Kit and method for determining total amount of free amino acids in tea leaves

Abstract

The invention discloses a determination kit and a determination method for the total amount of free amino acids in tea leaves, and belongs to the technical field of food detection. In order to solve the problems of low detection efficiency, high cost and large sample demand of the existing method for determining the total amount of free amino acids in tea leaves, the determination kit provided by the invention comprises a solution A (1 mg / ml glutamic acid standard solution), a solution B (phosphate buffer with pH of 8.0) and a solution C (2% ninhydrin solution containing 0.8 mg / mL stannous chloride). The corresponding determination method comprises the steps of sample treatment, dry matter content determination, standard curve making, sample detection and content calculation. The microplate reader is adopted for detection, a plurality of samples can be measured at the same time at one time, the detection time is only about 5 min, the required sample amount is only 50 mg, the reagent dosage is greatly reduced, the detection efficiency is remarkably improved, the detection cost is reduced, and the method is suitable for rapid detection of a large batch of tea samples and has important significance on tea quality judgment.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, and more specifically to a kit and method for determining the total free amino acid content in tea. Background Technology

[0002] Tea [Camellia sinensis (L.) O. Kuntze] is one of the world's most popular beverages. Tea contains abundant amino acids; 26 have been identified and recognized to date. In addition to the 20 protein-derived amino acids found in free amino acids, 6 non-protein-derived amino acids have also been detected. All naturally occurring amino acids are L-configured. Amino acids in tea are often classified into aromatic amino acids, hydroxy amino acids, and sulfur-containing amino acids based on the substituents on their side chains. The 6 non-protein-derived amino acids in tea do not exist in proteins and belong to plant secondary metabolites. Amino acids in tea are not only the basic building blocks of proteins but also important components of bioactive peptides, enzymes, and other bioactive molecules. The composition, content, degradation products, and transformation products of amino acids in tea directly affect the quality of tea. Amino acids participate in the formation of tea aroma during processing; the volatile aldehydes and other products they transform into are components of tea aroma. Furthermore, some amino acids themselves possess a certain aroma, while others are closely related to the taste and aroma of tea, and are important components constituting the quality of tea.

[0003] The total amount of free amino acids in tea is an important indicator for evaluating tea quality, and rapid detection of amino acid content is of great significance for tea quality assessment. Currently, the main methods for determining the total free amino acid content in tea include high-performance liquid chromatography (HPLC), automated amino acid analysis, and spectrophotometry. HPLC and automated amino acid analyzers are costly and involve complex sample pretreatment, making them unsuitable for rapid determination of total amino acid content. The current national standard method for determining the total free amino acid content in tea is the ninhydrin spectrophotometric method. Its main principle is based on the reaction of amino acids with ninhydrin in a pH 8.0 phosphate buffer under heating conditions to form a purple complex. The intensity of the purple color is positively correlated with the free amino acid content. This purple complex has a maximum absorbance at a wavelength of 570 nm and can be detected by spectrophotometry. However, this method has limitations in terms of the limited number of samples that can be measured at one time and its slow testing speed, making it unsuitable for large-scale sample determination. Currently, there are no commercially available kits specifically for amino acid detection in tea, hindering the improvement of efficiency in tea quality testing. Summary of the Invention

[0004] In view of this, and to overcome the shortcomings of the prior art, the present invention provides a kit and method for determining the total free amino acid content in tea. Using this kit and method to determine the total free amino acid content in tea can significantly improve the detection efficiency of amino acid content in large batches of tea samples and significantly reduce detection costs.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] First, this invention provides a kit for determining the total free amino acids in tea leaves, comprising:

[0007] Solution A: 1 mg / mL glutamic acid standard solution;

[0008] Solution B: Phosphate buffer solution with pH=8.0;

[0009] Solution C: a 2% (w / w) ninhydrin solution, wherein the ninhydrin solution contains 0.8 mg / mL stannous chloride;

[0010] The kit was stored at 4 °C.

[0011] Preferably, the kit also includes a 96-well microplate.

[0012] Furthermore, the kit also includes 12-well microplate strips.

[0013] This invention also provides a method for determining the total free amino acids in tea leaves, using the kit described above, and comprising the following steps:

[0014] (1) After crushing the tea sample, dry it to constant weight, record the weight of the tea before and after drying, and calculate the dry matter content of the tea sample.

[0015] (2) Preparation of standard curve: Add solution B, solution C, H2O and gradient volume of solution A in proportion, react in a water bath at 100 ℃ for 10 min, cool to room temperature and measure absorbance using an enzyme-linked immunosorbent assay (ELISA) reader, plot the standard curve with amino acid content as the abscissa and absorbance as the ordinate and obtain the standard curve equation.

[0016] (3) Weigh 0.05 g of tea sample, add 1 mL of distilled water, extract in boiling water bath for 10 min, centrifuge at 10000 rpm for 10 min, and take the supernatant;

[0017] (4) Take 100 μL of the supernatant prepared in step (3), add 400 μL of H2O, 250 μL of solution B and 250 μL of solution C, react in a water bath at 100 ℃ for 10 min, cool to room temperature and measure the absorbance with an enzyme-linked immunosorbent assay reader.

[0018] (5) Substitute the absorbance value obtained in step (4) into the standard curve equation obtained in step (2) to obtain the corresponding amino acid content C, and calculate the total free amino acid N in the sample:

[0019] N=(C×V1) / (V2×m×w×1000)

[0020] Where N is the total free amino acids, mg / g; V1 is the total volume of the extract, μL; V2 is the volume of the extract used for the determination, μL; m is the mass of the tea sample, g; and w is the dry matter content of the tea sample, %.

[0021] Preferably, the method for determining the dry matter content of the tea sample in step (1) is as follows:

[0022] The aluminum box was dried at 110 ℃ to constant weight, cooled and weighed M1. Then, tea samples were added to the aluminum box and the total weight was weighed M2. The aluminum box was then dried at 110 ℃ to constant weight again, cooled and weighed M3. The dry matter content w = (M3−M1) / (M2−M1)×100%.

[0023] Preferably, in step (2), based on a total volume of 1 mL, the volume ratio of solution B and solution C is 1:1, both being 250 μL. The gradient volumes of solution A and H2O are specifically as follows:

[0024] Solution A: 0 μL, H₂O: 500 μL;

[0025] Solution A 50 μL, H2O 450 μL;

[0026] Solution A 75 μL, H2O 425 μL;

[0027] Solution A 100 μL, H2O 400 μL;

[0028] Solution A 125 μL, H2O 375 μL;

[0029] Solution A 150 μL, H2O 350 μL;

[0030] Solution A 175 μL, H2O 325 μL;

[0031] Solution A 200 μL, H2O 300 μL.

[0032] Preferably, in steps (2) and (4), when measuring the absorbance, the diluted reaction solution is added to the enzyme label strip, and then the enzyme label strip is placed in the enzyme label plate for detection. The scanning wavelength of the enzyme labeler is 570 nm.

[0033] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a solution with the following beneficial effects:

[0034] This invention can simultaneously measure multiple samples, with a sample detection time of approximately 5 minutes. Compared with the spectrophotometer method, it greatly shortens the sample detection time and improves the detection efficiency of total amino acid content in tea samples.

[0035] This invention requires only 50 mg of dried tea sample. Compared with other testing methods, the required sample amount is small, and this kit can also be used to detect tea samples that are difficult to obtain or have small sample quantities.

[0036] The amount of various reagents required for testing has been greatly reduced, and all consumables are common consumables on the market, with low cost. The sample extraction and testing methods are simple, which significantly reduces the testing cost of amino acids in tea. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0038] Figure 1 The standard curves for amino acid determination in tea from Examples 1-2 are shown below.

[0039] Figure 2 The amino acid content of fresh tea leaves from different varieties in Examples 1-2;

[0040] Figure 3 The amino acid content of the finished tea products from different varieties of tea in Example 2;

[0041] Figure 4 This is the standard curve for amino acid determination in tea as shown in Comparative Example 1.

[0042] Figure 5 This is a comparison of the amino acid content measured by the method used in Comparative Example 1 and the method used in Example 1. Detailed Implementation

[0043] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0044] The kit for determining the total free amino acids in tea includes the following reagents:

[0045] Solution A: 1 mg / mL glutamate standard solution, 1 mL × 5 tubes, stored at 4 ℃;

[0046] Solution B: Phosphate buffer solution with pH=8.0, 100 ml × 1 bottle, stored at 4 ℃;

[0047] Solution C: 2% ninhydrin solution (containing 0.8 mg / mL stannous chloride), 100 mL × 1 bottle, stored at 4 °C.

[0048] The method for determining the total free amino acid content in tea using the above-mentioned kit is based on the principle that amino acids react with ninhydrin in a phosphate buffer (pH 8.0) under heating conditions to form a purple complex. The intensity of the purple color is positively correlated with the free amino acid content. Measuring the absorbance of this purple complex at 570 nm reflects the total free amino acid content in the tea. The specific steps of this method are as follows:

[0049] Preparation of instruments and supplies;

[0050] Prepare an ELISA reader, benchtop centrifuge, pipette, 96-well ELISA plate, 12-strand ELISA strips, liquid nitrogen, distilled water, mortar and pestle, and aluminum box.

[0051] Tea samples for testing:

[0052] Fresh tea samples of different varieties: Tea samples (Yinghong No. 9, Lingtou Dancong, Jin Xuan) were picked at the Yingde Tea Base of the Tea Research Institute of Guangdong Academy of Agricultural Sciences. The tea samples were immediately frozen with liquid nitrogen after picking and then stored in an ultra-low temperature freezer at -80℃ after being transported back to the laboratory.

[0053] Dry tea samples of different types: Yinghong No. 9 black tea sample, Lingtou Dancong oolong tea sample, and Jinxuan green tea sample were processed and preserved by the Tea Research Institute of Guangdong Academy of Agricultural Sciences.

[0054] Example 1

[0055] Determination of total free amino acid content in fresh tea leaf samples:

[0056] Extraction of fresh tea leaf samples: Fresh tea leaves were ground into powder in a pre-cooled mortar with liquid nitrogen. Liquid nitrogen was added in time during the grinding process to prevent the sample from oxidizing and turning brown. 200 mg of tea powder was weighed into a 1.5 mL centrifuge tube, and 1 mL of distilled water was added to the centrifuge tube with a pipette. The centrifuge tube was placed in a 100 ℃ water bath and extracted for 10 min. After cooling to room temperature, the sample was centrifuged at 10000 rpm at 25 ℃ for 10 min. The supernatant was collected for analysis.

[0057] Determination of dry matter content in tea samples:

[0058] Dry the aluminum box in a 110℃ oven until constant weight. After cooling to room temperature in a desiccator, weigh the aluminum box (M1). Add the ground tea sample powder to the aluminum box and weigh the total weight of the sample and aluminum box (M2). Transfer the aluminum box to a 110℃ oven and dry until constant weight. After cooling to room temperature in a desiccator, weigh it (M3). Calculate the dry matter content of the tea sample using the following formula:

[0059] w=(M3−M1) / (M2−M1)×100%

[0060] Preparation of the standard curve for the determination of total free amino acids:

[0061] Add reagents according to Table 1:

[0062] Table 1. Required reagent solution volume (μL) for each reaction tube

[0063] serial number 0 1 2 3 4 5 6 7 Solution B 250 250 250 250 250 250 250 250 Solution C 250 250 250 250 250 250 250 250 <![CDATA[H2O]]> 500 450 425 400 375 350 325 300 Solution A 0 50 75 100 125 150 175 200

[0064] The reaction was carried out in a 100 ℃ water bath for 10 min, then cooled to room temperature. The reaction solution was diluted 10 times with water. The enzyme-labeled strip was placed in an ELISA plate, and 200 μL of the diluted solution was added to the strip. The absorbance was measured using an ELISA reader with a scanning wavelength of 570 nm. Based on the OD values ​​measured after the reaction of each amino acid gradient concentration with ninhydrin solution, a standard curve was plotted with the amino acid content (µg) standard series as the x-axis and the 570 nm optical density value as the y-axis. Figure 1 As shown, the equation of the standard curve is obtained;

[0065] Determination of amino acid content in samples:

[0066] Take 100 μL of supernatant, add 400 μL of H2O, 250 μL of solution B, and 250 μL of solution C, react in a 100 ℃ water bath for 10 min, cool to room temperature, dilute the reaction solution 10 times with water, and measure the absorbance using an enzyme-linked immunosorbent assay (ELISA) reader.

[0067] Calculation of total free amino acid content in tea samples:

[0068] The OD value obtained after reacting tea sample extract with ninhydrin solution is used as the Y value of the amino acid standard curve. The corresponding amino acid content (C) (X value of the standard curve) is calculated using the standard curve equation. The total free amino acid content in the tea sample is calculated using the following formula:

[0069] N = (C × V1) / (V2 × m × w × 1000);

[0070] in:

[0071] N: The total amount of free amino acids in the tea sample, expressed in milligrams per gram (mg / g);

[0072] C: The amino acid content calculated from the amino acid standard curve, in micrograms (ug).

[0073] V1: Total volume of tea sample extract, in microliters (μL).

[0074] V2: The volume of extract used for sample determination, in microliters (μL).

[0075] m: Sample mass, in grams (g);

[0076] w: Dry matter content of the sample,%.

[0077] Example 2

[0078] Determination of total free amino acid content in dried tea samples

[0079] Extraction of dried tea samples:

[0080] The dried tea sample was ground into powder using a mortar and pestle. 50 mg of tea powder was weighed into a 1.5 mL centrifuge tube, and 1 mL of distilled water was added to the centrifuge tube using a pipette. The centrifuge tube was placed in a 100 ℃ water bath and extracted for 10 min. After cooling to room temperature, it was centrifuged at 10000 rpm at 25 ℃ for 10 min. The supernatant was collected for analysis.

[0081] Determination of dry matter content in tea samples;

[0082] Dry the aluminum box in a 110 ℃ oven until constant weight. After cooling to room temperature in a desiccator, weigh the aluminum box (M1). Add the ground tea sample powder from step 2 to the aluminum box and weigh the total weight of the sample and aluminum box (M2). Transfer the aluminum box to a 110 ℃ oven and dry until constant weight. After cooling to room temperature in a desiccator, weigh it (M3). Calculate the dry matter content of the tea sample using the following formula:

[0083] w=(M3−M1) / (M2−M1)×100%

[0084] Preparation of the standard curve for the determination of total free amino acids:

[0085] Add reagents according to Table 1 in Example 1, react in a 100 ℃ water bath for 10 min, cool to room temperature, dilute the reaction solution 10 times with water, place the enzyme label strip in the enzyme label plate, and add 200 μL of the diluted solution to the enzyme label strip. Measure the absorbance using an enzyme-linked immunosorbent assay (ELISA) reader with a scanning wavelength set to 570 nm. Based on the OD values ​​measured after reacting each amino acid gradient concentration with the ninhydrin solution, plot a standard curve with the amino acid content (µg) standard series as the x-axis and the 570 nm optical density value as the y-axis, as shown below. Figure 1 The equation of the standard curve is obtained;

[0086] Determination of amino acid content in samples:

[0087] Take 100 μL of supernatant, add 400 μL of H2O, 250 μL of solution B, and 250 μL of solution C, react in a 100 ℃ water bath for 10 min, cool to room temperature, dilute the reaction solution 10 times with water, and measure the absorbance using an ELISA reader.

[0088] Determination of amino acid content in samples:

[0089] The OD value obtained after reacting tea sample extract with ninhydrin solution is used as the Y value of the amino acid standard curve. The corresponding amino acid content (C) (X value of the standard curve) is calculated using the standard curve equation. The total free amino acid content in the tea sample is calculated using the following formula:

[0090] N = (C × V1) / (V2 × m × w × 1000);

[0091] in:

[0092] N: The total amount of free amino acids in the tea sample, expressed in milligrams per gram (mg / g);

[0093] C: The amino acid content calculated from the amino acid standard curve, in micrograms (ug).

[0094] V1: Total volume of tea sample extract, in microliters (μL).

[0095] V2: The volume of extract used for sample determination, in microliters (μL).

[0096] m: Sample mass, in grams (g);

[0097] w: Dry matter content of the sample,%.

[0098] Comparative Example 1

[0099] Traditional methods for determining the total free amino acid content in fresh tea leaf samples:

[0100] Extraction of fresh tea leaf samples: Fresh tea leaves were ground into powder in a pre-cooled mortar with liquid nitrogen. Liquid nitrogen was added in time during the grinding process to prevent the sample from oxidizing and turning brown. 1000 mg of tea powder was weighed into a 10 mL centrifuge tube. 5 mL of distilled water was added to the centrifuge tube with a pipette. The centrifuge tube was placed in a 100 ℃ water bath and extracted for 10 min. After cooling to room temperature, the mixture was centrifuged at 10000 rpm at 25 ℃ for 10 min. The supernatant was collected. The precipitate was extracted again with 4 mL of distilled water. The supernatants from the two extractions were combined and the volume was adjusted to 10 mL.

[0101] Determination of dry matter content in tea samples:

[0102] Dry the aluminum box in a 110℃ oven until constant weight. After cooling to room temperature in a desiccator, weigh the aluminum box (M1). Add the ground tea sample powder to the aluminum box and weigh the total weight of the sample and aluminum box (M2). Transfer the aluminum box to a 110℃ oven and dry until constant weight. After cooling to room temperature in a desiccator, weigh it (M3). Calculate the dry matter content of the tea sample using the following formula:

[0103] w=(M3−M1) / (M2−M1)×100%

[0104] Preparation of the standard curve for the determination of total free amino acids:

[0105] Add reagents according to Table 2:

[0106] Table 2. Required reagent solution volume (μL) for each reaction tube

[0107] serial number 0 1 2 3 4 5 6 7 Phosphate buffer at pH 8.0 500 500 500 500 500 500 500 500 2% ninhydrin solution (containing 0.8 mg / mL stannous chloride) 500 500 500 500 500 500 500 500 <![CDATA[H2O]]> 1000 950 925 900 875 850 825 800 1 mg / ml glutamate standard solution 0 50 75 100 125 150 175 200

[0108] The reaction was carried out in a 100 ℃ water bath for 10 min, cooled to room temperature, and the volume was adjusted to 10 mL with water. The absorbance of the reaction solution was measured using a UV spectrophotometer with the absorption wavelength set to 570 nm. Based on the OD values ​​measured after the reaction of each amino acid gradient concentration with ninhydrin solution, a standard curve was plotted with the amino acid content (ug) standard series as the x-axis and the 570 nm optical density value as the y-axis, as shown below. Figure 4 As shown, the equation of the standard curve is obtained;

[0109] Determination of amino acid content in samples:

[0110] Take 200 μL of supernatant, add 400 μL of H2O, 500 μL of pH 8.0 phosphate buffer, and 500 μL of 2% ninhydrin solution (containing 0.8 mg / mL stannous chloride), react in a 100 ℃ water bath for 10 min, cool to room temperature, add water to make up to 10 mL, and measure the absorbance using a UV spectrophotometer;

[0111] Calculation of total free amino acid content in tea samples:

[0112] The OD value obtained after reacting tea sample extract with ninhydrin solution is used as the Y value of the amino acid standard curve. The corresponding amino acid content (C) (X value of the standard curve) is calculated using the standard curve equation. The total free amino acid content in the tea sample is calculated using the following formula:

[0113] N = (C × V1) / (V2 × m × w × 1000);

[0114] in:

[0115] N: The total amount of free amino acids in the tea sample, expressed in milligrams per gram (mg / g);

[0116] C: Amino acid content calculated from the amino acid standard curve, in micrograms (μg).

[0117] V1: Total volume of tea sample extract, in microliters (μL).

[0118] V2: The volume of extract used for sample determination, in microliters (μL).

[0119] m: Sample mass, in grams (g);

[0120] w: Dry matter content of the sample,%.

[0121] Experiments have shown that the detection method of the present invention is fast and effective, and can effectively prevent experimental errors caused by inconsistent reaction times between amino acids and ninhydrin in the sample due to excessively long testing time. Moreover, the detection method has good repeatability and high accuracy.

[0122] The various embodiments described in this specification are presented in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A kit for determining the total free amino acids in tea, characterized in that, include: Solution A: 1 mg / ml glutamic acid standard solution; Solution B: Phosphate buffer solution with pH=8.0; Solution C: a 2% (w / w) ninhydrin solution, wherein the ninhydrin solution contains 0.8 mg / mL stannous chloride; The kit was stored at 4 °C.

2. The kit for determining the total free amino acids in tea according to claim 1, characterized in that, The kit also includes a 96-well microplate.

3. A kit for determining the total free amino acids in tea according to claim 1 or 2, characterized in that, The kit also includes 12-well microplates.

4. A method for determining the total free amino acids in tea, characterized in that, The kit according to any one of claims 1-3 includes the following steps: (1) After crushing the tea sample, dry it to constant weight, record the weight of the tea before and after drying, and calculate the dry matter content of the tea sample. (2) Preparation of standard curve: Add solution B, solution C, H2O and gradient volume of solution A in proportion, react in a water bath at 100 ℃ for 10 min, cool to room temperature and measure absorbance using an enzyme-linked immunosorbent assay (ELISA) reader, plot the standard curve with amino acid content as the abscissa and absorbance as the ordinate and obtain the standard curve equation. (3) Weigh 0.05 g of tea sample, add 1 mL of distilled water, extract in boiling water bath for 10 min, centrifuge at 10000 rpm for 10 min, and take the supernatant; (4) Take 100 μL of the supernatant prepared in step (3), add 400 μL of H2O, 250 μL of solution B and 250 μL of solution C, react in a water bath at 100℃ for 10 min, cool to room temperature and measure the absorbance with an enzyme-linked immunosorbent assay reader. (5) Substitute the absorbance value obtained in step (4) into the standard curve equation obtained in step (2) to obtain the corresponding amino acid content C, and calculate the total free amino acid N in the sample: N=(C×V1) / (V2×m×w×1000) Where N is the total free amino acids, mg / g; V1 is the total volume of the extract, μL; V2 is the volume of the extract used for the determination, μL; m is the mass of the tea sample, g; and w is the dry matter content of the tea sample, %.

5. The method for determining the total free amino acids in tea according to claim 4, characterized in that, The method for determining the dry matter content of the tea sample in step (1) is as follows: The aluminum box was dried at 110 ℃ to constant weight, cooled and weighed M1. Then, tea samples were added to the aluminum box and the total weight was weighed M2. The box was then dried at 110 ℃ to constant weight again, cooled and weighed M3. The dry matter content w = (M3−M1) / (M2−M1)×100%.

6. The method for determining the total free amino acids in tea according to claim 4, characterized in that, In step (2), the total volume is 1 mL, where the volume ratio of solution B and solution C is 1:1, both being 250 μL. The gradient volumes of solution A and H2O are as follows: Solution A: 0 μL, H₂O: 500 μL; Solution A 50 μL, H2O 450 μL; Solution A 75 μL, H2O 425 μL; Solution A 100 μL, H2O 400 μL; Solution A 125 μL, H2O 375 μL; Solution A 150 μL, H2O 350 μL; Solution A 175 μL, H2O 325 μL; Solution A 200 μL, H2O 300 μL.

7. The method for determining the total free amino acids in tea according to claim 4, characterized in that, In steps (2) and (4), when measuring absorbance, the diluted reaction solution is added to the enzyme label strip, and then the enzyme label strip is placed on the enzyme label plate for detection. The scanning wavelength of the enzyme labeler is 570 nm.

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