Method for identifying characteristic indicator substance, hainan camellia oil and liangguang camellia oil

Abstract

The application discloses a characteristic indicator substance for identifying Hainan camellia oil and Liangguang camellia oil, and belongs to the technical field of camellia oil type identification. The characteristic indicator substance is specific volatile matter 2,3-Butanediol. The application further discloses a method for identifying Hainan camellia oil and Liangguang camellia oil based on the characteristic indicator substance, which comprises the following steps: detecting the content of specific volatile matter 2,3-Butanediol in a to-be-detected sample and performing integral processing on data, and then performing normalization processing on the integral data; when the value of specific volatile matter 2,3-Butanediol in the to-be-detected sample after normalization processing is greater than 10, it is determined that the to-be-detected sample is Hainan camellia oil; and when the value of specific volatile matter 2,3-Butanediol in the to-be-detected sample after normalization processing is less than or equal to 10, it is determined that the to-be-detected sample is Liangguang camellia oil. In the application, 2,3-Butanediol is used as the characteristic indicator substance, and the content of 2,3-Butanediol in a to-be-detected sample is detected, so that Hainan camellia oil and Liangguang camellia oil can be quickly identified.

Description

[0001] This application is a divisional application of the patent application filed on May 23, 2024, with application number 202410645965X, entitled "A characteristic indicator substance and a method for identifying Hainan camellia oil and Guangdong and Guangxi camellia oil". Technical Field

[0002] This invention belongs to the technical field of camellia oil identification, specifically relating to a characteristic indicator substance and a method for identifying Hainan camellia oil and Guangdong and Guangxi camellia oil based on the characteristic indicator substance. Background Technology

[0003] Camellia oleifera, belonging to the genus Camellia in the family Theaceae, is a unique natural woody high-end edible oil variety native to China. It is considered one of the world's four major woody oilseed tree species, along with oil palm, olive, and coconut. As an important oil crop in the hilly regions of southern China, the camellia oleifera tree has abundant ecological and economic value and is widely cultivated in Jiangxi, Fujian, Hainan, Guangxi, and Guangdong provinces. Growing in warm and humid climates, its seeds (camellia seeds) are rich in oil, exhibiting particularly outstanding oil extraction performance. It is widely used in the production and processing of camellia oil to produce high-quality vegetable oil. Compared to olive oil, camellia oil's oil composition is mainly composed of monounsaturated fatty acids and oleic acid, showing a very high degree of similarity, and is internationally known as "Oriental olive oil." Camellia oil is a unique edible oil rich in functional active ingredients such as polyphenols, squalene, and phytosterols, possessing not only a mellow aroma but also a pleasant taste.

[0004] Hainan camellia oil, locally known as "mountain pomelo oil," is rich in unsaturated fatty acids and has long been considered of superior quality. It is golden in color and possesses a unique aroma. Further research has revealed that Hainan mountain pomelo oil contains higher concentrations of tea saponins, tea polyphenols, and α-tocopherols, giving it greater health benefits. This superior quality stems not only from Hainan's unique ecological environment and soil conditions (such as selenium-rich soils) but also from the fact that, through long-term natural adaptation, Hainan camellia is believed to have evolved into a distinct geographical variety or new species different from inland camellia.

[0005] In recent years, with consumers' increasing focus on health foods, the demand for camellia oil, a plant oil with multiple health benefits, has been growing rapidly. However, due to the high market price and widespread popularity of Hainan camellia oil, a large number of counterfeit products have emerged, posing challenges to consumer rights protection and market order. Therefore, a reliable and accurate characteristic indicator and identification method are needed to effectively distinguish genuine Hainan camellia oil. Summary of the Invention

[0006] In view of one or more of the above-mentioned defects or improvement needs of 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. Using 2,3-Butanediol as a characteristic indicator substance, Hainan camellia oil and Guangdong and Guangxi camellia oil can be quickly identified by detecting the content of 2,3-Butanediol in the sample to be tested.

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

[0008] According to a second aspect of the invention, a specific volatile compound 2,3-Butanediol is provided as a characteristic indicator for identifying 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-Guangxi camellia oil based on characteristic indicator substances is provided, comprising the following steps:

[0010] The content of the specific volatile compound 2,3-Butanediol in the sample was detected, the data were integrated, and then the integrated data were normalized.

[0011] If the value of the specific volatile compound 2,3-Butanediol in the sample to be tested after normalization is greater than 10, then the sample to be tested is determined to be Hainan camellia oil.

[0012] If the value of the specific volatile compound 2,3-Butanediol in the sample to be tested after normalization is less than or equal to 10, then the sample to be tested is determined to be camellia oil from Guangdong and Guangxi.

[0013] As a further improvement of the present invention, all samples to be tested are mixed in equal amounts and used as quality control samples together with the samples to be tested. The specific volatile compound 2,3-Butanediol in the samples to be tested and the quality control samples are integrated and quantified, and then the integrated data are normalized to ensure the quality of the test.

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

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

[0016] As a further improvement of the present invention

[0017] When performing quantitative analysis using Agilent MassHunter MS software, ions with high peak intensity and specificity are selected as the quantitative ions. A 1-minute window is set before and after a given RT to prevent peak drift, and the data are manually integrated and corrected; and / or,

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

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

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

[0021] The heating program is as follows: hold at 40℃ for 3 minutes; increase the temperature to 160℃ at a rate of 2℃ / min; after reaching 160℃, increase the temperature to 300℃ at a rate of 50℃ / min to reach the final temperature, and hold for 3 minutes.

[0022] The injection mode was splitless, the sample loading volume was 2 ml, the injection temperature was 270℃, and a 0.75 mm liner was used. High-purity He was used as the carrier gas and high-purity N2 as the collision gas, with flow rates of 1.0 mL / min and 1.5 mL / min, respectively. Volatile substances were detected using a full scan method, and then these signals were converted to multiple reaction monitoring mode. Volatile substances were adsorbed using HS-SPME fiber.

[0023] As a further improvement of the present invention, the mass spectrometry conditions are as follows: the ion source is EI; the MS stage 4 temperature is set to 150℃, the ion source temperature is 230℃; the collision energy is 70eV; the injection port temperature is set to 270℃, 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 scan range is m / z 50-650.

[0024] In summary, the technical solutions conceived by this invention have the following beneficial effects compared with the prior art:

[0025] This invention is the first to propose using 2,3-Butanediol as a characteristic indicator to distinguish between Hainan camellia oil and Guangdong and Guangxi camellia oil. By detecting the content of 2,3-Butanediol in the camellia oil sample, Hainan camellia oil and Guangdong and Guangxi camellia oil can be quickly and accurately distinguished. It has the advantages of simple operation, low cost, and high detection efficiency. Furthermore, the content of 2,3-Butanediol can be qualitatively and quantitatively detected using gas chromatography and mass spectrometry, which are existing technologies. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0027] This invention provides a characteristic indicator for distinguishing Hainan camellia oil from Guangdong and Guangxi camellia oil. The characteristic indicator is a specific volatile compound, 2,3-Butanediol, used to differentiate between the two types of camellia oil. The applicant has discovered for the first time that 2,3-Butanediol, as a substance generated from a biochemical reaction, exhibits different content levels in Hainan and Guangdong / Guangxi camellia oils. It is present at a stable, high concentration in genuine Hainan camellia oil, while its content is extremely low or undetectable in Guangdong / Guangxi camellia oil. Therefore, 2,3-Butanediol can serve as an effective characteristic indicator. By detecting the content of 2,3-Butanediol in the camellia oil sample, Hainan and Guangdong / Guangxi camellia oils can be quickly and accurately distinguished.

[0028] This invention uses 2,3-Butanediol as a characteristic indicator for distinguishing Hainan camellia oil and Guangdong and Guangxi camellia oil. The content of the specific volatile compound 2,3-Butanediol in the test sample is detected, and the data is integrated and then normalized.

[0029] More preferably, equal amounts of all test samples are taken and mixed together, and this mixture is used as a quality control sample (QC sample) for testing together with the test samples. Then, the specific volatile compound 2,3-Butanediol in the test samples and QC samples is integrated and quantified, and the integrated data are normalized. Simultaneously, the QC sample is inspected to ensure the quality of the test.

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

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

[0032] The heating program is as follows: hold at 40℃ for 3 minutes; increase the temperature to 160℃ at a rate of 2℃ / min; after reaching 160℃, increase the temperature to 300℃ at a rate of 50℃ / min to reach the final temperature, and hold for 3 minutes.

[0033] The injection mode was splitless, the sample loading volume was 2 ml, the injection temperature was 270℃, and a 0.75 mm liner was used. High-purity He was used as the carrier gas and high-purity N2 as the collision gas, with flow rates of 1.0 mL / min and 1.5 mL / min, respectively. Volatile substances were detected using a full scan method, and then these signals were converted to multiple reaction monitoring mode. Volatile substances were adsorbed using HS-SPME fiber.

[0034] The preferred mass spectrometry conditions were as follows: EI ion source; MS stage 4 temperature set to 150℃, ion source temperature set to 230℃; collision energy set to 70 eV; injection port temperature set to 270℃, injection volume set to 1 μL, split ratio set to 10:1; carrier gas high-purity He (99.999%) flow rate set to 1.0 mL / min; collision gas 99.999% high-purity N2 flow rate set to 1.5 mL / min; mode set to full scan (m / z 50-650).

[0035] When processing gas chromatography and mass spectrometry data, the area normalization method is used. First, the volatile substance 2,3-Butanediol in the sample is qualitatively identified, and then quantitative software is used to perform quantitative analysis and normalization on it and the quality control sample (QC sample). The data processing method is existing technology. Exemplarily, the data processing procedure for gas chromatography and mass spectrometry in this embodiment of the invention is as follows:

[0036] Import the data of volatile metabolites into Qualitative Navigator B.08.00 software. Qualitative analysis was performed when the similar retention time (Rt) signal was 5.35. According to the NIST database, the signal was identified as 2,3-Butanediol, and the CAS number was 24347-58-8.

[0037] Then, the 2,3-Butanediol metabolite was quantitatively analyzed using Agilent MassHunter (MS) software: ions with high peak intensity and specificity were selected as quantitative ions, and a 1-minute window was set before and after a given RT to prevent peak drift. The data were manually integrated and corrected.

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

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

[0040] Unless otherwise specified, the methods described in the following examples are conventional methods.

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

[0042] (1) Selection of experimental materials

[0043] Camellia oleifera species from the Camellia section of the genus *Camellia* were collected from tropical and subtropical regions of China, including 11 populations from Hainan Province and 4 populations from Guangdong Province and Guangxi Zhuang Autonomous Region. Each population was subjected to three biological replicates, totaling 45 samples (i.e., the Camellia oleifera species used in this embodiment are all tropical Camellia oleifera, differing only in their growing regions).

[0044] (2) Obtaining oil products

[0045] Select plump, healthy camellia seeds from Hainan and Guangdong provinces that were born in the current year. Crack the shells, remove the skins, and grind the seeds. Place them in an oven and dry them with hot air until they reach a constant weight (75℃). After the camellia seeds have cooled to room temperature, press them using an oil press (LYF-898 screw oil press). After the crude oil obtained from pressing has settled, collect the supernatant oil.

[0046] Take 2 ml of each of the samples to be tested and mix them together. Use this mixture as a quality control sample (QC sample) and test it together with the samples to be tested.

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

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

[0049] The heating program is as follows: 40℃, 3 minutes, at a rate of 2℃ / min, the temperature is increased to 160℃. After reaching 160℃, the temperature is increased to 300℃ at a rate of 50℃ / min, which is the final temperature, and then held for 3 minutes.

[0050] The injection mode was splitless, with a sample loading volume of 2 ml and an injection temperature of 270°C. An internal 0.75 mm imported liner (Agilent Technologies) was used. High-purity He (99.999%) was used as the carrier gas, and high-purity N2 (99.999%) as the collision gas, with flow rates of 1.0 mL / min and 1.5 mL / min, respectively. Volatile substances were detected using a full-scan method, and these signals were then converted to multiple reaction monitoring (MRM) mode. (Simultaneously, after sample testing, QC samples were inspected under the above conditions to ensure test quality.)

[0051] To ensure method reproducibility, HS-SPME fibers with approximately 70 cycles were used for volatile substance adsorption.

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

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

[0054] Import the volatile metabolite data of the test sample and QC into Qualitative Navigator B.08.00 software. A peak was found in the test sample at a similar retention time (Rt) of 5.35 (peak shift not exceeding 0.2 min). According to the NIST database, this signal was identified as 2,3-Butanediol when the CAS number was 24347-58-8.

[0055] Based on the qualitative results, 2,3-Butanediol metabolites and QC were quantitatively analyzed using Agilent MassHunter quantitative analysis (MS) software. Ions with high peak intensity and specificity were selected as quantitative ions. A 1-minute 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 process the quantitative data of the test samples and QC. The specific normalization parameters were set as follows: Span was set to 0.57, the minimum size of the data points was 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 from different sampling points.

[0059]

[0060] Experimental results show that 2,3-Butanediol is an effective characteristic indicator for distinguishing between Hainan camellia oil and Guangdong-Guangxi camellia oil. Based on this quantitative result, it can be determined whether a sample is Hainan camellia oil. When the normalized value of 2,3-Butanediol in the sample is greater than 10, it is identified as Hainan camellia oil. Conversely, if the normalized value of 2,3-Butanediol in the sample does not reach this value (less than or equal to 10), it is identified as Guangdong-Guangxi camellia oil.

[0061] This invention utilizes GC-MS analysis to detect and identify camellia oil, employing gas chromatography (GC) combined with mass spectrometry (MS) to analyze the compound components of the oil. In GC analysis, an HP-5MS capillary column is used for compound separation. The temperature program is set to 40°C for 3 minutes, then increased to 160°C at a rate of 2°C / min, and finally increased to 300°C at a rate of 50°C / min and held for 3 minutes. The injection mode is splitless, with a sample volume of 2 ml and an injection temperature of 270°C. High-purity He and N2 are used as carrier and collision gases, respectively, at flow rates of 1.0 mL / min and 1.5 mL / min. Furthermore, HS-SPME fibers are used to adsorb volatile substances during the analysis to ensure method repeatability. Simultaneously, after sample testing, quality control samples (QC samples) are inspected to ensure the quality of the analysis.

[0062] Furthermore, the GC-MS data were analyzed and processed using Qualitative Navigator B.08.00 software. Peak signals were qualitatively identified based on the NIST database. A peak with an Rt signal of 5.35 and CAS number 24347-58-8 was identified as 2,3-Butanediol. Quantitative analysis was performed using Agilent MassHunter MS software, and normalization was performed using LOWESS-Normalization-Tool software. Based on the normalized results, if the normalized integral value of 2,3-Butanediol in the sample was greater than 10, it was identified as Hainan camellia oil; conversely, if the content in the sample did not reach this value, it was identified as camellia oil from Guangdong Province and Guangxi Zhuang Autonomous Region.

[0063] This invention identifies Hainan camellia oil and Guangdong and Guangxi camellia oil by detecting the 2,3-Butanediol content in the sample and performing integral and normalization processing on the data. It has the advantages of simple operation, low cost, and high detection efficiency. The application of the characteristic indicator substance for Hainan camellia oil provided by this invention will not only improve consumers' awareness and selection ability regarding camellia oil quality, but also provide important technical support for establishing a sound standardized management system for camellia oil.

[0064] Those skilled in the art will readily understand that the above description is merely 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 within the scope of protection of the present invention.

Claims

1. A method for identifying Hainan camellia oil and Guangdong-Guangxi camellia oil based on characteristic indicator substances, characterized in that, Includes the following steps: All test samples were mixed in equal amounts and used as quality control samples. The mixture was then tested together with the test samples. The specific volatile compound 2,3-Butanediol in the test samples and quality control samples was integrated and quantified. The integrated data were then normalized to ensure the quality of the test. If the value of the specific volatile compound 2,3-Butanediol in the test sample after normalization is greater than 10, then the test sample is determined to be Hainan camellia oil. If the value of the specific volatile compound 2,3-Butanediol in the sample to be tested after normalization is less than or equal to 10, then the sample to be tested is determined to be camellia oil from Guangdong and Guangxi. The content of the specific volatile compound 2,3-Butanediol in the test sample and quality control sample was detected by gas chromatography-mass spectrometry. The conditions for gas chromatography are: An HP-5MS capillary column was used, 30m × 0.25mm × 0.25μm; The heating program is as follows: hold at 40℃ for 3 minutes; increase the temperature to 160℃ at a rate of 2℃ / min; after reaching 160℃, increase the temperature to 300℃ at a rate of 50℃ / min to reach the final temperature, and hold for 3 minutes. The injection mode was splitless, the sample loading volume was 2 ml, the injection temperature was 270℃, and a 0.75 mm liner was used. High-purity He was used as the carrier gas and high-purity N2 as the collision gas, with flow rates of 1.0 mL / min and 1.5 mL / min, respectively. Volatile substances were detected using a full scan method, and then these signals were converted to multiple reaction monitoring mode. HS-SPME fiber was used for the adsorption of volatile substances. The conditions for mass spectrometry are: The ion source was EI; the MS stage 4 temperature was set to 150℃, the ion source temperature to 230℃, the collision energy to 70 eV, the injection port temperature to 270℃, the injection volume to 1 μL, and the split ratio to 10:1; the carrier gas was high-purity He, with a flow rate of 1.0 mL / min; the collision gas was high-purity N2, with a flow rate of 1.5 mL / min; the mode was full scan, and the scan range was m / z 50-650.

2. The method for identifying Hainan camellia oil and Guangdong-Guangxi camellia oil based on characteristic indicator substances according to claim 1, characterized in that, First, the volatile substance 2,3-Butanediol was qualitatively analyzed using Qualitative Navigator B.08.00 software. Then, it was quantitatively analyzed using Agilent MassHunter MS software, and the data were integrated. Finally, the integrated data were normalized using LOWESS-Normalization-Tool software.

3. The method for identifying Hainan camellia oil and Guangdong-Guangxi camellia oil based on characteristic indicator substances according to claim 2, characterized in that, When performing quantitative analysis using Agilent MassHunter MS software, ions with high peak intensity and specificity are selected as the quantitative ions. A 1-minute window is set before and after a given RT to prevent peak drift, and the data are manually integrated and corrected; and / or, When using the LOWESS-Normalization-Tool software for normalization, the specific parameters are set as follows: Span is set to 0.57, the minimum size of the data points is 0.32, and the maximum size is set to 1.

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