Methyl esterification extraction method and detection method of fatty acid in tea

Abstract

The invention provides a methyl esterification extraction method and a detection method of fatty acid in tea, and belongs to the technical field of food detection. The invention provides a methyl esterification extraction method of fatty acid in tea leaves, which comprises the following steps: sequentially drying and crushing the tea leaves to obtain a tea leaf freeze-dried powder sample; mixing the tea freeze-dried powder sample, a sulfuric acid methanol solution and an antioxidant alcohol solution, and carrying out fatty acid methyl esterification reaction to obtain a fatty acid methyl esterified tea sample; and mixing the fatty acid methyl esterified tea sample, an inorganic salt aqueous solution and an extracting agent, and extracting to obtain fatty acid methyl ester. The fatty acid methyl esterification reaction is carried out on fatty acid in the tea leaves by combining the sulfuric acid methanol solution with the antioxidant alcohol solution, the condition is mild, oxidative degradation of the fatty acid in the treatment process can be effectively avoided, and the conversion rate and extraction efficiency of fatty acid methyl esterification are remarkably improved by combining the extraction step; and the stability and repeatability of a detection result are ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food detection, and in particular to a method for methyl esterification extraction and detection of fatty acids in tea. BACKGROUND

[0002] Tea is one of the most widely consumed natural beverages worldwide, containing a variety of nutrients and functional ingredients, including tea polyphenols, amino acids, alkaloids, and lipids. Among them, fatty acids, as an important material basis in tea, are not only closely related to the nutritional value of tea, but also play an important role in aroma formation, flavor retention, and storage quality changes. Therefore, accurate detection of fatty acid components in tea is of great significance for tea quality evaluation, processing technology optimization, and functional product development.

[0003] Currently, the detection of fatty acids mainly relies on gas chromatography or gas chromatography-mass spectrometry technology. Since fatty acids usually exist in esterified or combined form, pretreatment and methyl esterification are required before detection. The sample processing methods in related technologies mainly include Soxhlet extraction, chloroform-methanol system extraction, or petroleum ether extraction, followed by methyl esterification treatment with reagents such as sulfuric acid methanol and sodium methoxide. However, the existing methods still have certain deficiencies in the detection of tea fatty acids. On the one hand, the complex tea matrix is easily disturbed by impurities such as tea polyphenols, pigments, and sugars, causing unstable detection results. On the other hand, traditional solvents (including chloroform-methanol, petroleum ether, and diethyl ether) have a long extraction time, large solvent consumption, and large fluctuations in fatty acid recovery. In addition, some methyl esterification methods require harsh reaction conditions, such as high temperature or strong acid and alkali environment, which can easily lead to fatty acid degradation or byproduct generation, affecting the accuracy of qualitative and quantitative analysis.

[0004] Therefore, how to establish a high-efficiency fatty acid pretreatment process suitable for tea samples, simplify the operation steps, and improve the methyl esterification efficiency and detection sensitivity has become a technical problem to be solved in the current tea fatty acid analysis and detection field. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a method for methyl esterification extraction and detection of fatty acids in tea. The methyl esterification extraction method of the present application has mild conditions, which can effectively avoid the oxidative degradation of fatty acids during the processing process, and has high conversion rate and extraction efficiency of fatty acid methyl esterification.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides a method for methyl esterification extraction of fatty acids in tea, comprising the following steps: The tea is sequentially dried and crushed to obtain a tea lyophilized powder sample; The tea leaf freeze-dried powder sample, the methanol sulfuric acid solution and the antioxidant alcohol solution are mixed to carry out a fatty acid methylation reaction, so as to obtain a fatty acid methylated tea leaf sample; The fatty acid methylated tea leaf sample, the inorganic salt aqueous solution and the extractant are mixed to carry out extraction, so as to obtain a fatty acid methyl ester.

[0007] Preferably, the volume percentage concentration of sulfuric acid in the methanol sulfuric acid solution is 3-5%.

[0008] Preferably, the dosage ratio of the methanol sulfuric acid solution to tea leaf is 2-4 mL:100 mg.

[0009] Preferably, the antioxidant in the antioxidant alcohol solution includes one or more of butylated hydroxytoluene, tertiary butyl-4-hydroxyanisole and propyl gallate.

[0010] Preferably, the volume percentage concentration of antioxidant in the antioxidant alcohol solution is 0.1-0.5%.

[0011] Preferably, the dosage ratio of the antioxidant alcohol solution to tea leaf is 0.2-0.4 mL:100 mg.

[0012] Preferably, the temperature of the fatty acid methylation reaction is 90-100℃, and the time is 1-2 h.

[0013] Preferably, the extractant includes one or more of n-hexane, isooctane and petroleum ether.

[0014] The present application also provides a method for detecting methyl esterification of fatty acids in tea leaves, including the following steps: The fatty acid methyl ester prepared by the methyl esterification extraction method is detected by gas chromatography.

[0015] Preferably, the conditions of the gas chromatography detection include: the carrier gas is nitrogen, the carrier gas flow rate is 1.0 mL / min, the injection port temperature is 270℃; the detector temperature is 280℃; the split ratio is 10:1, the injection amount is 1.0 μL, the chromatographic column uses an HP-88 capillary column, and the temperature rising program is: the initial temperature is 100℃, maintained for 3 min, increased to 180℃ at a rate of 10℃ / min, maintained for 6 min, increased to 200℃ at a rate of 1℃ / min, maintained for 20 min, and increased to 230℃ at a rate of 4℃ / min, maintained for 5.5 min.

[0016] The application provides a methyl esterification extraction method of fatty acids in tea leaves, which comprises the following steps: sequentially drying and crushing tea leaves to obtain a tea freeze-dried powder sample; mixing the tea freeze-dried powder sample, a methyl sulfate solution and an antioxidant alcohol solution to perform a fatty acid methyl esterification reaction, so as to obtain a fatty acid methyl esterified tea sample; mixing the fatty acid methyl esterified tea sample, an inorganic salt aqueous solution and an extractant to perform extraction, so as to obtain a fatty acid methyl ester.

[0017] Compared with the prior art, the application has the following beneficial effects: The application provides a methyl esterification extraction method of fatty acids in tea leaves, which can effectively avoid the oxidative degradation of fatty acids in the processing process by using a methyl sulfate solution combined with an antioxidant alcohol solution for fatty acid methyl esterification reaction of fatty acids in tea leaves, and can significantly improve the conversion rate and extraction efficiency of fatty acid methyl esterification, thereby ensuring the stability and repeatability of the detection results.

[0018] The application also provides a methyl esterification detection method of fatty acids in tea leaves, which has the following specific advantages: (1) Method optimization: the use of a methyl sulfate solution and the introduction of an antioxidant can effectively avoid the oxidation of fatty acids and improve the methyl esterification efficiency; (2) High detection sensitivity: the gradient temperature program of gas chromatography can separate a plurality of saturated fatty acids and unsaturated fatty acids with high separation degree, and the detection results are more accurate; (3) Strong applicability: 37 kinds of fatty acid methyl esters in tea leaves can be detected at the same time, the coverage is wide, and the method is suitable for analysis of various tea samples; (4) Reliable quantification: the use of external standard method for quantification can improve the accuracy and comparability of data, and meet the needs of scientific research and industrial application; (5) Great popularization value: the method is simple and the results are stable, and can be applied to tea quality evaluation, nutritional component research and functional component analysis.

[0019] The data of the embodiments show that the methyl esterification extraction method of the application can detect 37 kinds of fatty acid methyl esters in tea leaves at the same time, has the advantages of high conversion rate, good repeatability, good separation effect and accurate quantification, and the application has the advantages of simple operation, high stability and wide applicability, and provides reliable technical support for tea quality evaluation, origin tracing and processing process monitoring. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The figure is the gas chromatography detection result spectrum of 37 kinds of fatty acid methyl esters in Example 1. Figure 2 The figure is the gas chromatography detection result spectrum of 37 kinds of fatty acid methyl esters in Comparative Example 1. DETAILED DESCRIPTION

[0021] The application provides a methyl esterification extraction method of fatty acids in tea leaves, which comprises the following steps: The tea leaves are sequentially dried and crushed to obtain a tea leaf freeze-dried powder sample; The tea leaf freeze-dried powder sample, a methanol sulfuric acid solution and an antioxidant alcohol solution are mixed to perform a fatty acid methylation reaction to obtain a fatty acid methylated tea leaf sample; The fatty acid methylated tea leaf sample, an inorganic salt aqueous solution and an extractant are mixed to perform extraction to obtain a fatty acid methyl ester.

[0022] In the present application, the raw materials used are all commercially available products in the art unless otherwise specified.

[0023] The present application sequentially dries and crushes the tea leaves to obtain a tea leaf freeze-dried powder sample.

[0024] In the present application, the drying is preferably freeze-drying, and the present application does not have special limitations on the specific parameters of the freeze-drying, which can be performed in a manner well known to those skilled in the art.

[0025] In the present application, the crushing is preferably followed by sieving.

[0026] In the present application, the particle size of the preferred tea leaf freeze-dried powder sample is preferably 150-250 μm, and can be specifically 150, 200 or 250 μm.

[0027] After obtaining the tea leaf freeze-dried powder sample, the present application mixes the tea leaf freeze-dried powder sample, a methanol sulfuric acid solution and an antioxidant alcohol solution to perform a fatty acid methylation reaction to obtain a fatty acid methylated tea leaf sample.

[0028] In the present application, the volume percentage concentration of sulfuric acid in the methanol sulfuric acid solution is preferably 3-5%, and can be specifically 3%, 4% or 5%.

[0029] In the present application, the dosage ratio of the methanol sulfuric acid solution to tea leaves is preferably 2-4 mL:100 mg, and can be specifically 2 mL:100 mg, 3 mL:100 mg or 4 mL:100 mg.

[0030] In the present application, the antioxidant in the antioxidant alcohol solution preferably includes one or more of butylated hydroxytoluene, tertiary butyl-4-hydroxyanisole (BHA) and propyl gallate (PG).

[0031] In the present application, the volume percentage concentration of the antioxidant in the antioxidant alcohol solution is preferably 0.1-0.5%, and can be specifically 0.1%, 0.2%, 0.3%, 0.4% or 0.5%.

[0032] In the present application, the ratio of the antioxidant alcohol solution to the tea leaves is preferably 0.2-0.4 mL:100 mg, and can be specifically 0.2 mL:100 mg, 0.3 mL:100 mg or 0.4 mL:100 mg.

[0033] In the present application, the alcohol solvent in the antioxidant alcohol solution preferably includes methanol and / or ethanol.

[0034] In the present application, the temperature of the fatty acid methyl esterification reaction is preferably 90-100℃, and can be specifically 90℃, 95℃ or 100℃, and the time is preferably 1-2 h, and can be specifically 1 h, 1.5 h or 2 h.

[0035] In the present application, the fatty acid methyl ester obtained by the fatty acid methyl esterification reaction preferably includes methyl butyrate, methyl hexanoate, methyl octanoate, methyl linoleate, methyl undecanoate, methyl dodecanoate, methyl tridecanoate, methyl tetradecanoate, methyl cis-9-tetradecenoate, methyl pentadecanoate, methyl cis-10-pentadecenoate, methyl hexadecanoate, methyl cis-9-hexadecenoate, methyl heptadecanoate, methyl cis-10-heptadecenoate, methyl octadecanoate, methyl trans-9-octadecenoate, methyl cis-9-octadecenoate, methyl trans, trans-9,12-octadecadienoate, methyl cis, cis-9,12-octadecadienoate, methyl eicosanoate, methyl cis, cis, cis-6,9,12-octadecatrienoate, methyl cis, cis, cis-9,12,15-octadecatrienoate, methyl cis-11-eicosenoate, methyl heneicosanoate, methyl cis, cis-11,14-eicosadienoate, methyl docosanoate, methyl cis, cis, cis-8,11,14-eicosatrienoate, methyl cis-11,14,17-eicosatrienoate, methyl cis-13-docosenoate, methyl cis-5,8,11,14-eicosatetraenoate, methyl tricosanoate, methyl cis-13,16-docosadienoate, methyl cis-5,8,11,14,17-eicosapentaenoate, methyl tetracosanoate, methyl cis-15-tetracosenoate and methyl cis-4,7,10,13,16,19-docosahexaenoate, a total of 37 fatty acid methyl esters.

[0036] In the present application, the methyl sulfate solution and the antioxidant alcohol solution are sequentially added to the tea leaf freeze-dried powder sample, and after being fully vortexed and uniformly mixed, the fatty acid methyl esterification reaction is carried out in a constant-temperature water bath, to obtain the fatty acid methyl esterified tea sample. The methyl sulfate solution is added first to ensure that the fatty acid methyl esterification reaction is complete, and then the antioxidant alcohol solution is added to protect the generated fatty acid methyl ester from oxidation, thereby improving the detection stability and accuracy.

[0037] After obtaining the fatty acid methylated tea sample, the present application mixes the fatty acid methylated tea sample, the inorganic salt aqueous solution and the extractant for extraction to obtain the fatty acid methyl ester.

[0038] In the present application, the inorganic salt aqueous solution preferably comprises one or more of sodium chloride aqueous solution, saturated potassium chloride aqueous solution and saturated ammonium chloride aqueous solution, and the inorganic salt aqueous solution serves to increase the ionic strength of the water phase, so that the fatty acid methyl ester is more easily introduced into the extractant (organic phase), and the separation is facilitated.

[0039] In the present application, the mass percentage concentration of sodium chloride in the sodium chloride aqueous solution is preferably 0.5-0.9%, and can be 0.5%, 0.6%, 0.7%, 0.8% or 0.9% in particular.

[0040] In the present application, the dosage ratio of the inorganic salt aqueous solution to the tea is preferably 4-8 mL:100 mg, and can be 4 mL:100 mg, 5 mL:100 mg, 6 mL:100 mg, 7 mL:100 mg or 8 mL:100 mg in particular.

[0041] In the present application, the extractant preferably comprises one or more of n-hexane, isooctane and petroleum ether, and the extractant serves to extract the fatty acid methyl ester, and the extractant has a low boiling point and is easy to volatilize and remove.

[0042] In the present application, the boiling range of the petroleum ether is preferably 40-60℃.

[0043] In the present application, the dosage ratio of the extractant to the tea is preferably 2-4 mL:100 mg, and can be 2 mL:100 mg, 3 mL:100 mg or 4 mL:100 mg in particular.

[0044] In the present application, the inorganic salt aqueous solution and the extractant are preferably added to the fatty acid methylated tea sample in sequence, and after vortex mixing, the supernatant is collected by centrifugation, and then the supernatant is dried by nitrogen blowing to obtain the fatty acid methyl ester, the inorganic salt aqueous solution is added first to separate the water phase and the organic phase clearly, and then the extractant is added to effectively extract the fatty acid methyl ester, thereby ensuring the extraction efficiency.

[0045] In the present application, the number of extractions is preferably 1-2 times.

[0046] The present application does not have special limitations on the specific parameters of vortex mixing, centrifugal collection and nitrogen blowing, and the methods well known to those skilled in the art can be used.

[0047] After obtaining the fatty acid methyl ester, the present application preferably uses n-hexane to redissolve to obtain a sample to be tested for subsequent methyl ester detection.

[0048] In the present application, the ratio of the amount of n-hexane to tea is preferably 200-400 μL: 50-100 mg.

[0049] The mechanism of the improved conversion rate of fatty acid methyl esterification in the present application is as follows: Low concentration sulfuric acid protonates carboxyl in methanol, improves carbonacyl electropositivity, promotes methanol nucleophilic attack, reduces activation energy, and improves reaction rate. Reducing acidity can significantly reduce the double bond isomerization / polymerization of unsaturated fatty acids and the dehydration condensation of sugars and pigments, reducing "consumption" or degradation, thus effectively improving the conversion rate. Antioxidants capture peroxide chain reaction free radicals (ROO· / RO·), block PUFA (polyunsaturated fatty acids) peroxidation, and make unsaturated FAME (fatty acid methyl ester) not be oxidatively cleaved or generate a tailing peak, so that the true conversion rate and the measurable peak area increase.

[0050] The mechanism of the improved extraction efficiency is as follows: Salting-out effect (addition of inorganic salt aqueous solution): increase the ionic strength of the water phase, reduce the solubility of FAME in the water phase, and tilt the distribution coefficient to n-hexane, thereby improving the extraction rate and making the layering clearer.

[0051] Low-boiling nonpolar solvent (extractant): high solubility and volatile and easy to remove, reducing residue and loss.

[0052] Mixing and constant temperature bath: improve mass transfer and diffusion, avoid matrix embedding, meet the Arrhenius relationship (mild warming makes k↑ while side reactions are still controlled), and comprehensively improve the apparent yield.

[0053] The present application also provides a method for detecting methyl esterification of fatty acids in tea, comprising the following steps: The fatty acid methyl ester prepared by the methyl esterification extraction method described in the above technical solution is subjected to gas chromatography detection.

[0054] In the present application, the conditions for gas chromatography detection preferably include: the carrier gas is nitrogen, the carrier gas flow rate is 1.0 mL / min, the injection port temperature is 270℃; the detector temperature is 280℃; the split ratio is 10:1, the injection amount is 1.0 μL, the chromatographic column uses an HP-88 capillary column, and the temperature rising program is: the initial temperature is 100℃, maintained for 3 min, increased to 180℃ at a rate of 10℃ / min, maintained for 6 min, increased to 200℃ at a rate of 1℃ / min, maintained for 20 min, and increased to 230℃ at a rate of 4℃ / min, maintained for 5.5 min.

[0055] In the present application, the purity of the nitrogen is preferably ≥99.999%.

[0056] In the present application, the specification of the HP-88 capillary column is preferably 100 m x 0.25 mm x 0.20 μm.

[0057] The application also provides a method for detecting methyl esterification of fatty acids in tea leaves, preferably quantitative analysis is realized by an external standard method, which can accurately determine the content of various fatty acids in tea leaves, and provides reliable technical support for tea quality evaluation, origin tracing and processing process monitoring.

[0058] The technical solutions in the application will be clearly and completely described below in combination with the embodiments in the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application.

[0059] The fresh leaves of Eurya japonica, the tea leaves of Eurya japonica, the fresh leaves of Xinyang Maojian tea and the tea leaves of Xinyang Maojian tea used in the application are all purchased from the local market in Xinyang City, Henan Province.

[0060] Example 1 Creation and verification of the detection method 1 Experimental method 1.1 Preparation of sample solution In the application, the tea leaves are sequentially subjected to freeze-drying, crushing and sieving to obtain a tea freeze-dried powder sample (150 μm). 50 mg of the freeze-dried powder sample of the tea to be tested is placed in a digestion tube, 1 mL of 5% (v / v) sulfuric acid methanol solution and 200 μL of butyl hydroxytoluene methanol solution (0.2 vol%) are sequentially added, and vortex mixing is performed for 30 s to fully mix. The sample is placed in a 95℃ constant temperature water bath for 1 h to perform a fatty acid methyl esterification reaction, and then naturally cooled to room temperature. 2 mL of 0.9 wt% NaCl aqueous solution and 1 mL of n-hexane are added, vortex mixing is performed for 30 s, and centrifugation is performed at 3000 g for 5 min. The upper organic phase is collected, and the extraction is repeated once. The upper liquid is combined. The solvent is gently blown dry with nitrogen. Finally, 400 μL of n-hexane is used for redissolution, and the sample is transferred into an injection bottle for standby.

[0061] 1.2 Preparation of standard solution The 37 kinds of fatty acid methyl ester mixed standard are prepared into a standard stock solution with a total mass concentration of 10 mg / mL with n-hexane, and stored in a-20℃ refrigerator.

[0062] 1.3 Drawing of standard curve The 37 kinds of fatty acid methyl ester mixed standard solution is prepared into 7-point series standard working solutions with concentrations in the range of 0.2-3 μg / mL, and the experiment detection is sequentially performed in the order from low concentration to high concentration. The standard curve is drawn according to the experimental results.

[0063] 1.4 Gas chromatography conditions The gas chromatography conditions are as follows: the carrier gas is high-purity nitrogen (purity≥99.999%), the carrier gas flow rate is 1.0 mL / min, the injection port temperature is 270°C, the detector temperature is 280°C, the split ratio is 10:1, and the injection amount is 1.0 μL. The chromatographic column is an HP-88 capillary column with a specification of 100 m x 0.25 mm x 0.20 μm. The temperature rising program is as follows: the initial temperature is 100°C, maintained for 3 min, increased to 180°C at a rate of 10°C / min, maintained for 6 min, increased to 200°C at a rate of 1°C / min, maintained for 20 min, and increased to 230°C at a rate of 4°C / min, maintained for 5.5 min.

[0064] 2. Results and analysis 2.1 Gas chromatogram of 37 kinds of fatty acid methyl ester mixed standard Under the experimental conditions, the experimental results of the gas chromatography detection of 37 kinds of fatty acid methyl ester mixed standard are as shown in Table 1. Figure 1 The use of gas chromatography alone effectively separates 37 kinds of fatty acid methyl esters.

[0065] 2.2 Standard curve parameters and detection limit and quantification limit of 37 kinds of fatty acid methyl esters Seven-point series of 37 kinds of fatty acid methyl ester standard working solutions with concentrations in the range of 0.2-3 μg / mL are prepared, and the corresponding linear regression equation and correlation coefficient are obtained according to the experimental results. The correlation coefficient is between 0.99965 and 0.99999. The reproducibility experiment (n=10) of 0.5 μg / mL 37 kinds of fatty acid methyl ester solution is carried out, the detection limit is calculated by three times the standard deviation, and the quantification limit is calculated by ten times the standard deviation. The detection limit of 37 kinds of fatty acid methyl esters is between 0.011 and 0.063 μg / mL, and the quantification limit is between 0.033 and 0.189 μg / mL. The detailed results are shown in Table 1.

[0066] Table 1 Standard curve parameters and detection limit and quantification limit of 37 kinds of fatty acid methyl esters

[0067] Example 2 Analysis of fatty acid components in tea samples The fatty acid components in the tea samples were detected and analyzed by the method in Example 1, and the detection results are shown in Table 2. A total of 21 fatty acids were detected in the fresh leaves and the freeze-dried powder samples of the Eurya japonica tea, among which C16:0, C18:1n9c, C18:2n6c and C18:3n3 were the main free fatty acids, accounting for 95.20wt% in the fresh leaves of Eurya japonica and 95.29wt% in the freeze-dried powder samples of Eurya japonica tea. A total of 22 fatty acids were detected in Xinyang Maocan, among which C16:0, C18:0, C18:1n9c, C18:2n6c and C18:3n3 were the main fatty acids, accounting for 93.94wt% in the fresh leaves of Xinyang Maocan and 94.39wt% in the freeze-dried powder samples of Xinyang Maocan.

[0068] Table 2 Types and contents of fatty acids detected in two tea samples (mg / g)

[0069] Comparative Example 1 Effect of different temperature programs on the separation degree of fatty acid methyl esterification The standard sample was detected by the following procedure: the carrier gas was high-purity nitrogen (purity ≥ 99.999%), the carrier gas flow rate was 1.0 mL / min, the injection port temperature was 270℃, the detector temperature was 280℃, the split ratio was 10:1, and the injection amount was 1.0 μL. The chromatographic column was an HP-88 capillary column with a specification of 100 m x 0.25 mm x 0.20 μm. The temperature program was as follows: the initial temperature was 140℃, maintained for 5 min, then increased to 240℃ at a rate of 4℃ / min, and maintained for 20 min. The results are shown in Table 1. Figure 2 As shown in Table 1, C18:3n3 and C18:3n6 cannot be completely separated. In addition, C20:3n3, C22:1n9 and C20:4n6 cannot be separated. However, the temperature program in the present application can completely separate these fatty acids.

[0070] Example 3 Effect of different antioxidants on the total content of fatty acid methyl esterification To verify the effect of different antioxidants in the method of the present application, different antioxidants (butylated hydroxytoluene, propyl gallate, vitamin E, ascorbic acid and gallic acid) were selected for experiments, and a control group without adding antioxidants was set. The experimental steps of each group were the same as those of Example 1, except that the types of antioxidants added were changed. The fatty acid methyl esters obtained were detected by gas chromatography, and the total peak area of the fatty acid methyl esterization products was calculated. The results are shown in Table 3. All experimental groups with antioxidants showed an increase in the total content of fatty acid methyl esters compared with the control group. Among them, butylated hydroxytoluene, butylated hydroxytoluene and propyl gallate showed more significant effects, with the total content of fatty acid methyl esters increasing by more than 13%; while the effects of vitamin E, ascorbic acid and gallic acid were relatively general, and could slow down the oxidative degradation of fatty acids to some extent.

[0071] Table 3 Effect of different antioxidants on the total content of fatty acid methyl esterization

[0072] The above description is only the preferred embodiments of the present application, and does not limit the present application in any form. It should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for methyl esterification extraction of fatty acids in tea leaves, characterized by, The method comprises the following steps: drying and crushing the tea leaves in sequence to obtain a tea leaf freeze-dried powder sample; mixing the tea leaf freeze-dried powder sample, a methanol-sulfuric acid solution and an antioxidant alcohol solution to perform a fatty acid methylation reaction, thereby obtaining a fatty acid methylated tea leaf sample; mixing the fatty acid methylated tea leaf sample, an inorganic salt aqueous solution and an extractant to perform extraction, thereby obtaining fatty acid methyl esters.

2. The methyl esterification extraction method according to claim 1, characterized by, The volume percentage concentration of sulfuric acid in the methanol-sulfuric acid solution is 3-5%.

3. The methyl esterification extraction method according to claim 1 or 2, characterized by, The dosage ratio of the methanol-sulfuric acid solution to the tea leaves is 2-4 mL:100 mg.

4. The methyl esterification extraction method according to claim 1, characterized by, The antioxidant in the antioxidant alcohol solution comprises one or more of butylated hydroxytoluene, tertiary butyl-4-hydroxyanisole and propyl gallate.

5. The methyl esterification extraction method according to claim 1 or 4, characterized by, The volume percentage concentration of the antioxidant in the antioxidant alcohol solution is 0.1-0.5%.

6. The methyl esterification extraction method according to claim 5, characterized by, The dosage ratio of the antioxidant alcohol solution to the tea leaves is 0.2-0.4 mL:100 mg.

7. The methyl esterification extraction method according to claim 1, characterized by, The temperature of the fatty acid methylation reaction is 90-100℃, and the time is 1-2 h.

8. The methyl esterification extraction method according to claim 1, characterized by, The extractant comprises one or more of n-hexane, isooctane and petroleum ether.

9. A method for detecting methyl esterification of fatty acids in tea leaves, characterized by, The method comprises the following steps: The fatty acid methyl esters obtained by the methyl esterification extraction method according to any one of claims 1-8 are subjected to gas chromatography detection.

10. The method of methyl ester formation detection according to claim 9, wherein, The gas chromatography detection conditions comprise: the carrier gas is nitrogen, the carrier gas flow rate is 1.0 mL / min, the injection port temperature is 270℃, the detector temperature is 280℃, the split ratio is 10:1, the injection amount is 1.0 μL, the chromatographic column is an HP-88 capillary column, and the temperature rising program is: the initial temperature is 100℃, maintained for 3 min, then increased to 180℃ at a rate of 10℃ / min, maintained for 6 min, then increased to 200℃ at a rate of 1℃ / min, maintained for 20 min, then increased to 230℃ at a rate of 4℃ / min, maintained for 5.5 min.

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