50°C / min as the final temperature, and maintain for 3 minutes; The injection mode was splitless mode, the sample volume was 2 ml, the injection temperature was 270 °C, and a 0.75 mm liner was built in. High-purity He was used as the carrier gas and high-purity N2 was used as the collision gas, with flow rates of 1.0 mL / min and 1.5 mL / min, respectively. Volatiles were detected using full scan mode, and then these signals were converted to multiple reaction monitoring mode. HS-SPME fiber was used for adsorption of volatile substances. The conditions for mass spectrometry were: The ion source was EI; the MS 4th pole temperature was set to 150°C, and the ion source temperature was 230°C; the collision energy was 70eV; the injection port temperature was set to 270°C, the injection volume was 1μL, and the split ratio was set to 10:1; the carrier gas was high-purity He with a flow rate of 1.0mL / min; the collision gas was high-purity N2 with a flow rate of 1.5mL / min; the mode was full scan, and the scanning range was m / z 50-650.

2. The method for identifying Hainan camellia oil and Guangdong and Guangxi camellia oil based on characteristic indicator substances according to claim 1, characterized in that: Firstly, Qualitative Navigator B.08.00 software was used to qualitatively characterize the volatile substance 2,3-Butanediol, and then Agilent MassHunter MS quantitative software was used to quantitatively analyze it and integrate the data. Finally, LOWESS-Normalization-Tool software was used to normalize the integrated data.

3. The method for identifying Hainan camellia oil and Guangdong and Guangxi camellia oil based on characteristic indicator substances according to claim 2, characterized in that: When using Agilent MassHunter MS quantitative software for quantitative analysis, select ions with high peaks and specificity as quantitative ions, set a 1-min range window before and after a given RT to prevent peak drift, and perform manual integration correction on the data; and / or, When using LOWESS-Normalization-Tool software for normalization processing, the specific parameters are set as follows: Span is set to 0.57, the minimum size of the data point is 0.32, and the maximum size is set to 1.

Images