Method for rapidly and preliminarily screening amino acid content and tea polyphenol content based on single tea tree

Abstract

The invention discloses a method for rapidly and preliminarily screening amino acid and tea polyphenol content based on a single tea tree plant, and relates to the technical field of excellent single plant screening methods. Relatively portable instruments and equipment are utilized, primary screening of single tea trees with the content difference of amino acid and tea polyphenol is rapidly completed in a fresh tea leaf collecting place, the method can also be applied to early identification in the new tea variety breeding process, the number of fresh tea leaves used for detection is greatly reduced, the screening process of high-quality single tea trees is accelerated, and the screening efficiency is improved. The material cost and the time cost are reduced.

Description

Technical Field

[0001] The invention relates to the technical field of excellent plant single-plant screening methods, and in particular to a method for rapid primary screening based on the amino acid and tea polyphenol content of a single tea tree. Background Art

[0002] Catechin, also known as catechin acid and catechin essence (Caredhieacid), is a kind of phenolic active substances extracted from natural plants such as tea leaves, with a molecular formula of C15H14O6 and a relative molecular mass of 290.28. It is a white needle-shaped crystal (water-acetic acid), with a melting point of 212-216°C, soluble in hot water, ethanol, glacial acetic acid, acetone, slightly soluble in cold water and ether, and almost insoluble in benzene, chloroform and petroleum ether.

[0003] Catechins account for about 75% to 80% of the content of tea polyphenols. They have a variety of pharmacological effects such as anti-tumor, anti-oxidation, anti-bacteria, and protection of heart and brain organs. They are also one of the sources of the bitter taste of tea. Amino acids are organic compounds containing alkaline amino groups and acidic carboxyl groups. Compounds formed when the hydrogen atoms on the carbon atoms of carboxylic acids are replaced by amino groups.

[0004] Tea contains about 1% to 4% amino acids, of which 26 have been found, including 20 protein amino acids and 6 non-protein amino acids, of which the most important are theanine, glutamic acid, aspartic acid and arginine. Theanine, in particular, is almost unique to tea, accounting for 1% to 2% of the dry matter of tea and about 50% of all amino acids. The amino acids in tea are also one of the main sources of the fresh taste of tea. Tea resources with high tea polyphenols and high amino acid content have great potential application value in improving my country's tea varieties.

[0005] Therefore, in the process of field investigation of tea germplasm resources, screening, identification and creation of excellent germplasm resources, there is a great need for a rapid screening method for tea polyphenols and high amino acid content that can be used under simple field equipment conditions.

[0006] However, to date, the identification of tea polyphenols and amino acid content in tea both at home and abroad has mostly adopted the high performance liquid chromatography method. This method is affected by the development period and living environment of the tea tree, has high requirements for detection equipment, takes a long time to detect and requires a certain amount of tea leaves. It is unable to achieve rapid identification of wild resources, especially a large number of individual plants.

[0007] Therefore, a new solution to the above problems needs to be proposed. Summary of the invention

[0008] The purpose of the present invention is to provide a method for rapid preliminary screening of amino acid and tea polyphenol contents of individual tea trees, which uses relatively portable instruments and equipment to quickly complete the preliminary screening of individual tea trees with differences in amino acid and tea polyphenol contents at the site where fresh tea leaves are collected. The method can also be applied to early identification in the process of breeding new tea varieties, with the purpose of greatly reducing the amount of fresh tea leaves used for testing, speeding up the screening process of high-quality individual tea trees, reducing material costs and time costs, so as to solve the technical problems raised in the background technology.

[0009] To achieve the above object, the present invention provides the following technical solution: a method for rapid initial screening of amino acid content of a single tea plant, comprising at least the following steps:

[0010] S1: Use a puncher to punch holes in fresh tea leaves to take samples, and grind the samples into a 1.5 mL centrifuge tube;

[0011] S2: Add boiling water into the centrifuge tube, extract in boiling water for a certain period of time, and then centrifuge to obtain the extraction mother liquor;

[0012] S3: Add phosphate buffer, ninhydrin colorimetric agent and extraction mother solution to the eight-tube connection in sequence, extract in a boiling water bath for a certain period of time, and observe and compare the color changes. The darker the color, the higher the amino acid content.

[0013] Furthermore, the sampling site for punching holes in fresh tea leaves with a hole puncher is the second leaf, and the mass of the sample is 0.03±0.005g.

[0014] Furthermore, the 1.5 mL centrifuge tube in S1 is a conical centrifuge tube, and a high-speed tissue grinder is used for grinding in the 1.5 mL centrifuge tube, and the model of the high-speed tissue grinder is OSE-Y30.

[0015] Furthermore, liquid nitrogen needs to be added to assist in the grinding process in S1, and the grinding time is 2-3 minutes.

[0016] Furthermore, the volume of boiling water added to the centrifuge tube in S2 is 0.75 mL, the extraction time in S2 in boiling water is 30 min, the extraction in boiling water is shaken once every 5 min, the boiling water used is ultrapure water, the oscillator used for the oscillation and shaking is a portable vortex mixer, and the oscillation and shaking time is 15 s.

[0017] Furthermore, the centrifugation time in S2 is 3 minutes, a portable low-speed centrifuge is used for the centrifugation, and the rotation speed during the centrifugation process is 2000 rpm.

[0018] Furthermore, the eight-tube connection in S3 is a 200 μL eight-tube connection, the amount of the phosphate buffer added is 40 μL, the amount of the ninhydrin color developer added is 40 μL, the amount of the extraction mother solution added is 80 μL, and the boiling water bath time is 15 min.

[0019] A method for rapid initial screening of tea polyphenol content based on a single tea tree, comprising at least the following steps:

[0020] Step 1: Use a puncher to punch holes in fresh tea leaves to take samples, take 0.03±0.005g of the sample, put it into a 1.5mL centrifuge tube and grind it;

[0021] Step 2: Add 0.75 mL of 70% methanol aqueous solution to the centrifuge tube, and the 70% methanol aqueous solution needs to be preheated in a 70° C. water bath before adding to the centrifuge tube;

[0022] Step 3: After mixing well in the centrifuge tube, transfer to a 70°C water bath and extract for 10 minutes. During the extraction process, shake once every 5 minutes and centrifuge for 3 minutes after extraction to obtain the extraction mother liquor.

[0023] Step 4: In an eight-tube series, add 10% Folin phenol and the mother solution in turn, shake well, let react for 5 minutes, then add 7.5% sodium carbonate solution, let stand for 10 minutes, and observe the color change. The darker the color, the higher the tea polyphenol content.

[0024] Furthermore, the 1.5 mL centrifuge tube in the step 1 is a conical centrifuge tube, and the grinding in the 1.5 mL centrifuge tube is performed using a high-speed tissue grinder, the model of the high-speed tissue grinder is OSE-Y30, and liquid nitrogen needs to be added to assist in the grinding process in the step 1, and the crushing time is 2-3 minutes.

[0025] Furthermore, the eight-tube connection in step 2 is a 200 μL eight-tube connection, the amount of the 10% Folin phenol reagent added is 100 μL, the amount of the extraction mother solution added is 2.5 μL, and the amount of the 7.5% sodium carbonate solution added is 80 μL.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention can greatly reduce the number of fresh tea leaves used for testing through the design of an overall method, and adopts relatively portable instruments and equipment to achieve rapid preliminary screening at the tea tree fresh leaf collection site, thereby speeding up the screening process of high-quality individual plants and reducing material costs and time costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0029] Figure 1 This is a schematic diagram of the color differences of 59 tea plant strains after rapid screening of amino acid content in an embodiment of the present invention;

[0030] Figure 2 This is a schematic diagram of the color differences of 59 tea tree plants after rapid screening of tea polyphenols content in an embodiment of the present invention. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0032] The reagents used in this example can be purchased from a conventional biochemical reagent store unless otherwise specified. The HPLC directional determination in this example was repeated three times, and the results were averaged.

[0033] This example is a preliminary screening of wild tea tree plants with high amino acid and tea polyphenol content in Chashu Village, Delong Town, Nanchuan District, Chongqing.

[0034] 1. Test materials

[0035] 59 tea tree plants were selected as research materials.

[0036] 2. Rapid screening of tea leaves with high amino acid and tea polyphenol content

[0037] 2.1 Rapid screening of tea leaves with high amino acid content

[0038] 0.03 g of fresh leaves were taken with a corker, ground in a 1.5 mL centrifuge tube, and extracted in boiling water for 30 min (shaken every 5 min), and then centrifuged in a centrifuge for 3 min to obtain the mother liquor; 40 μL of phosphate buffer, 40 μL of ninhydrin colorimetric agent, and 80 μL of the mother liquor were added to an eight-tube tube, and the mixture was placed in a boiling water bath for 15 min. After cooling for 10 min, the color change was observed and photographed with a camera ( Figure 1Adobe Photoshop 2022 and Adobe Color were used for color extraction and RGB conversion. According to the RGB values, the 59 individual plants were divided into three categories: relatively high, relatively medium, and relatively low amino acid content. The number of individual plants in each category was 15, 35, and 9, respectively (Table 1).

[0039] Table 1 Rapid screening and classification results of amino acid content

[0040]

[0041] 2.2 Rapid screening of tea leaves with high tea polyphenol content

[0042] Take 0.03g of fresh leaves with a corker, grind in a 1.5mL centrifuge tube, add 0.75mL of 70% methanol aqueous solution (preheated in a 70℃ water bath), mix well and transfer to a 70℃ water bath, soak for 10min (shake every 5min), centrifuge for 3min after extraction to obtain the mother liquor; add 100μL of Folin phenol reagent and 2.5μL of the mother liquor to an eight-tube tube, shake well, react for 5min, then add 80μL of sodium carbonate solution, let stand for 10min, observe and compare the color changes and take pictures with a camera ( Figure 2 AdobePhotoshop 2022 and Adobe Color were used for color extraction and RGB conversion. According to the RGB values, the 59 individual plants were divided into three categories: relatively high, relatively medium, and relatively low tea polyphenol content. The number of individual plants in each category was 25, 23, and 11, respectively (Table 2).

[0043] Table 2 Rapid screening and classification results of tea polyphenols content

[0044]

[0045] 3. 16 individual plant samples were further quantitatively determined using high-speed liquid chromatography (HPLC) to verify the rapid screening results. The rapid screening individual plant results were compared with the liquid phase results, and the comparison results are as follows (Table 3, Table 4):

[0046] Table 3 Comparison of HPLC determination results of amino acids of 16 tea plant strains with rapid screening results

[0047]

[0048] Table 4 Comparison of HPLC determination results of tea polyphenols in 16 tea plant strains with rapid screening results

[0049]

[0050]

[0051] As shown in Table 3, under the conditions of the two methods, single plants No. 8, 17, 34, 37, and 58 all showed relatively high amino acid content, and the consistency between the rapid screening method and the HPLC test results reached 93.75%. As shown in Table 4, under the conditions of the two methods, single plants No. 5, 8, 11, 17, 18, 19, 35, 36, 37, 47, and 55 all showed relatively high tea polyphenol content, and the consistency between the rapid screening method and the HPLC test results reached 87.5%.

[0052] It can be seen from Tables 3 and 4 that the method for quickly screening individual plants with high amino acid and high tea polyphenol contents in fresh tea leaves of the present invention can quickly screen out individual plants with high amino acid and high tea polyphenol contents in fresh tea leaves, and compared with the results of HPLC quantitative determination, it can be seen that the present invention has high accuracy, can speed up the screening process for high-quality individual plants, and can reduce material costs and time costs.

[0053] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for rapid initial screening of amino acid content in a single tea plant, characterized in that: At least the following steps are included: S1: Use a puncher to punch holes in fresh tea leaves to take samples, and grind the samples into a 1.5 mL centrifuge tube; S2: Add boiling water into the centrifuge tube, extract in boiling water for a certain period of time, and then centrifuge to obtain the extraction mother liquor; S3: Add phosphate buffer, ninhydrin colorimetric agent and extraction mother solution to the eight-tube connection in sequence, extract in a boiling water bath for a certain period of time, and observe and compare the color changes. The darker the color, the higher the amino acid content.

2. The method according to claim 1, characterized in that: The sampling site for punching holes in fresh tea leaves using a hole puncher is the second leaf, and the mass of the sample is 0.03±0.005g.

3. A method for rapid initial screening of amino acid content in a single tea plant according to claim 1, characterized in that: The 1.5 mL centrifuge tube in S1 is a conical centrifuge tube, and a high-speed tissue grinder is used for grinding in the 1.5 mL centrifuge tube. The model of the high-speed tissue grinder is OSE-Y30.

4. The method according to claim 1, characterized in that: Liquid nitrogen needs to be added to assist the grinding process in S1, and the grinding time is 2-3 minutes.

5. The method according to claim 1, characterized in that: The volume of boiling water added to the centrifuge tube in S2 is 0.75 mL, the extraction time in S2 in boiling water is 30 min, the extraction in boiling water is shaken once every 5 min, the boiling water used is ultrapure water, the oscillator used for the oscillation is a portable vortex mixer, and the oscillation time is 15 s.

6. The method according to claim 1, characterized in that: The centrifugation time in S2 is 3 minutes, a portable low-speed centrifuge is used for the centrifugation, and the rotation speed during the centrifugation process is 2000 rpm.

7. The method according to claim 1, characterized in that: The eight-tube connection in S3 is a 200 μL eight-tube connection, the amount of the phosphate buffer added is 40 μL, the amount of the ninhydrin color developer added is 40 μL, the amount of the extraction mother solution added is 80 μL, and the boiling water bath time is 15 min.

8. A method for rapid initial screening of tea polyphenol content in a single tea plant, characterized in that: At least the following steps are included: Step 1: Use a puncher to punch holes in fresh tea leaves to take samples, take 0.03±0.005g of the sample, put it into a 1.5mL centrifuge tube and grind it; Step 2: Add 0.75 mL of 70% methanol aqueous solution to the centrifuge tube, and the 70% methanol aqueous solution needs to be preheated in a 70° C. water bath before adding to the centrifuge tube; Step 3: After mixing well in the centrifuge tube, transfer to a 70°C water bath and extract for 10 minutes. During the extraction process, shake once every 5 minutes and centrifuge for 3 minutes after extraction to obtain the extraction mother liquor. Step 4: In an eight-tube series, add 10% Folin phenol and the mother solution in turn, shake well, let react for 5 minutes, then add 7.5% sodium carbonate solution, let stand for 10 minutes, and observe the color change. The darker the color, the higher the tea polyphenol content.

9. The method for rapid initial screening of tea polyphenol content based on a single tea plant according to claim 8, characterized in that: The 1.5 mL centrifuge tube in step 1 is a conical centrifuge tube. The 1.5 mL centrifuge tube is ground using a high-speed tissue grinder, the model of which is OSE-Y30. Liquid nitrogen is required to assist in the grinding process in step 1, and the grinding time is 2-3 minutes.

10. The method for rapid initial screening of tea polyphenols content based on a single tea plant according to claim 8, characterized in that: The eight-tube connection in step 2 is a 200 μL eight-tube connection, the amount of the 10% Folin phenol reagent added is 100 μL, the amount of the extraction mother solution added is 2.5 μL, and the amount of the 7.5% sodium carbonate solution added is 80 μL.

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