A method for detecting theanine, a new food raw material for aiding sleep, in food

Abstract

The present invention relates to a method for detecting theanine, a new food raw material for sleep aid in food, wherein the food is but not limited to milk and dairy products, meat and meat products, cereals and their products, fruits and their products, nuts and seeds and their products, water, food and frozen drinks, alcohol, candy, chocolate and jelly, condiments, products with cereals, beans or potato powder (granules) as main raw materials, special dietary foods and food matrices such as tea, and the new food raw material for sleep aid is tea theanine. The method comprises the following steps: (1) preparing a standard stock solution and a standard working solution; (2) preparing a sample solution to be tested; (3) high performance liquid chromatography analysis conditions; (4) establishing a standard working curve; and (5) result analysis. The method can complete the separation of 17 common amino acids and aminobutyric acid in 15 minutes, has high sensitivity, high stability and high reliability for detecting theanine in food, and has excellent promotion and application value.

Description

Technical Field

[0001] The invention relates to a method for detecting food raw materials, in particular to a method for detecting theanine, a new food raw material for aiding sleep, in food. Background Art

[0002] Theanine from tea leaves, with a molecular formula of C7H 14 N2O3, an amide compound, is a unique amino acid in tea, accounting for 1%-2% of the weight of dry tea, and its main component is L-theanine. Theanine was first isolated from green tea in 1950 and is a characteristic amino acid of tea. There are more than 20 kinds of amino acids in tea, and theanine accounts for about 50% to 60% of the total amino acids in tea. Theanine was first extracted from tea, but now theanine extracted from natural tea is not the mainstream source on the market. At present, the production of chemically synthesized theanine accounts for nearly 2 / 3 of the international theanine market, theanine produced by biological fermentation accounts for about 25%, and natural theanine has retreated to third place, accounting for less than 10%. Theanine is a special amino acid contained in tea. Studies have shown that theanine has the effects of lowering blood pressure, calming the nerves, improving sleep, and promoting brain function, so it is also known as the "brain amino acid". It is added to food as a functional ingredient to develop functional foods that relieve nervous tension and improve intelligence.

[0003] At present, there are quite a few foods in the Chinese market that contain tea theanine, mainly in common foods such as beverages, dairy products, and candies. The actual content of these foods that claim to contain tea theanine is completely unknown, and the quality of the products varies. Some businesses may use inferior products as good ones and engage in fraudulent sales.

[0004] At present, the relevant standards for tea theanine in food are only GB / T 23193-2017 "Determination of theanine in tea by high performance liquid chromatography", GB / T 30987-2020 "Determination of free amino acids in plants", group standard T / GZTSS1-2021 "Determination of theanine and theobromine content in tea by near-infrared diffuse reflectance spectroscopy", and ISO 19563:2017 "Determination of theanine in tea and instant tea in solid form using high-performance liquid chromatography". Patents such as CN 114965745 A, CN 107202836 B, CN 117571866A are only applicable to tea or solid instant tea matrices. They are not applicable to common matrices on the market that are added with tea theanine, such as candy, dairy products, beverages, etc., and cannot meet the detection requirements of tea theanine in complex foods. Patent application CN118243815A announced a method for determining the content of L-theanine, which uses water to extract L-theanine in compressed candies and uses a liquid chromatograph to measure it at a wavelength of 195nm to 203nm. This technology is only applicable to compressed candies with simpler food matrices. L-theanine peaks at 2.3min. The detection wavelength of the ultra-low wavelength band of 195nm to 203nm cannot effectively resist interference, and thus cannot meet the detection requirements of a variety of complex food matrices in the announcement. At the same time, only water extraction is used, and L-theanine is unstable in aqueous solution. Without adding a stabilizer, the stability of L-theanine in the sample during the extraction process and the sample test on the machine cannot be guaranteed, and thus the accuracy of the measurement results cannot be effectively guaranteed.

[0005] At present, there is no standard or research method for the determination of tea theanine in more than 10 categories of food. In order to protect the legitimate rights and interests of consumers and combat the bad hype and conceptual addition of functional components by enterprises, it is necessary to establish a detection method for tea theanine in food as soon as possible. Summary of the invention

[0006] The purpose of the present invention is to overcome the problems existing in the existing theanine detection and provide a method for detecting theanine, a new food raw material for sleep aids in food. The present invention innovatively adopts a compound stabilizer as an extracting solution to extract theanine from tea leaves in a complex food matrix. After separation by a hydrophilic chromatographic column, a post-column online reaction is carried out with an o-phthalaldehyde (OPA) derivative solution, and detection is carried out using a fluorescence detector. High-sensitivity, high-stability and high-reliability detection of theanine from tea leaves in food can be completed within 15 minutes.

[0007] At the same time, the present invention adopts OPA online post-column derivatization to perform derivatization, avoiding the conventional complicated and cumbersome derivatization operation process and the shortcomings of insufficient use of derivatization agent that may be caused by pre-column derivatization, and is suitable for the detection of tea theanine in complex food matrices including soy sauce samples containing high levels of free amino acids.

[0008] The detection method of the present invention uses a common high performance liquid chromatograph equipped with a fluorescence detector as an analytical instrument, rather than expensive instruments such as a liquid-mass spectrometer and an amino acid analyzer, which makes it easier to popularize and promote the application of the method. At the same time, the chromatographic column used is an ordinary and inexpensive C18 chromatographic column, rather than an expensive ion exchange chromatographic column, which greatly reduces the detection cost.

[0009] The specific plan is as follows:

[0010] A method for detecting theanine, a new food raw material for aiding sleep, in food comprises the following steps:

[0011] a. Obtain theanine standard working solution;

[0012] b. Prepare the sample solution to be tested: weigh the sample to be tested, add the extract, which is an aqueous solution containing ascorbic acid and disodium ethylenediaminetetraacetate, vortex disperse, place in an ultrasonic cleaner for ultrasonic extraction, take out and cool to room temperature after the extraction is completed, dilute with the extract and make up to the scale, mix, centrifuge, take the supernatant and filter with a microporous membrane, collect the filtrate for high performance liquid chromatography determination;

[0013] c. High performance liquid chromatography detection: using a high performance liquid chromatograph, the detector is a fluorescence detector; the chromatographic column is a C18 column; the filtrate collected in step b is sent to the high performance liquid chromatograph, an eluent is added for gradient elution, the eluate is collected and a derivatizing agent is added, the derivatizing agent is o-phthalaldehyde, and the derivatized solution is subjected to fluorescence detection;

[0014] d. Drawing of standard curve: Detect according to the chromatographic conditions of step c, with the concentration of tea theanine standard working solution as the abscissa and the corresponding peak area as the ordinate, and perform linear regression to obtain the standard curve equation;

[0015] e. Result analysis: The retention time measured in step c is used to qualitatively determine whether theanine is present in the test sample. If present, the peak area of ​​theanine in the filtrate is used to quantify the content of theanine in the test sample according to the standard curve prepared in step d.

[0016] Furthermore, the food comprises at least one of milk and dairy products, meat and meat products, cereals and their products, fruits and their products, nuts and seeds and their products, water, frozen drinks, alcohol, candy, chocolate, jelly, condiments, products made from cereals, beans or potatoes, dietary foods, and tea.

[0017] Furthermore, the method for preparing the theanine standard working solution in step a includes:

[0018] Prepare a standard stock solution, the specific steps of preparing the standard stock solution are: accurately weigh an appropriate amount of theanine standard product converted to 100% by mass according to purity, accurate to 0.01 mg, dissolve it in a 25 mL brown volumetric flask, dissolve it with the extract and make up to 25 mL, and prepare a standard stock solution with a mass concentration of 1000 mg / L; store it at 0°C to 4°C, and the validity period is 3 months;

[0019] Prepare a standard intermediate solution, the specific steps of preparing the standard intermediate solution are: accurately pipette 10.0 mL of theanine standard stock solution of tea into a 100 mL brown volumetric flask, dilute to the mark with the extract, mix well, and prepare a standard intermediate solution with a mass concentration of 100 mg / L; store at 0°C to 4°C, with a validity period of 1 month;

[0020] To prepare standard working solutions, 0.010 mL, 0.020 mL, 0.050 mL, 0.10 mL, 0.50 mL, 1.00 mL and 2.00 mL of tea theanine standard intermediate solution were respectively pipetted into 10 mL brown volumetric flasks, the volume was made up to the mark with the extract, and the solution was mixed to obtain theanine standard working solutions. The theanine concentrations of the theanine standard working solutions were 0.100 mg / L, 0.200 mg / L, 0.500 mg / L, 1.00 mg / L, 5.00 mg / L, 10.0 mg / L and 20.0 mg / L, respectively.

[0021] Furthermore, the specific steps of preparing the sample solution to be tested in step b are:

[0022] For liquid, semi-solid and solid samples, weigh 1g, accurate to 0.001g, put the sample in a 50mL stoppered colorimetric tube, add 30mL of the extract, vortex disperse, place in an ultrasonic cleaner for ultrasonic extraction for 30min, mix once every 10min, take out after extraction, cool to room temperature, dilute with the extract and make up to the scale, mix, centrifuge at 12000r / min for 3min, take the supernatant and filter with a microporous membrane, and measure the filtrate on a high performance liquid chromatograph; or,

[0023] For chocolate, candy and tea samples, weigh 1g, accurate to 0.001g, put the sample in a 50mL stoppered colorimetric tube, add 30mL of the extract, vortex disperse, place in a constant temperature water bath at 80-100℃ for 30min to 60min, mix once every 10min, take out after extraction, cool to room temperature, dilute with the extract and make up to the scale, mix, centrifuge at 12000r / min for 3min, take the supernatant and filter with a microporous filter membrane, and measure the filtrate on a high performance liquid chromatograph;

[0024] Preferably, for a sample containing protein, acetic acid is used to precipitate the protein, and after the protein is removed, the extracting solution is added for extraction;

[0025] Preferably, the concentration of ascorbic acid in the extract is 0.01-0.03 mol / L, and the concentration of disodium ethylenediaminetetraacetate is 0.001-0.003 mol / L.

[0026] Furthermore, in step c, the C18 chromatographic column adopts a RX-C18 column with a specification of 4.6 mm×250 mm inner diameter and a particle size of 5 μm;

[0027] Optionally, the detection column temperature is 40°C, the flow rate is 1 mL / min, and the injection volume is 10 μL;

[0028] Optionally, detection wavelengths: excitation wavelength: 348 nm, emission wavelength: 450 nm.

[0029] Further, in step c, for samples with a free amino acid content not lower than that of soy sauce, mobile phase A: water; mobile phase B: 0.05 mol / L disodium hydrogen phosphate solution; mobile phase C: methanol, and the gradient elution procedure is as follows:

[0030]

[0031] Furthermore, for samples with free amino acid content lower than that of soy sauce, mobile phase A: water; mobile phase C: methanol, the gradient elution program is as follows:

[0032]

[0033] Furthermore, the derivatizing agent in step c is a solution of o-phthalaldehyde, the flow rate is 0.1 mL / min, and the derivatization temperature is 80° C. The o-phthalaldehyde solution is prepared by weighing 0.40 g of o-phthalaldehyde and dissolving it in 20 mL of methanol, diluting it with 4.0 g / L sodium tetraborate decahydrate solution to 1000 mL, filtering it with a microporous filter membrane, and adding 0.80 mL of 2-mercaptoethanol to mix well;

[0034] Optionally, the correlation coefficient of the standard curve equation in step d is not less than 0.9995.

[0035] Furthermore, the detection method has anti-interference ability for 17 amino acids present in the food sample matrix, including alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and cysteine.

[0036] Furthermore, the average spiked recovery rate of the detection method is between 93.7% and 102.2%.

[0037] Beneficial effects: The present invention establishes a high performance liquid chromatography determination method for tea theanine, a new food raw material for sleep aids in food. The present invention innovatively uses a compound stabilizer, 0.01 mol / L ascorbic acid + 0.001 mol / L disodium ethylenediaminetetraacetic acid solution, as an extracting solution to extract tea theanine from a complex food matrix, and after separation by a hydrophilic chromatographic column, a post-column online reaction is carried out with an o-phthalaldehyde (OPA) derivative solution, and a fluorescence detector is used for detection, with a short detection time, high sensitivity, high stability, and high reliability.

[0038] The detection method of the present invention has good adaptability to a variety of complex matrices and is suitable for the detection of tea theanine in complex food matrices including soy sauce samples containing high levels of free amino acids. It has good anti-interference ability for 17 similar amino acids, aminobutyric acid and its hydrolyzate L-glutamine and other components, so the detection accuracy is high.

[0039] In short, the present invention provides a new technical idea for the analysis and detection of theanine in tea, a new food raw material for sleep aids, in foods by relevant inspection agencies and internal laboratories of enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solution of the present invention, the accompanying drawings will be briefly introduced below. Obviously, the accompanying drawings in the following description only relate to some embodiments of the present invention, but are not intended to limit the present invention.

[0041] Figure 1 This is the separation of tea theanine and other amino acids when the mobile phase is water and methanol provided by an embodiment of the present invention, wherein: A. chromatograms of 17 amino acids (alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, cystine); B. chromatogram of tea theanine standard solution; C. chromatogram of γ-aminobutyric acid; chromatogram of DL-glutamine;

[0042] Figure 2It is the separation of tea theanine and other amino acids when the mobile phase is 0.05 mol / L disodium hydrogen phosphate solution and methanol provided by an embodiment of the present invention, wherein: A. chromatograms of 17 amino acids (alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, cystine); B. chromatogram of tea theanine standard solution; C. chromatogram of γ-aminobutyric acid; chromatogram of DL-glutamine;

[0043] Figure 3 The blank matrix and spiked (1.0 mg / L) chromatograms of milk powder provided by one embodiment of the present invention, wherein A. the blank matrix chromatogram of milk powder spiked (1.0 mg / L); B. the blank matrix chromatogram of milk powder;

[0044] Figure 4 The blank matrix and spiked (1.0 mg / L) chromatograms of yogurt provided by one embodiment of the present invention, wherein A. the blank matrix chromatogram of yogurt spiked (1.0 mg / L); B. the blank matrix chromatogram of yogurt;

[0045] Figure 5 The blank matrix and spiked (1.0 mg / L) chromatograms of sausage provided by an embodiment of the present invention, wherein A is the chromatogram of the blank matrix of sausage spiked (1.0 mg / L); B is the chromatogram of the blank matrix of sausage;

[0046] Figure 6 The blank matrix and spiked (1.0 mg / L) chromatograms of Eight Treasure Porridge provided by an embodiment of the present invention are shown in A. The blank matrix and spiked (1.0 mg / L) chromatogram of Eight Treasure Porridge; B. The blank matrix chromatogram of Eight Treasure Porridge;

[0047] Figure 7 The blank matrix and spiked (1.0 mg / L) chromatograms of candied fruit provided by one embodiment of the present invention, wherein A. the blank matrix chromatogram of candied fruit spiked (1.0 mg / L); B. the blank matrix chromatogram of candied fruit;

[0048] Figure 8 The blank matrix and spiked (1.0 mg / L) chromatograms of melon seeds provided by an embodiment of the present invention include: A. melon seeds blank matrix spiked (1.0 mg / L) chromatogram; B. melon seeds blank matrix chromatogram;

[0049] Fig. 9 The blank matrix and spiked (1.0 mg / L) chromatograms of a beverage provided by an embodiment of the present invention, wherein A. Beverage blank matrix spiked (1.0 mg / L) chromatogram; B. Beverage blank matrix chromatogram;

[0050] Fig.10The blank matrix and spiked (1.0 mg / L) chromatograms of wine provided by one embodiment of the present invention, wherein A. blank matrix spiked (1.0 mg / L) chromatogram of wine; B. blank matrix chromatogram of wine;

[0051] Fig.11 The chromatograms of chocolate blank matrix and spiked (1.0 mg / L) provided by one embodiment of the present invention include: A. chromatogram of chocolate blank matrix spiked (1.0 mg / L); B. chromatogram of chocolate blank matrix;

[0052] Fig.12 The blank matrix and spiked (1.0 mg / L) chromatograms of candy provided by one embodiment of the present invention, wherein A. the chromatogram of the blank matrix of candy spiked (1.0 mg / L); B. the chromatogram of the blank matrix of candy;

[0053] Fig.13 The blank matrix and spiked (1.0 mg / L) chromatograms of bread provided by an embodiment of the present invention, wherein A. Bread blank matrix spiked (1.0 mg / L) chromatogram; B. Bread blank matrix chromatogram;

[0054] Fig.14 The blank matrix and spiked (1.0 mg / L) chromatograms of infant rice cereal provided by one embodiment of the present invention include: A. blank matrix spiked (1.0 mg / L) chromatogram of infant rice cereal; B. blank matrix chromatogram of infant rice cereal;

[0055] Fig.15 A. a soy sauce blank matrix and a spiked (1.0 mg / L) chromatogram provided by an embodiment of the present invention, wherein the soy sauce blank matrix spiked (1.0 mg / L) chromatogram; B. a soy sauce blank matrix chromatogram;

[0056] Fig.16 This is an actual sample chromatogram provided by an embodiment of the present invention, wherein A. Jasmine honey tea beverage B. Green tea beverage C. Original black tea beverage D. Jingjiu E. Cranberry γ-aminobutyric acid solid beverage F. Tieguanyin G. Dahongpao H. Zhengshan Xiaozhong. DETAILED DESCRIPTION

[0057] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. In the examples, those without specifying specific techniques or conditions are carried out according to the techniques or conditions described in the literature in this area or according to the product specification. Those without specifying the manufacturer of reagents or instruments used are all conventional products that can be obtained commercially. In the following examples, if not clearly stated, "%" refers to weight percentage.

[0058] Example 1

[0059] A method for detecting theanine, a new food raw material for aiding sleep, in food comprises the following steps:

[0060] a. Obtain theanine standard working solution;

[0061] b. Prepare the sample solution to be tested: weigh the sample to be tested, add the extract, which is an aqueous solution containing ascorbic acid and disodium ethylenediaminetetraacetate, vortex disperse, place in an ultrasonic cleaner for ultrasonic extraction, take out and cool to room temperature after the extraction is completed, dilute with the extract and make up to the scale, mix, centrifuge, take the supernatant and filter with a microporous membrane, collect the filtrate for high performance liquid chromatography determination;

[0062] c. High performance liquid chromatography detection: using a high performance liquid chromatograph, the detector is a fluorescence detector; the chromatographic column is a C18 column, the detection wavelength: excitation wavelength: 348nm, emission wavelength: 450nm; the filtrate collected in step b is sent to a high performance liquid chromatograph, an eluent is added for gradient elution, the eluate is collected and a derivatizing agent is added, the derivatizing agent is o-phthalaldehyde, and the derivatized solution is subjected to fluorescence detection;

[0063] d. Drawing of standard curve: Detect according to the chromatographic conditions of step c, with the concentration of tea theanine standard working solution as the abscissa and the corresponding peak area as the ordinate, and perform linear regression to obtain the standard curve equation;

[0064] e. Result analysis: The retention time measured in step c is used to qualitatively determine whether theanine is present in the test sample. If present, the peak area of ​​theanine in the filtrate is used to quantify the content of theanine in the test sample according to the standard curve prepared in step d.

[0065] Specifically, step a includes:

[0066] Preparation of tea theanine standard stock solution (1000mg / L): Accurately weigh an appropriate amount of theanine standard product (accurate to 0.01mg) converted to 100% by mass according to purity, dissolve it in a 25mL brown volumetric flask, dissolve it with the extract and make up to 25mL to prepare a standard stock solution with a mass concentration of 1000mg / L. Store at 0℃~4℃, with a validity period of 3 months.

[0067] Preparation of tea theanine standard intermediate solution (100 mg / L): Accurately pipette 10.0 mL of tea theanine standard stock solution into a 100 mL brown volumetric flask, dilute to scale with the extract, mix well, and prepare a standard intermediate solution with a mass concentration of 100 mg / L. Store at 0℃~4℃, valid for 1 month.

[0068] Prepare tea theanine standard series working solution: pipette 0.010mL, 0.020mL, 0.050mL, 0.10mL, 0.50mL, 1.00mL and 2.00mL of tea theanine standard intermediate solution into a 10mL brown volumetric flask, dilute to the mark with the extract, and mix well. The mass concentrations of the tea theanine standard series working solutions are 0.100mg / L, 0.200mg / L, 0.500mg / L, 1.00mg / L, 5.00mg / L, 10.0mg / L and 20.0mg / L. Prepare immediately before use.

[0069] Step b includes:

[0070] Prepare the extract: weigh 0.37 g of disodium ethylenediaminetetraacetate and 1.76 g of L(+)-ascorbic acid into a beaker, add 800 mL of water and dissolve by ultrasonic, dilute with water to 1000 mL, and mix well.

[0071] Prepare 0.05 mol / L disodium hydrogen phosphate solution: weigh 17.9 g disodium hydrogen phosphate dodecahydrate, add 800 mL water and ultrasonically dissolve, dilute with water to 1000 mL, mix well, pass through a microporous filter membrane (0.45 μm, aqueous phase, the same below) and set aside.

[0072] Prepare sodium tetraborate decahydrate solution (4.0 g / L): weigh 4.0 g of sodium tetraborate decahydrate and dissolve it in water to dilute it to 1000 mL.

[0073] Prepare the o-phthalaldehyde (OPA) derivative solution: weigh 0.40 g of o-phthalaldehyde and dissolve it in 20 mL of methanol, dilute it with 4.0 g / L sodium tetraborate decahydrate solution and dilute it to 1000 mL, filter it with a microporous filter membrane, add 0.80 mL of 2-mercaptoethanol and mix well, and set aside for use. Prepare it before use.

[0074] Preparation and storage of the test samples before testing: Take at least 200g of representative samples, mix the obtained representative samples, put them into a clean container, seal and mark them. Use a homogenizer to homogenize the obtained semi-solid representative samples containing pulp, pomace, etc., mix them evenly, put them into a clean container, seal and mark them. Use a high-speed grinder to grind the obtained solid representative samples, mix them evenly, put them into a clean container, seal and mark them. Candy, chocolate and other samples with high viscosity need to be frozen and then crushed evenly by a high-speed grinder. Keep the samples in a refrigerator below 0℃~4℃.

[0075] Pre-treatment of the test sample:

[0076] For liquid, semi-solid and solid samples, weigh 1g (accurate to 0.001g) of sample into a 50mL stoppered colorimetric tube, add 30mL of extracting solution, vortex disperse, place in an ultrasonic cleaner for ultrasonic extraction for 30min, mix once every 10min, take out after extraction, cool to room temperature, dilute with extracting solution and make up to scale, mix, centrifuge at 12000r / min for 3min, take the supernatant and filter it with a microporous membrane, and measure the filtrate by high performance liquid chromatography.

[0077] Note: If the sample with high protein content is not completely precipitated, add appropriate amount of acetic acid to precipitate the protein.

[0078] For chocolate, candy and tea samples, weigh 1g (accurate to 0.001g) of sample into a 50mL stoppered colorimetric tube, add 30mL of extracting solution, vortex disperse, place in a constant temperature water bath at 80℃ for 30min (60min for tea sample), mix every 10min, take out after extraction, cool to room temperature, dilute with extracting solution and make up to scale, mix, centrifuge at 12000r / min for 3min, take the supernatant and filter with microporous membrane, and measure the filtrate by high performance liquid chromatography.

[0079] For the blank test, the same pretreatment method as above is used except that no sample is added.

[0080] Step c comprises: Chromatographic column: RX-C18 column, column length 250 mm, inner diameter 4.6 mm, particle size 5 μm, or equivalent.

[0081] For foods with different free amino acid contents, the gradient elution is different. When detecting foods other than soy sauce with high free amino acid content, the mobile phase is: mobile phase A: water; mobile phase C: methanol. The gradient elution program is shown in Table 1. When detecting foods with high free amino acid content such as soy sauce, the mobile phase is: mobile phase A: water; mobile phase B: 0.05 mol / L disodium hydrogen phosphate solution; mobile phase C: methanol. The gradient elution program is shown in Table 2.

[0082] Other chromatographic conditions and derivatization conditions were the same, including: the derivatization agent was o-phthalaldehyde (OPA) derivatization solution. Derivatization temperature: 80°C. Derivatization agent flow rate: 0.1 mL / min. Column temperature: 40°C. Flow rate: 1.0 mL / min. Detection wavelength: excitation wavelength: 348 nm, emission wavelength: 450 nm. Injection volume: 10 μL.

[0083] Table 1 Gradient elution procedure for foods with low free amino acids

[0084]

[0085] Table 2 Gradient elution procedure for foods with high free amino acid content

[0086]

[0087] Step d includes:

[0088] The standard series working solutions were injected into the HPLC, the corresponding peak areas were measured, and the standard curve was drawn with the mass concentration of theanine in the standard series working solutions as the horizontal axis and the peak area as the vertical axis. The HPLC chromatogram of the derivative of theanine standard solution of tea leaves is shown in Figure 1 and Figure 2 .in Figure 1 The separation of tea theanine and other amino acids when the mobile phase is water and methanol. Figure 2 This is the separation of tea theanine and other amino acids when the mobile phase is 0.05 mol / L disodium hydrogen phosphate solution and methanol.

[0089] It can be seen that the method provided by the present invention can distinguish theanine from 17 kinds of amino acids (alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, cystine), and can also achieve good separation from aminobutyric acid and its hydrolysis product L-glutamine.

[0090] Step e includes: injecting the sample solution to be tested into a liquid chromatograph, obtaining the peak area according to the same chromatographic conditions, and obtaining the mass concentration of tea theanine in the test solution according to the standard curve. The response values ​​of tea theanine in the standard working solution and the sample solution to be tested should be within the linear response range of the instrument. If the content exceeds the linear range of the standard curve, it needs to be appropriately diluted and re-measured.

[0091] Specifically, the content of theanine in tea leaves in the test sample is calculated according to formula (1):

[0092]

[0093] Where:

[0094] X——the content of tea theanine in the test sample, in milligrams per kilogram (mg / kg);

[0095] ρ——The mass concentration of tea theanine in the test sample solution, in milligrams per liter (mg / L);

[0096] ρ0——The mass concentration of tea theanine in the blank solution of the test sample, in milligrams per liter (mg / L);

[0097] V——The fixed volume of the sample to be tested, in milliliters (mL);

[0098] m——the sampling amount of the sample to be tested, in grams (g);

[0099] 1000——Unit conversion factor.

[0100] The result is rounded to 3 significant digits.

[0101] To ensure precision, the absolute difference between two independent measurement results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean.

[0102] The above method was used to test a variety of representative complex food sample matrices. The test results are shown in Figures 3 to 15 The experimental results show that no interference occurs near the peak of the target theanine, indicating that the present invention can meet the requirements for accurate determination of tea theanine in various complex food matrices.

[0103] In summary, the present invention has good specificity and strong anti-interference ability, and can cope with the influence of various complex food matrices.

[0104] Example 2

[0105] It was found in the experiment that theanine from tea leaves is unstable in water and can decompose to varying degrees within 6 to 12 hours. In order to solve the instability of theanine from tea leaves during the detection process, the present invention adds ascorbic acid and disodium ethylenediaminetetraacetate as stabilizers to water, and investigates the extraction effects of different extraction solutions on theanine in food: 0.01 mol / L ascorbic acid solution, 0.01 mol / L ascorbic acid + 0.01 mol / L disodium ethylenediaminetetraacetate solution, and 0.01 mol / L ascorbic acid + 0.001 mol / L disodium ethylenediaminetetraacetate solution.

[0106] The experimental method is: taking solid beverage samples, adding different extraction solutions respectively, extracting according to the method in Example 1 and then performing chromatographic detection.

[0107] The experimental results show that the combination of ascorbic acid and disodium EDTA can play a good synergistic antioxidant role and eliminate the influence of heavy metal ions. It can also ensure the stability of theanine even under high-temperature extraction conditions of 100°C. However, too high concentration of disodium EDTA solution will cause large fluctuations in the detection baseline.

[0108] Therefore, the present invention finally selects 0.01 mol / L ascorbic acid+0.001 mol / L disodium ethylenediaminetetraacetic acid solution as the extracting solution.

[0109] The standard solution was taken, and the compound stabilizer used in the present invention was added as an extracting solution, and then the theanine content was tested. The results are shown in Tables 3, 4 and 5.

[0110] It can be seen from Table 3, Table 4 and Table 5 that tea theanine in all standard solutions can maintain good stability for more than 48 hours.

[0111] Table 3 Results of stability test of standard working solution

[0112]

[0113] A.0.100mg / L standard solution tea theanine concentration

[0114] B. 1.00mg / L standard solution tea theanine concentration

[0115] C. 20.0mg / L standard solution tea theanine concentration

[0116] Table 4 100 mg / L standard solution tea theanine concentration (intermediate solution) stability results

[0117]

[0118] Table 5 1000mg / L standard solution tea theanine concentration (stock solution) stability results

[0119]

[0120] Example 3

[0121] Food matrices are complex and may contain interfering substances such as proteins. This embodiment uses a sample containing protein for testing, uses different pretreatment methods, and then determines the content of theanine.

[0122] The specific experimental process is as follows: weigh 1g (accurate to 0.001g) of the sample into a 50mL stoppered colorimetric tube, add 30mL of the extract, vortex disperse, place in an ultrasonic cleaner for ultrasonic extraction for 30min, mix every 10min, take out after the extraction is completed, cool to room temperature, add 2mL each of 200g / L zinc acetate and 100g / L potassium ferrocyanide, or 4mL of acetic acid solution, or 4mL of other precipitants, respectively, precipitate protein, dilute with the extract and make up to the scale, mix, centrifuge at 12000r / min for 3min, take the supernatant and filter it with a microporous membrane, and determine the filtrate by high performance liquid chromatography.

[0123] Through research, it was found that when the common potassium ferrocyanide-zinc acetate solution was used as the protein removal system, the spiked recovery rate of tea theanine in the protein-containing sample was less than 70%, which could not meet the determination requirements. After investigating the effects of other different precipitants, this embodiment finally selected acetic acid solution as the protein removal system, which could obtain a satisfactory precipitation effect and effectively ensure a good spiked recovery rate of tea theanine.

[0124] Example 4

[0125] Theanine has no fluorescence characteristics and has weak ultraviolet absorption. When detected by ultraviolet detector, its ultraviolet spectrum lacks characteristic peaks. When detected in the low-wave band around 210nm, due to the high polarity of theanine and the rapid peak emission, it is easily interfered by a large amount of impurities in food and has low sensitivity.

[0126] Patent application CN118243815A announced a method for directly determining the content of L-theanine in compressed candy without derivatization. The L-theanine content is as high as 2500 mg / kg. The test of samples with lower content is easily affected by the interference of sample matrix. According to literature reports, the main derivatization reagents used for amino acid detection are: 6-aminoquinoline-N-hydroxysuccinimidyl carbamate (ACCQ-TAQ), phenyl isothiocyanate (PITC), 2,4-dinitrofluorobenzene (DNFB), dansyl chloride (DNS-CL), ninhydrin, o-phthalaldehyde (OPA), etc. Each derivatization reagent has its own advantages and disadvantages.

[0127] For example, the derivative product of 6-aminoquinolyl-N-hydroxysuccinimidyl formate is stable and does not interfere with experimental determination, but the reagent is expensive and the operation is complicated. The derivative product of phenyl isothiocyanate is stable, but generally requires a long reaction under heating conditions. At the same time, it is volatile and highly toxic, and there are many derivative by-products, which have a great impact on the life of the chromatographic column. The 2,4-dinitrofluorobenzene pre-column derivatization method has the advantages of more accurate analysis results, good repeatability, simple operation, and low cost, but excessive derivatization reagents have a certain impact on the analysis results, and the derivative by-products are easy to interfere with the results. The derivative product of dansyl chloride is stable, but the derivatization time takes two hours. At the same time, when dealing with different complex food matrices, especially samples containing high levels of free amino acids, the amount of derivatization reagents is easy to be insufficient, and excessive amounts will lead to more by-products, which will have a great impact on the accuracy of the measurement results. The ninhydrin method has the advantages of rapid derivatization, stable derivative products, and the ability to detect most amino acids. However, the instrument is expensive, the preparation of the mobile phase is cumbersome, the chromatographic column is expensive and easily contaminated, and it is unable to effectively separate theanine from other amino acids.

[0128] Therefore, the pre-column derivatization method generally has the problems of cumbersome operation, long derivatization time, many by-products of the derivatization agent, which affect the accuracy of the measurement results, and the pre-column derivatization generally requires manual derivatization, and the efficiency and accuracy of the derivatization are limited by the professional level of the operator. In particular, the OPA pre-column derivatization method requires online pre-column derivatization with instruments from specialized manufacturers such as Agilent to obtain accurate results due to the poor stability of the derivatized products (which has a great impact on the accuracy of the measurement results).

[0129] The present invention adopts OPA online post-column derivatization technology. The so-called OPA online post-column derivatization refers to the target after separation by the chromatographic column directly undergoing a derivatization reaction with the derivatization agent o-phthalaldehyde on the instrument to generate a compound with ultraviolet absorption and entering the detector for detection along with the mobile phase. OPA online post-column derivatization can not only effectively avoid the conventional complex and cumbersome derivatization operation process and the shortcomings of insufficient derivatization agent usage, more by-products, or excessive derivatization agent leading to shortened chromatographic column life caused by pre-column derivatization, but also can instantly complete the derivatization operation of tea theanine in food matrix online, and can effectively cope with the detection of tea theanine in complex food matrices including soy sauce containing high content of free amino acids and other complex food samples.

[0130] Example 5

[0131] The composition of food matrices is complex, and different chromatographic columns have different separation effects on complex matrices. On the one hand, complex matrices contain interferents similar to theanine. On the other hand, conventional chromatographic columns usually have good applicability to one matrix and cannot be applicable to multiple different matrices at the same time. Therefore, conventional chromatographic columns cannot achieve accurate detection of theanine in complex matrices.

[0132] The present invention compares four different hydrophilic chromatographic columns, including the PR-C18 chromatographic column with red (4.6×250mm, 5μm), the AQ-C18 chromatographic column with red (4.6×250mm, 5μm), the CAQ-C18 chromatographic column with orange (4.6×250mm, 5μm) and the RX-C18 chromatographic column with blue (4.6×250mm, 5μm), and comprehensively considers the separation conditions and the pH range tolerance of the columns, and finally selects the RX-C18 chromatographic column with blue (4.6×250mm, 5μm).

[0133] On this basis, we further explored and optimized the composition and proportion of various mobile phases, such as water, 0.025mol / L disodium hydrogen phosphate solution, and 0.05mol / L disodium hydrogen phosphate solution. We then formed two mobile phases with different compositions and different gradient elution procedures to cope with complex food samples with different free amino acid contents. In other words, for foods with high free amino acid contents such as soy sauce, a special elution procedure different from other food matrices is required, otherwise accurate detection cannot be achieved.

[0134] Finally, under the above optimized chromatographic conditions, the chromatogram of tea theanine in the actual positive sample is shown in Fig.16 It can be seen that the present invention can complete the detection of tea theanine in food in 15 minutes and has good anti-interference performance.

[0135] Example 6

[0136] The method of the present invention carefully optimizes the important parameters of derivatization, including the flow rate of the derivatization reagent, the derivatization reaction time, and the effective use time of the derivatization reagent. The determined optimization conditions include:

[0137] The derivatization agent is o-phthalaldehyde (OPA) derivatization solution, flow rate: 0.1mL / min. Derivatization temperature: 80℃. The o-phthalaldehyde (OPA) derivatization solution is prepared immediately before use: weigh 0.40g o-phthalaldehyde and dissolve it in 20mL methanol, dilute it with 4.0g / L sodium tetraborate decahydrate solution and dilute it to 1000mL, filter it with a microporous filter membrane, add 0.80mL 2-mercaptoethanol and mix well for use.

[0138] The above-mentioned optimized conditions make the method of the present invention have high sensitivity. In the method of the present invention, when the sampling amount is 1.00 g and the volume is fixed to 50 mL, the detection limit of the method is 1.50 mg / kg; the quantitative limit is 5.00 mg / kg.

[0139] Therefore, the method of the present invention can effectively meet the requirement for accurate determination of tea theanine in different content ranges added to foods.

[0140] Example 7

[0141] The detection method in Example 1 was used to investigate the spiked recoveries of blank matrix samples of milk powder, yogurt, sausage, eight-treasure porridge, candied fruit, melon seeds, beverages, wine, chocolate, candy, soy sauce, bread, and infant rice cereal after extraction with the extracting solution. The results are shown in Table 6.

[0142] Table 6 Recovery and relative standard deviation of tea theanine spiked in food

[0143]

[0144]

[0145] It can be seen that the above samples all obtained good spike recovery rates, with an average spike recovery rate of 93.7% to 102.2%, which can fully meet the requirements for accurate determination of tea theanine in food.

[0146] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0147] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0148] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for detecting theanine, a new food raw material for aiding sleep, in food, characterized by: The following steps are involved: a. Obtain theanine standard working solution; b. Prepare the sample solution to be tested: weigh the sample to be tested, add the extract, which is an aqueous solution containing ascorbic acid and disodium ethylenediaminetetraacetate, vortex disperse, place in an ultrasonic cleaner for ultrasonic extraction, take out and cool to room temperature after the extraction is completed, dilute with the extract and make up to the scale, mix, centrifuge, take the supernatant and filter with a microporous membrane, collect the filtrate for high performance liquid chromatography determination; c. High performance liquid chromatography detection: using a high performance liquid chromatograph, the detector is a fluorescence detector; the chromatographic column is a C18 column; the filtrate collected in step b is sent to the high performance liquid chromatograph, an eluent is added for gradient elution, the eluate is collected and a derivatizing agent is added, the derivatizing agent is o-phthalaldehyde, and the derivatized solution is subjected to fluorescence detection; d. Drawing of standard curve: Detect according to the chromatographic conditions of step c, with the concentration of tea theanine standard working solution as the abscissa and the corresponding peak area as the ordinate, and perform linear regression to obtain the standard curve equation; e. Result analysis: The retention time measured in step c is used to qualitatively determine whether theanine is present in the test sample. If present, the peak area of ​​theanine in the filtrate is used to quantify the content of theanine in the test sample according to the standard curve prepared in step d.

2. The method for detecting theanine, a new food raw material for aiding sleep, in food according to claim 1, is characterized by: The food comprises at least one of milk and dairy products, meat and meat products, cereals and their products, fruits and their products, nuts and seeds and their products, water, frozen drinks, alcohol, candy, chocolate, jelly, condiments, products made from cereals, beans or potatoes, dietary foods, and tea.

3. The method for detecting theanine in food as a new food raw material for aiding sleep according to claim 1, characterized in that: The method for preparing the theanine standard working solution in step a comprises: Prepare a standard stock solution, the specific steps of preparing the standard stock solution are: accurately weigh an appropriate amount of theanine standard product converted to 100% by mass according to purity, accurate to 0.01 mg, dissolve it in a 25 mL brown volumetric flask, dissolve it with the extract and make up to 25 mL, and prepare a standard stock solution with a mass concentration of 1000 mg / L; store it at 0°C to 4°C, and the validity period is 3 months; Prepare a standard intermediate solution, the specific steps of preparing the standard intermediate solution are: accurately pipette 10.0 mL of theanine standard stock solution of tea into a 100 mL brown volumetric flask, dilute to the mark with the extract, mix well, and prepare a standard intermediate solution with a mass concentration of 100 mg / L; store at 0°C to 4°C, with a validity period of 1 month; To prepare standard working solutions, 0.010 mL, 0.020 mL, 0.050 mL, 0.10 mL, 0.50 mL, 1.00 mL and 2.00 mL of tea theanine standard intermediate solution were respectively pipetted into 10 mL brown volumetric flasks, the volume was made up to the mark with the extract, and the solution was mixed to obtain theanine standard working solutions. The theanine concentrations of the theanine standard working solutions were 0.100 mg / L, 0.200 mg / L, 0.500 mg / L, 1.00 mg / L, 5.00 mg / L, 10.0 mg / L and 20.0 mg / L, respectively.

4. The method for detecting theanine in food as a new food raw material for aiding sleep according to claim 2, characterized in that: The specific steps of preparing the sample solution to be tested in step b are: For liquid, semi-solid and solid samples, weigh 1g, accurate to 0.001g, put the sample in a 50mL stoppered colorimetric tube, add 30mL of the extract, vortex disperse, place in an ultrasonic cleaner for ultrasonic extraction for 30min, mix once every 10min, take out after extraction, cool to room temperature, dilute with the extract and make up to the scale, mix, centrifuge at 12000r / min for 3min, take the supernatant and filter with a microporous membrane, and measure the filtrate on a high performance liquid chromatograph; or, For chocolate, candy and tea samples, weigh 1g, accurate to 0.001g, put the sample in a 50mL stoppered colorimetric tube, add 30mL of the extract, vortex disperse, place in a constant temperature water bath at 80-100℃ for 30min to 60min, mix once every 10min, take out after extraction, cool to room temperature, dilute with the extract and make up to the scale, mix, centrifuge at 12000r / min for 3min, take the supernatant and filter with a microporous filter membrane, and measure the filtrate on a high performance liquid chromatograph; Preferably, for a sample containing protein, acetic acid is used to precipitate the protein, and after the protein is removed, the extracting solution is added for extraction; Preferably, the concentration of ascorbic acid in the extract is 0.01-0.03 mol / L, and the concentration of disodium ethylenediaminetetraacetate is 0.001-0.003 mol / L.

5. The method for detecting theanine in food as a new food raw material for aiding sleep according to claim 2, characterized in that: In step c, the C18 chromatographic column adopts a RX-C18 column with a specification of 4.6 mm×250 mm inner diameter and a particle size of 5 μm; Optionally, the detection column temperature is 40°C, the flow rate is 1 mL / min, and the injection volume is 10 μL; Optionally, detection wavelengths: excitation wavelength: 348 nm, emission wavelength: 450 nm.

6. The method for detecting theanine in food as a new food raw material for aiding sleep according to claim 5, characterized in that: In step c, for samples with a free amino acid content not lower than that of soy sauce, mobile phase A: water; mobile phase B: 0.05 mol / L disodium hydrogen phosphate solution; mobile phase C: methanol, and the gradient elution procedure is as follows:

7. The method for detecting theanine in food as a new food raw material for aiding sleep according to claim 5, characterized in that: For samples with free amino acid content lower than that of soy sauce, mobile phase A: water; mobile phase C: methanol, the gradient elution program is as follows:

8. The method for detecting theanine, a new food ingredient for aiding sleep, in food according to claim 6 or 7, characterized in that: The derivatizing agent in step c is a solution of o-phthalaldehyde, the flow rate is 0.1 mL / min, and the derivatization temperature is 80° C. The o-phthalaldehyde solution is prepared by weighing 0.40 g of o-phthalaldehyde and dissolving it in 20 mL of methanol, diluting it with 4.0 g / L sodium tetraborate decahydrate solution to 1000 mL, filtering it with a microporous filter membrane, and adding 0.80 mL of 2-mercaptoethanol to mix well; Optionally, the correlation coefficient of the standard curve equation in step d is not less than 0.9995.

9. The method for detecting theanine, a new food ingredient for aiding sleep, in food according to claim 8, characterized in that: The detection method has anti-interference ability for 17 kinds of amino acids present in the food sample matrix, including alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine and cystine.

10. The method for detecting theanine in food as a new food raw material for aiding sleep according to claim 8, characterized in that: The average recovery rate of the detection method is between 93.7% and 102.2%.

Images