Bitter tea bitter substance separation system

Abstract

The utility model relates to the technical field of tea analysis, and discloses a bitter substance separation system for bitter tea, which comprises a pretreatment system, a gradient elution system and a chromatographic separation system, the gradient elution system comprises a plurality of eluent accommodating cavities, opening and closing valves corresponding to the eluent accommodating cavities, a mixer for mixing eluents in proportion and an eluent flow path; a mobile phase A and a mobile phase B are respectively accommodated in the at least two eluent accommodating cavities, the mobile phase A is a solution of deionized water and formic acid with the volume percent of 0.1%, and the mobile phase B is a solution of acetonitrile and formic acid with the volume percent of 0.1%; the chromatographic separation system comprises a chromatographic separation column arranged in a column oven; the eluent flow path and the tea sample flow path are connected into the chromatographic separation column in parallel to separate bitter substances. The problem that a material base separation system for bitter taste formation of bitter tea is lacked in the prior art is solved.

Description

TECHNICAL FIELD

[0001] The utility model relates to tea analysis technical field especially relates to a bitter tea bitter taste material separation system. BACKGROUND

[0002] The taste of tea is greatly different due to factors such as its variety and processing method. Among them, part of germplasm resources represented by Jianghua bitter tea in tea tree resources has obvious bitterness, and is favored by part of people due to its unique bitter and astringent taste. For example, Jianghua bitter tea 37-2, there is no research on analyzing the taste of Jianghua bitter tea 37-2 based on metabolomics technology, especially the identification and analysis of key bitter substances and the separation of the material basis of bitterness formation have not been reported. UTILITY MODEL CONTENT

[0003] Therefore, the utility model aims at providing a bitter tea bitter taste material separation system to solve the problem that there is no separation system for the material basis of bitter tea bitterness in the prior art, so as to comprehensively understand the material basis of bitter tea bitterness, enrich the theory of tea flavor chemistry, and provide certain reference and guidance for the research and development of other bitter tea resources in China.

[0004] The utility model solves the above technical problem through the following technical means: a bitter tea bitter taste material separation system, which comprises a pretreatment system, a gradient elution system and a chromatographic separation system; the pretreatment system is used for crushing and centrifugal separation of tea sample, and comprises a tea sample flow path provided with a filter membrane; the gradient elution system comprises a plurality of eluent containing cavities, opening and closing valves corresponding to the eluent containing cavities, a mixer for mixing eluents in proportion and an eluent flow path; at least two eluent containing cavities contain mobile phase A and mobile phase B respectively, the mobile phase A is a solution with a volume percentage of 0.1% of deionized water and formic acid, and the mobile phase B is a solution with a volume percentage of 0.1% of acetonitrile and formic acid; the chromatographic separation system comprises a chromatographic separation column arranged in a column oven; the eluent flow path and the tea sample flow path are connected in parallel to the chromatographic separation column to realize bitter taste material separation.

[0005] Optionally, the packing diameter of the chromatographic separation column is 1.8 microns.

[0006] Optionally, the pore size of the filter membrane is 0.22 microns. Large molecular interference substances are removed.

[0007] Optionally, a multi-way valve is connected to the outlet end of the chromatographic separation column, and the outlet of the multi-way valve is respectively connected to a fraction collector and a detector. The fraction collector is used for collecting separated bitter substances, and the detector is used for qualitative or quantitative analysis of bitter substances.

[0008] Optionally, the detector is a mass spectrometer.

[0009] Optionally, the pretreatment system comprises a high-speed grinder for crushing the tea sample, a centrifuge tube for high-temperature extraction of the tea sample, and a centrifuge for high-speed centrifugal treatment of the tea sample after high-temperature extraction.

[0010] The present application has the following beneficial effects:

[0011] The present application has the following beneficial effects: BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 is a schematic diagram of the overall structure of the present application.

[0013] Among them, 11-filter membrane, 21-eluent containing cavity, 22-shutoff valve, 23-mixer, 31-column oven, 32-chromatographic separation column. DETAILED DESCRIPTION

[0014] The advantages and effects of the present application can be understood by the content disclosed in the specification. It should be noted that the drawings provided in the following examples are only used for illustrative purposes, and the representation is only a schematic diagram, not a physical drawing, and should not be understood as a limitation of the present application. In order to better illustrate the embodiments of the present application, some components in the drawings may be omitted, enlarged or reduced, and do not represent the actual product size; for those skilled in the art, it is understandable that some well-known structures and their descriptions may be omitted.

[0015] The same or similar reference numerals in the drawings of the embodiments of the present application correspond to the same or similar components, and in the description of the present application, it should be understood that if the terms "upper", "lower", "left", "right", "front", "back" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation, therefore the terms used to describe the positional relationship in the drawings are only used for illustrative purposes, and cannot be understood as a limitation of the present application. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0016] As Figure 1The utility model discloses a bitter tea bitter substance separation system, including pretreatment system, gradient elution system and chromatographic separation system.

[0017] In the embodiment, the pretreatment system comprises a high-speed grinder for crushing tea sample, a centrifugal tube for high-temperature extraction of tea sample, a centrifuge for high-speed centrifugal treatment of tea sample after high-temperature extraction and a tea sample flow path provided with a filter membrane, and it is understood that the high-speed grinder, the centrifugal tube and the centrifuge are common pretreatment equipment, and their specific structure and principle are not described again.

[0018] In the embodiment, the gradient elution system comprises a plurality of eluent containing cavities, opening and closing valves corresponding to the eluent containing cavities, a mixer for proportionally mixing eluent and an eluent flow path; the at least two eluent containing cavities contain mobile phase A and mobile phase B respectively; the mobile phase A is a solution with a volume percentage of 0.1% of deionized water and formic acid, and the mobile phase B is a solution with a volume percentage of 0.1% of acetonitrile and formic acid, that is, in the mobile phase A and the mobile phase B, formic acid is the solute, and the formic acid accounts for 0.1% of the total volume of the solution, and the elution effect is best.

[0019] In the embodiment, the liquid sample obtained after the tea sample is sequentially treated by the high-speed grinder, the centrifugal tube and the centrifuge is taken into the tea sample flow path provided with the filter membrane, the pore size of the filter membrane is 0.22 μm, and then the sample is quantitatively connected to the eluent flow path by the autosampler of the chromatographic system and then enters the chromatographic separation system, and the single sampling amount is 2 mL.

[0020] In the embodiment, in order to ensure the separation effect of the bitter substance, the elution program of the gradient elution system is specifically as follows: 0-0.5 min, 2% mobile phase B; 0.5-8 min, 15% mobile phase B; 8-13 min, 35% mobile phase B; 13-15 min, 70% mobile phase B; 15-16 min, 85% mobile phase B; 16-16.5 min, 2% mobile phase B; 16.5-20 min, 2% mobile phase B.

[0021] In the embodiment, the chromatographic separation system comprises a chromatographic separation column arranged in a column oven, and the chromatographic separation column is selected from an Acquity UPLC HSS T3 chromatographic column (2.1 mm*100 mm, 1.8 μm, Waters, Manchester, UK) for chromatographic separation of the compound, the packing diameter of the chromatographic separation column is 1.8 μm, the column oven controls the temperature of the chromatographic column to be 40 DEG C, the flow rate of the chromatographic column is set to be 0.4 mL / min, and the sampling amount is 3 μL; the eluent flow path and the tea sample flow path are connected in parallel to the chromatographic separation column to realize separation of the bitter substance.

[0022] In this embodiment, the outlet end of the chromatographic separation column is connected with a multi-way valve, and the outlet of the multi-way valve is connected with a fraction collector and a detector. The detector is a mass spectrometer, and a Q-Exactive series mass spectrometer (Thermo) is selected as an example for mass spectrometry analysis, and positive ion and negative ion modes are used for detection. In the positive ion and negative ion modes, the parameters are set as follows: the capillary voltage is set to 3500V and-3000V, respectively; the capillary temperature is set to 300 DEG C; the flow rate of the drying gas is 10 L / min; the temperature is kept at 325 DEG C; and the mass scanning range is m / z 100-1200.

[0023] In this embodiment, the bitter substances obtained by the fraction collector mainly include flavonoid substances and alkaloids, wherein the alkaloids (kukoamine, 1-methyladenine, etc.), flavonol compounds and flavones (catechin, gallocatechin, gallocatechingallate, epigallocatechingallate, epicatechingallate, epigallocatechingate, kaempferol, morin, etc.), and polymeric catechins (Theasinensin B, Theasinensin C, proanthocyanidin B3, etc.). Taking Jianghua Kucha as the main test sample, the metabolites of kukoamine, catechin, gallocatechingallate and proanthocyanidin B3 in the separated bitter substances are obviously up-regulated, that is, the main sources of the bitter substances of Jianghua Kucha, and it should be understood that the separation system of the utility model can also be used for the separation of bitter substances in other Kucha.

[0024] The working principle of the utility model is as follows:

[0025] Taking Jianghua Kucha 37-2 as an example, one bud and two leaves of fresh leaves are picked, frozen by liquid nitrogen, and then vacuum freeze-dried. The preliminarily processed tea sample is put into the pretreatment system, crushed by a high-speed grinder (IKA, Germany), and the excess sample can be stored at-80 DEG C for later use. 0.1g of the above tea powder sample is accurately weighed in a 15mL centrifuge tube, 10mL of 100 DEG C boiling water is added for extraction for 5min, the centrifuge tube is shaken up and down every 1min, the shaking can be realized by manual or common swinging structure, and then placed in a high-speed centrifuge for centrifugation for 20min at a speed of 5000r / min. The sample liquid separated by the centrifuge is taken, and the supernatant part is taken through a 0.22mu m filter membrane 11 into the tea sample flow path, the chromatographic separation column separates the compounds, the column temperature is controlled by a column temperature box to be 40 DEG C, the flow rate of the chromatographic column is set to be 0.4mL / min, and the sample amount is 3mu L; the eluent flow path and the tea sample flow path are connected in parallel to the chromatographic separation column to realize the separation of bitter substances.

[0026] After the sample was separated by a chromatographic column, mass spectrometry was performed by a mass spectrometer (Thermo) to detect in positive ion and negative ion modes by electrospray ionization (ESI), respectively. The original spectrum obtained by mass spectrometry was subjected to peak selection and peak alignment by Thermo Xcalibur 3.1 software (Thermo Fisher Company, USA), and the structure of the compound was identified according to the real standard substance, MS2 spectrum, mass accuracy, metabolomics database (Metlin and HMDB) and previous work. The bitter substances obtained by the fraction collector can be used for further purification or analysis and identification.

[0027] The above examples are only used to illustrate the technical solutions of the present application and are not limited. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the present application. The technical, shape and structure parts not described in detail should be covered in the scope of the claims of the present application.

Claims

1. A system for separating bitter substances from bitter tea, characterized in that, Includes a pretreatment system, a gradient elution system, and a chromatographic separation system; The pretreatment system is used to crush and centrifuge tea samples; the pretreatment system includes a tea sample flow path equipped with a filter membrane (11); The gradient elution system includes multiple eluent containers (21), on / off valves (22) corresponding to the eluent containers (21), a mixer (23) for mixing the eluent in proportion, and an eluent flow path; at least two of the eluent containers (21) contain mobile phase A and mobile phase B respectively; mobile phase A is a solution of deionized water and formic acid with a volume percentage of 0.1%, and mobile phase B is a solution of acetonitrile and formic acid with a volume percentage of 0.1%; The chromatographic separation system includes a chromatographic separation column (32) installed in a column oven (31); the eluent flow path and the tea sample flow path are connected in parallel to the chromatographic separation column (32) to achieve the separation of bitter substances.

2. The bitterness substance separation system for bitter tea according to claim 1, characterized in that: The diameter of the packing material of the chromatographic separation column (32) is 1.8 μm.

3. The bitterness substance separation system for bitter tea according to claim 1, characterized in that: The filter membrane (11) has a pore size of 0.22 μm.

4. The bitterness substance separation system for bitter tea according to claim 1, characterized in that: The outlet of the chromatographic separation column (32) is connected to a multi-port valve, and the outlet of the multi-port valve is connected to a fraction collector and a detector, respectively.

5. The bitterness substance separation system for bitter tea according to claim 4, characterized in that: The detector is a mass spectrometer.

6. The bitterness substance separation system for bitter tea according to claim 1, characterized in that: The pretreatment system also includes, in sequence, a high-speed grinder for crushing tea samples, centrifuge tubes for high-temperature extraction of tea samples, and a centrifuge for high-speed centrifugation of the tea samples after high-temperature extraction.

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