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CN-121994562-A - Kit and method for measuring total amount of free amino acids in tea

CN121994562ACN 121994562 ACN121994562 ACN 121994562ACN-121994562-A

Abstract

The invention discloses a kit and a method for measuring total amount of free amino acids in tea, 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 measuring the total amount of free amino acids of tea, the invention provides a measuring kit which comprises a solution A (1 mg/mL glutamic acid standard solution), a solution B (pH 8.0 phosphate buffer solution) and a solution C (2% ninhydrin solution containing 0.8 mg/mL stannous chloride). The corresponding measuring method comprises the steps of sample treatment, dry matter content measurement, standard curve preparation, sample detection and content calculation. The invention adopts the enzyme-labeled instrument to detect, can simultaneously detect a plurality of samples at one time, the detection time is only about 5 min, the required sample amount is only 50mg, the reagent amount is greatly reduced, the detection efficiency is obviously improved, the detection cost is reduced, the invention is suitable for the rapid detection of a large number of tea samples, and has important significance for tea quality judgment.

Inventors

  • CHEN YIYONG
  • HUANG YANFENG
  • LI JIANLONG
  • TANG JINCHI
  • ZHOU BO
  • ZHANG MAN
  • LIU JIAYU
  • CUI YINGYING
  • Nong Hongyan
  • HE BEI

Assignees

  • 广东省农业科学院茶叶研究所

Dates

Publication Date
20260508
Application Date
20260204

Claims (7)

  1. 1. A kit for determining total amount of free amino acids in tea leaves, which is characterized by comprising: 1 mg/ml glutamic acid standard solution; Solution B phosphate buffer at ph=8.0; the solution C is a ninhydrin solution with the mass fraction of 2%, and the ninhydrin solution contains 0.8 mg/mL stannous chloride; The kit was stored in a4 ℃ environment.
  2. 2. A kit for determining total amount of free amino acids in tea leaves as claimed in claim 1, wherein said kit further comprises a 96-well elisa plate.
  3. 3. A kit for determining total amount of free amino acids in tea leaves according to claim 1 or 2, wherein said kit further comprises a 12-well ELISA strip.
  4. 4. A method for determining total amount of free amino acids in tea leaves, which is characterized by adopting the kit as claimed in any one of claims 1 to 3 and comprising the following steps: (1) Crushing a tea sample, drying to constant weight, recording the weight of the tea before and after drying, and calculating the dry matter content of the tea sample; (2) The preparation of a standard curve, namely adding the solution B, the solution C, H 2 O and the solution A with gradient volume in proportion, carrying out water bath reaction at 100 ℃ for 10: 10min, cooling to room temperature, measuring a light absorption value by using an enzyme-labeled instrument, drawing the standard curve by taking the amino acid content as an abscissa and the light absorption value as an ordinate, and obtaining a standard curve equation; (3) Weighing 0.05 g tea sample, adding 1 mL distilled water, leaching in boiling water bath for 10 min, centrifuging at 10000 rpm for 10 min, and collecting supernatant; (4) Taking 100 mu L of the supernatant prepared in the step (3), adding 400 mu L H 2 O, 250 mu L of solution B and 250 mu L of solution C, reacting in a 100 ℃ water bath for 10min, cooling to room temperature, and measuring the absorbance value by using an enzyme-labeled instrument; (5) Substituting the absorbance value measured in the step (4) into the standard curve equation obtained in the step (2) to obtain the corresponding amino acid content C, and calculating the total amount N of free amino acids in the sample: N=(C×V 1 )/(V 2 ×m×w×1000) Wherein N is total amount of free amino acids, mg/g, V 1 is total volume of the extracting solution, μL, V 2 is total volume of the extracting solution used for measurement, μL, m is mass of tea leaf sample, g, and w is dry matter content of tea leaf sample.
  5. 5. The method for determining total amount of free amino acids in tea leaves according to claim 4, wherein the method for determining dry matter content of tea leaf sample in step (1) comprises: The aluminum box is dried at 110 ℃ to constant weight, the weight M 1 is weighed after cooling, then the total weight M 2 is weighed after the tea sample is added into the aluminum box, then the aluminum box is dried at 110 ℃ again to constant weight, the weight M 3 is weighed after cooling, and the dry matter content w= (M 3 −M 1 )/(M 2 −M 1 ) multiplied by 100%.
  6. 6. The method for determining total amount of free amino acids in tea leaves according to claim 4, wherein in the step (2), the volume ratio of the solution B to the solution C is 1:1, and the total volume is 250 μl, and the gradient volumes of the solutions a and H 2 O are specifically: Solution A0. Mu.L, H 2 O500. Mu.L; 50. Mu.L of solution A and 450. Mu.L of H 2 O; solution A75. Mu.L, H 2 O425. Mu.L; 100. Mu.L of solution A and 400. Mu.L of H 2 O; solution A125. Mu.L, H 2 O375. Mu.L; Solution A150. Mu.L, H 2 O350. Mu.L; 175. Mu.L of solution A, H 2 O325. Mu.L; 200. Mu.L of solution A and 300. Mu.L of H 2 O.
  7. 7. The method for measuring total free amino acids in tea leaves according to claim 4, wherein in the step (2) and the step (4), the diluted reaction solution is added to an ELISA plate for detection when the absorbance is measured, and the ELISA plate is placed in the ELISA plate for detection, wherein the scanning wavelength of the ELISA plate is 570 nm.

Description

Kit and method for measuring total amount of free amino acids in tea Technical Field The invention relates to the technical field of food detection, in particular to a kit and a method for measuring total amount of free amino acids in tea. Background Tea [ CAMELLIA SINENSIS (l.) o.kuntze ] is one of the most popular beverages in the world. The tea contains abundant amino acids, 26 kinds of tea have been found and identified, and besides 20 kinds of protein source amino acids are found in free amino acids, 6 kinds of non-protein source amino acids are detected. The amino acids existing in nature are all L-configuration. Amino acids in tea are often classified into aromatic amino acids, hydroxy amino acids, sulfur-containing amino acids, etc. according to the substituents on the side chains. The 6 non-protein amino acids in tea tree are not present in protein and belong to the secondary metabolic substances of plants. Amino acids in tea are not only the basic units of constituent proteins, but also important constituent components of active peptides, enzymes and other bioactive molecules. The composition, content of amino acids in tea leaves and their degradation products and conversion products also directly affect the quality of tea leaves. Amino acids participate in the formation of tea aroma during tea processing, and volatile aldehydes and other products converted from the amino acids are components of the tea aroma. In addition, some amino acids have certain fragrance, and some amino acids have close relation with the taste and fragrance of tea, so that the amino acids are one of important components for forming the quality of the tea. The total amount of the free amino acids in the tea is an important index for judging the quality of the tea, and the rapid detection of the amino acid content has important significance for judging the quality of the tea. The existing determination of the total amount of the free amino acids in the tea mainly comprises a high performance liquid chromatography, an automatic amino acid analysis method, a spectrophotometry method and the like, and the high performance liquid chromatography and the automatic amino acid analysis method have the disadvantages of high sample determination cost and complex sample pretreatment and are not suitable for the rapid determination of the total amino acid content. The current national standard determination method of the total free amino acid content of tea adopts an ninhydrin spectrophotometry method, and the main principle is that amino acid reacts with ninhydrin in a pH=8.0 phosphate buffer solution under the heating condition to generate a purple complex, wherein the purple depth is positively related to the free amino acid content. The violet complex has a maximum absorbance at wavelength 570 nm and can be detected using a spectrophotometer. However, this method has the disadvantage of limited number of samples to be measured at a time and slow sample measurement speed, and is not suitable for measuring a large number of samples. At present, a kit for detecting the amino acid of the tea leaves does not exist in the market, and the improvement of the efficiency of tea quality detection is restricted. Disclosure of Invention In view of the above, in order to remedy the above-mentioned shortcomings of the prior art, the present invention provides a kit and a method for measuring the total amount of free amino acids in tea. The method for measuring the total free amino acid content of the tea leaves by adopting the kit can obviously improve the detection efficiency of the amino acid content of a large number of tea leaf samples and obviously reduce the detection cost. In order to achieve the above purpose, the invention adopts the following technical scheme: Firstly, the invention provides a kit for determining total amount of free amino acids in tea, which comprises the following components: 1 mg/mL glutamic acid standard solution; Solution B phosphate buffer at ph=8.0; the solution C is a ninhydrin solution with the mass fraction of 2%, and the ninhydrin solution contains 0.8 mg/mL stannous chloride; The kit was stored in a4 ℃ environment. Preferably, the kit further comprises a 96-well elisa plate. Further, the kit also comprises a 12-linked hole ELISA strip. The invention also provides a method for measuring the total amount of free amino acids in tea, which adopts the kit according to the technical scheme and comprises the following steps: (1) Crushing a tea sample, drying to constant weight, recording the weight of the tea before and after drying, and calculating the dry matter content of the tea sample; (2) The preparation of a standard curve, namely adding the solution B, the solution C, H 2 O and the solution A with gradient volume in proportion, carrying out water bath reaction at 100 ℃ for 10: 10min, cooling to room temperature, measuring a light absorption value by using an enzyme-labeled instrument, drawing the standard cu