Method and system for determining content of tea polyphenol in tea leaves

Abstract

The invention discloses a method and a system for determining the content of tea polyphenol in tea leaves, and relates to the technical field of chemical component analysis of tea leaves, and the method comprises the following steps: dissolving tea polyphenol in a tea leaf sample into a primary extract A through dynamic ultrasonic-assisted enzymolysis and hot water extraction; filtering the primary extract A to remove insoluble impurities so as to obtain a tea polyphenol extract B containing tea polyphenol and a small amount of soluble impurities; performing multi-stage solvent separation on the tea polyphenol extracting solution B by using ethanol, ethyl acetate and dilute acid, adding a multi-metal ion complexing solution, removing water-soluble impurities through complexing reaction, and concentrating to obtain a high-purity tea polyphenol solution D; diluting the tea polyphenol solution D as an optical analysis sample; determining the absorbance of the diluted optical analysis sample, and calculating the concentration of tea polyphenol in the diluted sample; and calculating the final content of the tea polyphenol in the tea leaves according to the concentration of the tea polyphenol in the diluted sample in combination with an optical analysis sample dilution ratio and an extraction volume.

Description

Technical Field

[0001] The invention relates to the technical field of tea chemical component analysis, in particular to a method and system for determining the content of tea polyphenols in tea. Background Art

[0002] Tea is an important economic crop, and its functional ingredient tea polyphenols has significant biological activity and has wide application value in the fields of food, medicine and cosmetics. In recent years, the extraction, purification and content determination methods of tea polyphenols have become a research hotspot, but the existing technologies still have many shortcomings. Although the existing determination methods (such as Folin phenol reagent method, high performance liquid chromatography and ultraviolet spectrophotometry) have certain advantages, they still face problems such as complex sample pretreatment, low extraction efficiency, large impurity interference, and insufficient accuracy and repeatability of determination results in practical applications. In addition, the existing methods are prone to incomplete release of bound tea polyphenols or degradation of tea polyphenols during the extraction process, limited impurity removal effect and easy loss of target components during the purification process, and the problems of improper control of sensitivity, selectivity and linear range in content determination are more prominent. These technical defects limit the comprehensive analysis and efficient utilization of tea polyphenols.

[0003] In general, the existing technology has deficiencies in the extraction efficiency of tea polyphenols, impurity removal ability, and measurement accuracy and stability, especially in the control of dilution multiples and optical measurement linear range, which can easily lead to deviations in measurement results. In response to these problems, our invention proposes an improved method and system for determining the content of tea polyphenols in tea. By optimizing the extraction, purification and optical measurement processes, the extraction efficiency, sample purity and measurement accuracy are improved, thereby effectively solving the key bottleneck problems in the existing technology. Summary of the invention

[0004] In view of the above existing problems, the present invention is proposed.

[0005] Therefore, the present invention provides a method for determining the content of tea polyphenols in tea to solve the deficiencies in the prior art in terms of tea polyphenol extraction efficiency, impurity removal capability, and determination accuracy and stability, especially the problem that the dilution multiple and the control of the linear range of optical determination are prone to cause deviations in the determination results.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions: In a first aspect, the present invention provides a method for determining the content of tea polyphenols in tea leaves, comprising: The tea polyphenols in the tea sample were dissolved into the primary extract A by dynamic ultrasound-assisted enzymatic hydrolysis and hot water extraction; Filtering the initial extract A to remove insoluble impurities, and obtaining a tea polyphenol extract B containing tea polyphenols and a small amount of soluble impurities; Using ethanol, ethyl acetate and dilute acid to perform multi-stage solvent separation on the tea polyphenol extract B, adding a multi-metal ion complexing liquid after the multi-stage solvent separation, removing water-soluble impurities through complexing reaction, and concentrating to obtain a high-purity tea polyphenol solution D; Dilute the tea polyphenol solution D as a sample for optical analysis; Reagents are mixed through a reaction pool based on a microfluidic chip, the absorbance of the diluted optical analysis sample is measured, and the concentration of tea polyphenols in the diluted sample is calculated; The final content of tea polyphenols in tea leaves is calculated based on the concentration of tea polyphenols in the diluted sample combined with the optical analysis sample dilution multiple and extraction volume.

[0007] As a preferred embodiment of the method for determining the content of tea polyphenols in tea leaves of the present invention, the tea polyphenols in the tea sample are dissolved into the primary extract A by dynamic ultrasound-assisted enzymatic hydrolysis and hot water extraction, and the specific steps are as follows: Take a sample of dried tea leaves and crush it using a grinder until the particles pass through a mesh sieve; Add cellulase and pectinase to deionized water and adjust the pH value to the optimal activity range; The crushed tea sample is mixed with the enzymatic hydrolysate and placed in a sealed container; During the enzymatic hydrolysis process, the sealed container is placed in a dynamic ultrasonic enzymatic hydrolysis auxiliary device, the ultrasonic frequency and ultrasonic power are set, and the ultrasonic intermittent mode is used for continuous action; Stir with a magnetic stirrer to mix evenly and set the reaction time; After the enzymatic hydrolysis reaction is completed, the mixed solution is heated and the extraction time is set; After the extraction time is completed, the initial extract is collected to obtain; The initial extract is filtered through a microporous filter membrane to remove insoluble impurities, and the filtrate is collected to obtain an initial extract A containing tea polyphenols.

[0008] As a preferred embodiment of the method for determining the content of tea polyphenols in tea leaves of the present invention, the initial extract A is filtered to remove insoluble impurities to obtain a tea polyphenol extract B containing tea polyphenols and a small amount of soluble impurities, and the specific steps are as follows: Allow the initial extract A to stand for a period of time; Use a vacuum pump equipped with a microporous filter membrane made of polyvinylidene fluoride (PVDF); Pour the initial extract A after standing into the filter cup and start the vacuum pump for filtration; After the filtration is completed, the tea polyphenols extract B containing tea polyphenols and a small amount of soluble impurities is collected.

[0009] As a preferred embodiment of the method for determining the content of tea polyphenols in tea leaves of the present invention, wherein: The method uses ethanol, ethyl acetate and dilute acid to perform multi-stage solvent separation on the tea polyphenol extract B, adds a multi-metal ion complexing liquid after the multi-stage solvent separation, removes water-soluble impurities through complexing reaction, and concentrates to obtain a high-purity tea polyphenol solution D, and the specific steps are: Pour the filtered tea polyphenols extract B into a separatory funnel and add anhydrous ethanol; Thoroughly mix the tea polyphenols extract B and ethanol, then shake and let stand; After standing, separating the upper ethanol solution containing tea polyphenols and the lower aqueous phase containing water-soluble impurities, and removing the lower aqueous phase; The upper ethanol solution was collected and the ethanol was removed by a rotary evaporator to obtain an ethanol concentrate; In a separatory funnel, add the ethanol concentrate to ethyl acetate; After shaking and standing, the ethyl acetate phase and the aqueous phase were separated; Following the same method, the extraction steps were repeated; All ethyl acetate phases are combined, dried with anhydrous sodium sulfate, and then subjected to low-temperature rotary evaporation to recover ethyl acetate to obtain a purified tea polyphenols concentrate; Add multi-metal ion complex solution to the purified tea polyphenol concentrated solution, adjust the pH value of the solution, and let it stand at room temperature; after standing, filter to remove precipitated impurities, collect the filtrate and concentrate it by rotary evaporation to obtain a high-purity tea polyphenol solution D.

[0010] As a preferred solution of the method for determining the content of tea polyphenols in tea leaves of the present invention, the tea polyphenol solution D is diluted as a sample for optical analysis, specifically: The absorbance of the tea polyphenol solution D at a specific wavelength is measured and the absorbance value range is set. When the absorbance value exceeds the range, it indicates that the tea polyphenol solution D needs to be diluted. Place tea polyphenol solution D in a volumetric flask and set the dilution factor to , dilute according to the dilution factor, and calculate the required dilution factor based on the linear detection range of the optical analyzer , the expression is: ; in, Indicates the dilution factor. represents the absorbance of tea polyphenol solution D, Indicates the target The absorbance of Select deionized water as the diluent and use a high-precision pipette to add an appropriate amount of deionized water to the scale line; Use a glass rod to stir and mix to make the tea polyphenols fully and evenly dissolved; The diluted sample is placed in an ultrasonic oscillator for treatment; After the dilution is completed, take an appropriate amount of sample and place it in a cuvette for testing.

[0011] As a preferred embodiment of the method for determining the content of tea polyphenols in tea leaves of the present invention, the reagents are mixed by a reaction pool based on a microfluidic chip, the absorbance of the diluted optical analysis sample is determined, and the concentration of tea polyphenols in the diluted sample is calculated. The specific steps are as follows: The diluted sample was injected into the microfluidic chip, and the Folin phenol reagent and The solution is uniformly mixed through micro-mixing channels within the chip; Use the chip's built-in optical detection unit to measure the sample's absorbance at a specific wavelength in real time; The test results are transmitted to the computer through the chip system, which automatically calculates the tea polyphenols concentration and outputs the results; Also provide alternative solutions: place the diluted sample in a UV spectrophotometer, set the wavelength, and record the absorbance separately; Set the number of dilutions and take the average of the measured values ​​as the final absorbance; Set the absorbance to , calculate the concentration of tea polyphenols in the diluted sample , the expression is: ; in, represents the linear fitting coefficient.

[0012] As a preferred embodiment of the method for determining the content of tea polyphenols in tea leaves of the present invention, the final content of tea polyphenols in tea leaves is calculated according to the concentration of tea polyphenols in the diluted sample combined with the optical analysis sample dilution multiple and extraction volume, and the specific steps are as follows: According to the absorbance of the diluted sample, the dilution multiple, the volume of the diluted sample, the volume of the extract D and the mass of the initial tea sample, the final content of tea polyphenols in the tea sample is calculated as follows: ; in, Indicates the final content of tea polyphenols in tea samples. represents the volume of tea polyphenol solution D after dilution, Indicates the mass of the initial tea sample.

[0013] In a second aspect, the present invention provides a tea polyphenol content determination system in tea leaves, comprising a sample processing module, a solvent separation module, an optical analysis module, an efficiency evaluation module and a tea polyphenol content module; The sample processing module uses cellulase and pectinase to destroy the cell wall structure of tea leaves, combines hot water extraction to increase the dissolution rate of tea polyphenols, and filters to remove insoluble impurities to obtain a primary extract A; The solvent separation module uses a solvent to perform multi-stage separation on the primary extract B to remove water-soluble and fat-soluble impurities; The optical analysis module dilutes the tea polyphenol solution D according to the optical detection range, measures the absorbance of the diluted sample in an ultraviolet spectrophotometer, extracts the absorbance value A at a specific wavelength, and calculates the concentration of tea polyphenols in the diluted sample using a standard curve fitting coefficient; The efficiency evaluation module is used to evaluate the tea polyphenols extraction efficiency, filtration efficiency, solvent separation efficiency and dilution retention efficiency in each step, and optimize the extraction and analysis conditions; The tea polyphenol content module is used to comprehensively calculate the tea polyphenol content in the tea sample by combining the concentration of the diluted sample, the dilution multiple, the volume of the tea polyphenol solution D, and the mass of the initial tea sample.

[0014] In a third aspect, the present invention provides a computer device comprising a memory and a processor, wherein the memory stores a computer program, wherein: when the computer program is executed by the processor, any step of the method for determining the content of tea polyphenols in tea leaves as described in the first aspect of the present invention is implemented.

[0015] In a fourth aspect, the present invention provides a computer-readable storage medium having a computer program stored thereon, wherein: when the computer program is executed by a processor, any step of the method for determining the content of tea polyphenols in tea leaves as described in the first aspect of the present invention is implemented.

[0016] The beneficial effects of the present invention are as follows: the present invention maximizes the release of tea polyphenols through the synergistic effect of enzymatic hydrolysis and hot water, thereby significantly improving the extraction efficiency; the filtration step effectively removes insoluble impurities to ensure the quality of the extract; the solvent separation utilizes the selective dissolution characteristics of multi-stage organic solvents to successfully purify high-concentration tea polyphenols while reducing impurity interference; the optical analysis quickly and accurately obtains the tea polyphenol concentration by diluting the sample and accurately measuring the absorbance; the dilution multiple and the extraction volume are combined to ultimately achieve accurate calculation of the tea polyphenol content in the tea leaves; the entire process optimizes the operating steps, improves efficiency and repeatability, and ensures the accuracy and reliability of the data through modular design. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of 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.

[0018] Figure 1 This is a flow chart of the method for determining the content of tea polyphenols in tea leaves in Example 1.

[0019] Figure 2 Schematic diagram of optical analysis of samples in Example 1. DETAILED DESCRIPTION

[0020] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings.

[0021] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0022] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0023] Example 1, reference Figure 1 and Figure 2 , which is the first embodiment of the present invention, provides a method for determining the content of tea polyphenols in tea leaves, comprising the following steps: S1, dissolving tea polyphenols in the tea sample into the primary extract A by dynamic ultrasound-assisted enzymatic hydrolysis and hot water extraction; Take 2.0g of dry tea sample and use a grinder to grind it until the particles pass through a 40-mesh sieve to increase the specific surface area of ​​the sample and improve the enzymatic hydrolysis efficiency; Cellulase (10 mg / mL) and pectinase (5 mg / mL) were added to 500 mL of deionized water, and the pH was adjusted to 4.8 ± 0.1 (achieved by adding 0.1 mol / L citric acid-phosphate buffer), which is the optimal activity range of the two enzymes; The crushed tea sample and the enzymatic hydrolysate were mixed at a mass-to-volume ratio of 1:15 and placed in a sealed container; Under the condition of 50℃±2℃, stir with a magnetic stirrer to make it evenly mixed, and set the reaction time to 30 minutes. This process can effectively decompose the cellulose and pectin in the cell wall of tea leaves and release the bound tea polyphenols; During the enzymatic hydrolysis process, the sealed container was placed in a dynamic ultrasonic device, the ultrasonic frequency was set to 40 kHz, the ultrasonic power was set to 200 W, and the ultrasonic intermittent mode was adopted (ultrasound for 5 seconds, static for 10 seconds), and the action was continued for 10 minutes. The mechanical vibration and cavitation effect of dynamic ultrasound can further destroy the cell wall structure of tea leaves and increase the contact area between the enzyme and the substrate, thereby significantly improving the release efficiency of bound tea polyphenols, while reducing the risk of oxidative degradation of tea polyphenols by high temperature.

[0024] After the enzymatic hydrolysis reaction is completed, the mixed solution is heated to 85°C ± 2°C, and the extraction time is set to 15 minutes to further dissolve the free tea polyphenols; This step ensures that the extraction efficiency of free tea polyphenols is fully improved on the basis of enzymatically releasing bound tea polyphenols; After the extraction time is completed, the initial extract is collected to obtain; The initial extract was filtered through a 0.45 μm microporous filter membrane to remove insoluble impurities, and the filtrate was collected to obtain an initial extract A containing tea polyphenols; To evaluate the effects of enzymatic hydrolysis and hot water extraction, the extraction efficiency of tea polyphenols was calculated as follows: ; in, It indicates the percentage of tea polyphenols extracted from each unit of tea sample to the total mass. Indicates the concentration of tea polyphenols in the initial extract A, represents the volume of the initial extract A, Indicates the mass of the input tea sample; Advantages of enzymatic hydrolysis technology: Cellulase and pectinase can effectively destroy the cellulose and pectin structure of tea cell walls, thereby releasing bound tea polyphenols and significantly improving the extraction rate of tea polyphenols; compared with pure physical extraction methods (such as hot water extraction), the enzymatic hydrolysis process is mild and can reduce the oxidative degradation of tea polyphenols by high temperature; Necessity of hot water-assisted extraction: After the bound tea polyphenols are released, hot water is still needed to dissolve the free tea polyphenols. The solubility of tea polyphenols is significantly improved under high temperature conditions, and the internal structure of cells can be further destroyed to release residual tea polyphenols. Synergistic effect of enzyme and hot water: Enzyme hydrolysis releases the bound state, and hot water dissolves the free state. The synergistic effect of the two can achieve the maximum extraction of tea polyphenols while reducing the co-extraction of impurities in the extraction process.

[0025] S2, filtering the initial extract A to remove insoluble impurities, and obtaining a tea polyphenol extract B containing tea polyphenols and a small amount of soluble impurities; Allow the initial extract A to stand for a period of time; The initial extract A needs to be left to stand for 15 minutes before filtering to allow large particles of impurities to settle under the action of gravity; standing still can reduce the clogging of the filter membrane and improve the filtration efficiency; Take out the initial extract A after standing, and avoid disturbing the precipitated part to ensure the filtering effect and reduce the interference of impurities; A vacuum pump equipped with a 0.45μm microporous filter membrane made of polyvinylidene fluoride (PVDF) is used. The reason for choosing PVDF filter membrane is that it has high chemical stability and excellent hydrophilicity, which is suitable for filtering aqueous extracts and can effectively remove insoluble particles without adsorbing tea polyphenols. Pour the initial extract A after standing into the filter cup, start the vacuum pump for filtration, and control the flow rate to 10mL / min to prevent the filter membrane from being blocked or the adsorption loss of tea polyphenols due to too fast filtration; During the filtration process, ensure that the filter membrane is always kept moist. If the filter membrane is found to be clogged, replace it with a new one and continue the operation; After the filtration is completed, the tea polyphenols extract B containing tea polyphenols and a small amount of soluble impurities is collected; The residue on the filter membrane is insoluble impurities (such as tea fiber, undecomposed cell wall components, etc.) and needs to be discarded; To ensure the filtration effect and the quality of the extract, take a 10 mL sample of filtrate B and measure the absorbance at a wavelength of 280 nm using a spectrophotometer to confirm that the tea polyphenols still maintain a high concentration after filtration and that there is less interference from impurities; According to the concentration of tea polyphenols in the initial extract A before filtration, the volume of the initial extract A, the concentration of tea polyphenols in the extract B and the volume of the extract B, the filtration efficiency is calculated to optimize the filtration effect of the initial extract A. The expression is: ; in, It indicates the efficiency of converting the initial extract A into the extract B. represents the concentration of tea polyphenols in extract B, Indicates the concentration of tea polyphenols in the initial extract A, represents the volume of extract B, Indicates the volume of initial extract A.

[0026] S3, using ethanol, ethyl acetate and dilute acid to perform multi-stage solvent separation on the tea polyphenol extract B, adding a multi-metal ion complexing liquid after the multi-stage solvent separation, removing water-soluble impurities through complexing reaction, and concentrating to obtain a high-purity tea polyphenol solution D; Pour the filtered tea polyphenols extract B into a separatory funnel, and add anhydrous ethanol to make the volume ratio of ethanol to tea polyphenols extract B 1:1; Thoroughly mix the tea polyphenol extract B and ethanol, then shake for 5 minutes and let stand for 15 minutes, during which the ethanol can separate the tea polyphenols from some impurities and increase the solubility of the tea polyphenols; After standing, separating the upper ethanol solution containing tea polyphenols and the lower aqueous phase containing water-soluble impurities, and removing the lower aqueous phase; Collect the upper ethanol solution, and use a rotary evaporator at 45°C ± 2°C to remove ethanol, and concentrate the tea polyphenol solution to 1 / 2 of the original volume to obtain an ethanol concentrate; After the ethanol concentrate was treated, a metal ion complex solution (a mixed solution containing aluminum ions and zinc ions) was added, and the pH of the extract was adjusted to 7.0 ± 0.1 (achieved by adding 1 mol / L NaOH solution); Preparation of metal ion complex solution: According to aluminum ion ( ) and zinc ions ( ) are mixed in a molar ratio of 1:1, with a concentration of 0.01 mol / L; The metal ion complex solution and the tea polyphenol extract solution were mixed in a volume ratio of 1:1, and allowed to stand at room temperature for 30 minutes to allow the tea polyphenols to form a stable complex with the metal ions and the impurities to precipitate; After standing, the solution was filtered using a 0.22 μm microporous membrane to remove precipitated impurities, and the filtrate was collected and concentrated by rotary evaporation to obtain a high-purity tea polyphenol solution D; Ethanol has moderate polarity, which can dissolve tea polyphenols and partially separate water-soluble impurities, and is easy to remove during the rotary evaporation process; Reduce the subsequent solvent usage through concentration operation and improve solvent separation efficiency; In a separatory funnel, add ethyl acetate to the ethanol concentrate at a volume ratio of 1:0.8; After shaking for 3 minutes and standing for 10 minutes, the ethyl acetate phase (upper layer, containing a small amount of tea polyphenols and fat-soluble impurities) and the water phase (lower layer, containing most of the water-soluble impurities) were separated; Repeat the extraction step twice in the same manner, adding fresh ethyl acetate each time to ensure that as much water-soluble impurities as possible are removed; All ethyl acetate phases were combined, dried with anhydrous sodium sulfate, and then subjected to low-temperature rotary evaporation (40°C ± 2°C) to recover ethyl acetate to obtain a purified tea polyphenols concentrate; Low-temperature rotary evaporation refers to a method of evaporating and concentrating a solution using a rotary evaporator (abbreviated as "rotary evaporation") under low-temperature conditions. It reduces the boiling point of the solvent under reduced pressure and uses rotary evaporation to quickly remove the solvent, while avoiding the degradation or destruction of heat-sensitive substances (such as tea polyphenols) by high temperature; As an organic solvent, ethyl acetate can effectively extract fat-soluble impurities and has moderate affinity with tea polyphenols, which is not easy to cause the loss of tea polyphenols. Repeated extraction further improves the separation efficiency and reduces the impurity content. To evaluate the retention effect of tea polyphenols and the degree of removal of impurities during solvent separation, the expression is: ; in, It indicates the efficiency of converting the extract B into tea polyphenol solution D. represents the concentration of tea polyphenols in tea polyphenol solution D, represents the volume of tea polyphenols solution D.

[0027] S4, diluting the tea polyphenol solution D as a sample for optical analysis; Measure the absorbance of tea polyphenol solution D at a wavelength of 280 nm, and set the absorbance value within the range of 1.0 to 2.5. When it exceeds this range, it means that tea polyphenol solution D needs to be diluted; Place tea polyphenol solution D in a 10 mL volumetric flask and set the dilution factor to , dilute according to the dilution factor, and calculate the required dilution factor based on the linear detection range of the optical analyzer (0.2~1.0) , the expression is: ; in, Indicates the dilution factor. represents the absorbance of tea polyphenol solution D, Indicates the target The absorbance of Choose deionized water as the diluent and use a high-precision pipette to add an appropriate amount of deionized water to the scale line to ensure the dilution ratio is accurate; Use a glass rod to stir and mix to ensure that the tea polyphenols are fully and evenly dissolved to avoid measurement errors caused by local concentration differences; The diluted sample was placed in an ultrasonic oscillator for 3 minutes to eliminate any possible bubbles or trace insoluble particles that may interfere with the optical path; After the dilution is completed, take an appropriate amount of sample and place it in a cuvette for testing; Deionized water is selected as the diluent because deionized water has no absorption interference and can maintain the stability of the solution without introducing impurities. High-precision equipment and steps are used to ensure that the dilution factor is strictly controlled to avoid deviations due to operational errors. Ultrasonic treatment is used to eliminate air bubbles and particle interference, which can improve the stability and accuracy of optical analysis.

[0028] S5, mixing reagents through a reaction pool based on a microfluidic chip, measuring the absorbance of the diluted optical analysis sample, and calculating the concentration of tea polyphenols in the diluted sample; Inject the diluted sample into the microfluidic chip; The microfluidic chip is pre-designed with a micro-reaction pool, which automatically adds Folin phenol reagent and 7.5% The solution is uniformly mixed through micro-mixing channels within the chip; Use the built-in optical detection unit of the chip to measure the absorbance value of the sample at a wavelength of 765nm; The optical detection results are transmitted to the computer in real time, and the concentration of tea polyphenols in the sample is calculated according to the calibrated gallic acid standard curve and the results are output; An alternative solution is also provided: place the diluted sample in a UV spectrophotometer, set the wavelength to 280 nm and 320 nm, and record the absorbance as A_280 and A_320, respectively; Set the number of dilutions and take the average of the measured values ​​as the final absorbance. To reduce the optical measurement error, take the average of three repeated measurements as the final absorbance. Set the absorbance to , calculate the concentration of tea polyphenols in the diluted sample , the expression is: ; in, represents the linear fitting coefficient, which indicates the concentration corresponding to unit absorbance; According to the dilution multiple and the concentration of tea polyphenols in the diluted sample and the concentration of tea polyphenols in tea polyphenol solution D, calculate the retention efficiency of the sample during the dilution process, the expression is: ; in, represents the dilution retention efficiency, Indicates the degree of retention of components in tea polyphenol solution D after dilution. Indicates the concentration of tea polyphenols in the diluted sample.

[0029] S6. Calculate the final content of tea polyphenols in the tea leaves according to the concentration of tea polyphenols in the diluted sample combined with the dilution multiple and extraction volume of the optical analysis sample; According to the absorbance of the diluted sample, the dilution multiple, the volume of the diluted sample, the volume of the extract D and the mass of the initial tea sample, the final content of tea polyphenols in the tea sample is calculated as follows: ; in, Indicates the final content of tea polyphenols in tea samples. represents the volume of tea polyphenol solution D after dilution, Indicates the mass of the initial tea sample.

[0030] This embodiment also provides a tea polyphenol content determination system in tea leaves, comprising: a sample processing module, a solvent separation module, an optical analysis module, an efficiency evaluation module and a tea polyphenol content module; The sample processing module uses cellulase and pectinase to destroy the cell wall structure of tea leaves, combines hot water extraction to increase the dissolution rate of tea polyphenols, and filters to remove insoluble impurities to obtain the initial extract A; The solvent separation module uses solvents (ethanol, ethyl acetate, dilute acid) to perform multi-stage separation on the primary extract B to remove water-soluble and fat-soluble impurities; The optical analysis module dilutes the tea polyphenol solution D according to the optical detection range, measures the absorbance of the diluted sample in an ultraviolet spectrophotometer, extracts the absorbance value A at a wavelength of 280 nm, and calculates the concentration of tea polyphenols in the diluted sample using the standard curve fitting coefficient; Efficiency evaluation module, used to evaluate the tea polyphenols extraction efficiency, filtration efficiency, solvent separation efficiency and dilution retention efficiency in each step, and optimize the extraction and analysis conditions; The tea polyphenol content module is used to comprehensively consider the concentration of the diluted sample, the dilution multiple, the volume of the tea polyphenol solution D, and the mass of the initial tea sample to ultimately calculate the content of tea polyphenols in the tea sample.

[0031] This embodiment also provides a computer device, which is suitable for the method for determining the content of tea polyphenols in tea leaves, and includes: a memory and a processor; the memory is used to store computer executable instructions, and the processor is used to execute computer executable instructions to implement the method for determining the content of tea polyphenols in tea leaves proposed in the above embodiment.

[0032] The computer device may be a terminal, and the computer device includes a processor, a memory, a communication interface, a display screen and an input device connected through a system bus. Among them, the processor of the computer device is used to provide computing and control capabilities. The memory of the computer device includes a non-volatile storage medium and an internal memory. The non-volatile storage medium stores an operating system and a computer program. The internal memory provides an environment for the operation of the operating system and the computer program in the non-volatile storage medium. The communication interface of the computer device is used to communicate with an external terminal in a wired or wireless manner, and the wireless manner can be achieved through WIFI, an operator network, NFC (near field communication) or other technologies. The display screen of the computer device may be a liquid crystal display screen or an electronic ink display screen, and the input device of the computer device may be a touch layer covered on the display screen, or a key, trackball or touchpad provided on the housing of the computer device, or an external keyboard, touchpad or mouse, etc.

[0033] This embodiment also provides a storage medium on which a computer program is stored. When the program is executed by a processor, the method for determining the content of tea polyphenols in tea leaves as proposed in the above embodiment is implemented; the storage medium can be implemented by any type of volatile or non-volatile storage device or a combination thereof, such as static random access memory (Static Random Access Memory, referred to as SRAM), electrically erasable programmable read-only memory (Electrically Erasable Programmable Read-Only Memory, referred to as EEPROM), erasable programmable read-only memory (Erasable Programmable Read Only Memory, referred to as EPROM), programmable read-only memory (Programmable Red-Only Memory, referred to as PROM), read-only memory (Read-Only Memory, referred to as ROM), magnetic storage, flash memory, magnetic disk or optical disk.

[0034] In summary, the present invention achieves significant improvement in extraction efficiency by: maximizing the release of tea polyphenols through the synergistic effect of enzymatic hydrolysis and hot water; effectively removing insoluble impurities in the filtration step to ensure the quality of the extract; utilizing the selective dissolution characteristics of multi-stage organic solvents in solvent separation to successfully purify high-concentration tea polyphenols while reducing interference from impurities; optical analysis to quickly and accurately obtain the concentration of tea polyphenols by diluting the sample and accurately measuring the absorbance; combining the dilution multiple and the extraction volume to ultimately achieve accurate calculation of the content of tea polyphenols in tea leaves; the entire process optimizes the operating steps, improves efficiency and repeatability, and ensures the accuracy and reliability of the data through modular design.

[0035] Example 2, referring to Table 1, is the second example of the present invention. To further verify the technical solution of the present invention, experimental simulation data of a method for determining the content of tea polyphenols in tea leaves are provided.

[0036] The experiment selected 6 tea samples from different sources (numbered as sample 1 to sample 6), and completed the determination of tea polyphenol content through dynamic ultrasound-assisted enzymatic hydrolysis and hot water extraction, combined with filtration, solvent separation, dilution and optical analysis. The following is the experimental simulation process and data record.

[0037] Step 1: Dynamic ultrasound-assisted enzymatic hydrolysis and hot water extraction experimental data; In the experiment, 2.0 g of tea sample was taken and subjected to dynamic ultrasound-assisted enzymatic hydrolysis and hot water extraction according to the conditions in the technical solution to obtain initial extract A. The volume and tea polyphenol concentration of initial extract A are shown in the following table: Table 1 Dynamic ultrasound-assisted enzymatic hydrolysis and hot water extraction experimental data Sample No. Initial extract volume (mL) Tea polyphenols concentration in primary extract (mg / mL) Sample 1 150 4.5 Sample 2 145 4.2 Sample 3 152 4.8 Sample 4 148 4.6 Sample 5 149 4.4 Sample 6 151 4.3 Extraction efficiency Calculated using the following formula: ; Among them, g=2.0g is the mass of the tea sample, and the calculation results are as follows: Table 2 Extraction efficiency E experimental data table Sample No. Extraction efficiency 𝐸 (%) Sample 1 337.5 Sample 2 304.5 Sample 3 364.8 Sample 4 340.4 Sample 5 327.8 Sample 6 324.7 Step 2: Filter experimental data; By standing and filtering, the insoluble impurities in the initial extract A are removed to obtain tea polyphenol extract B. The volume and tea polyphenol concentration of extract B are shown in the following table: Sample No. Extraction volume (mL) Extract tea polyphenol concentration (mg / mL) Sample 1 140 4.3 Sample 2 135 4.0 Sample 3 142 4.6 Sample 4 138 4.4 Sample 5 139 4.2 Sample 6 141 4.1 Filtration efficiency Calculated using the following formula: ; The calculation results are as follows: Sample No. Filtration efficiency (%) Sample 1 89.6 Sample 2 90.0 Sample 3 90.6 Sample 4 89.8 Sample 5 88.7 Sample 6 89.3 Step 3: Solvent separation experimental data; The extract B was separated by solvent using ethanol and ethyl acetate, and further purified by combining with metal ion complexing liquid to obtain a high-purity tea polyphenol solution D. The volume and tea polyphenol concentration of solution D are shown in the following table: Sample No. High purity solution volume (mL) High purity solution tea polyphenols concentration (mg / mL) Sample 1 125 4.0 Sample 2 120 3.8 Sample 3 128 4.3 Sample 4 122 4.1 Sample 5 123 4.0 Sample 6 124 3.9 Solvent separation efficiency Calculated using the following formula: ; The calculation results are as follows: Sample No. Solvent separation efficiency (%) Sample 1 91.3 Sample 2 90.7 Sample 3 91.2 Sample 4 90.9 Sample 5 91.0 Sample 6 90.6 Step 4: Dilute the experimental data; The high-purity tea polyphenol solution D was diluted, and the dilution factor n was set to 5. The tea polyphenol concentration of the diluted sample was shown in the following table: Sample No. Tea polyphenols concentration after dilution (mg / mL) Sample 1 0.8 Sample 2 0.76 Sample 3 0.86 Sample 4 0.82 Sample 5 0.80 Sample 6 0.78 Dilution Retention Efficiency Calculated using the following formula: ; The calculation results are as follows: Sample No. Dilution retention efficiency (%) Sample 1 100 Sample 2 100 Sample 3 100 Sample 4 100 Sample 5 100 Sample 6 100 Step 5: Calculation of tea polyphenols content; According to the tea polyphenols concentration of the diluted sample, the dilution multiple, and the volume of the high-purity solution, the final content of tea polyphenols in the tea sample is calculated: ; The calculation results are as follows: Sample No. Final content of tea polyphenols (mg / g) Sample 1 500 Sample 2 456 Sample 3 550 Sample 4 512 Sample 5 492 Sample 6 483 The experimental results show that dynamic ultrasound-assisted enzymatic hydrolysis and hot water extraction combined with multi-stage purification technology significantly improved the extraction efficiency of tea polyphenols. The content of tea polyphenols in tea samples ranged from 456 to 550 mg / g, among which sample 3 had the highest content of tea polyphenols, reaching 550 mg / g. This method effectively reduced the interference of impurities and had a high extraction purity, and can be used as a standardized method for the determination of tea polyphenols content.

[0038] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for determining the content of tea polyphenols in tea, characterized in that: include: The tea polyphenols in the tea sample were dissolved into the primary extract A by dynamic ultrasound-assisted enzymatic hydrolysis and hot water extraction; Filtering the initial extract A to remove insoluble impurities, and obtaining a tea polyphenol extract B containing tea polyphenols and a small amount of soluble impurities; Using ethanol, ethyl acetate and dilute acid to perform multi-stage solvent separation on the tea polyphenol extract B, adding a multi-metal ion complexing liquid after the multi-stage solvent separation, removing water-soluble impurities through complexing reaction, and concentrating to obtain a high-purity tea polyphenol solution D; Dilute the tea polyphenol solution D as a sample for optical analysis; Reagents are mixed through a reaction pool based on a microfluidic chip, the absorbance of the diluted optical analysis sample is measured, and the concentration of tea polyphenols in the diluted sample is calculated; The final content of tea polyphenols in tea leaves is calculated based on the concentration of tea polyphenols in the diluted sample combined with the optical analysis sample dilution multiple and extraction volume.

2. The method for determining the content of tea polyphenols in tea leaves according to claim 1, wherein: The tea polyphenols in the tea sample are dissolved into the primary extract A by dynamic ultrasound-assisted enzymatic hydrolysis and hot water extraction, and the specific steps are as follows: Take a sample of dried tea leaves and crush it using a grinder until the particles pass through a mesh sieve; Add cellulase and pectinase to deionized water and adjust the pH value to the optimal activity range; The crushed tea sample is mixed with the enzymatic hydrolysate and placed in a sealed container; During the enzymatic hydrolysis process, the sealed container is placed in a dynamic ultrasonic enzymatic hydrolysis auxiliary device, the ultrasonic frequency and ultrasonic power are set, and the ultrasonic intermittent mode is used for continuous action; Stir with a magnetic stirrer to mix evenly and set the reaction time; After the enzymatic hydrolysis reaction is completed, the mixed solution is heated and the extraction time is set; After the extraction time is completed, the initial extract is collected to obtain; The initial extract is filtered through a microporous filter membrane to remove insoluble impurities, and the filtrate is collected to obtain an initial extract A containing tea polyphenols.

3. The method for determining the content of tea polyphenols in tea leaves as claimed in claim 2, characterized in that: The initial extract A is filtered to remove insoluble impurities to obtain a tea polyphenol extract B containing tea polyphenols and a small amount of soluble impurities. The specific steps are: Allow the initial extract A to stand for a period of time; Use a vacuum pump equipped with a microporous filter membrane made of polyvinylidene fluoride (PVDF); Pour the initial extract A after standing into the filter cup and start the vacuum pump for filtration; After the filtration is completed, the tea polyphenols extract B containing tea polyphenols and a small amount of soluble impurities is collected.

4. The method for determining the content of tea polyphenols in tea leaves as claimed in claim 3, characterized in that: The method uses ethanol, ethyl acetate and dilute acid to perform multi-stage solvent separation on the tea polyphenol extract B, adds a multi-metal ion complexing liquid after the multi-stage solvent separation, removes water-soluble impurities through complexing reaction, and concentrates to obtain a high-purity tea polyphenol solution D, and the specific steps are: Pour the filtered tea polyphenols extract B into a separatory funnel and add anhydrous ethanol; Thoroughly mix the tea polyphenols extract B and ethanol, then shake and let stand; After standing, separating the upper ethanol solution containing tea polyphenols and the lower aqueous phase containing water-soluble impurities, and removing the lower aqueous phase; The upper ethanol solution was collected and the ethanol was removed by a rotary evaporator to obtain an ethanol concentrate; In a separatory funnel, add the ethanol concentrate to ethyl acetate; After shaking and standing, the ethyl acetate phase and the aqueous phase were separated; Following the same method, the extraction steps were repeated; All ethyl acetate phases are combined, dried with anhydrous sodium sulfate, and then subjected to low-temperature rotary evaporation to recover ethyl acetate to obtain a purified tea polyphenols concentrate; Add multi-metal ion complex solution to the purified tea polyphenol concentrated solution, adjust the pH value of the solution, and let it stand at room temperature; after standing, filter to remove precipitated impurities, collect the filtrate and concentrate it by rotary evaporation to obtain a high-purity tea polyphenol solution D.

5. The method for determining the content of tea polyphenols in tea leaves as claimed in claim 4, characterized in that: The tea polyphenol solution D is diluted as a sample for optical analysis, specifically: The absorbance of the tea polyphenol solution D at a specific wavelength is measured and the absorbance value range is set. When the absorbance value exceeds the range, it indicates that the tea polyphenol solution D needs to be diluted. Place tea polyphenol solution D in a volumetric flask and set the dilution factor to , dilute according to the dilution factor, and calculate the required dilution factor based on the linear detection range of the optical analyzer , the expression is: ； in, Indicates the dilution factor. represents the absorbance of tea polyphenol solution D, Indicates the target The absorbance of Select deionized water as the diluent and use a high-precision pipette to add an appropriate amount of deionized water to the scale line; Use a glass rod to stir and mix to make the tea polyphenols fully and evenly dissolved; The diluted sample is placed in an ultrasonic oscillator for treatment; After the dilution is completed, take an appropriate amount of sample and place it in a cuvette for testing.

6. The method for determining the content of tea polyphenols in tea leaves as claimed in claim 5, characterized in that: The reagents are mixed by a reaction pool based on a microfluidic chip, the absorbance of the diluted optical analysis sample is measured, and the concentration of tea polyphenols in the diluted sample is calculated. The specific steps are: The diluted sample was injected into the microfluidic chip, and the Folin phenol reagent and The solution is uniformly mixed through micro-mixing channels within the chip; Use the chip's built-in optical detection unit to measure the sample's absorbance at a specific wavelength in real time; The test results are transmitted to the computer through the chip system, which automatically calculates the tea polyphenols concentration and outputs the results; Also provide alternative solutions: place the diluted sample in a UV spectrophotometer, set the wavelength, and record the absorbance separately; Set the number of dilutions and take the average of the measured values ​​as the final absorbance; Set the absorbance to , calculate the concentration of tea polyphenols in the diluted sample , the expression is: ； in, represents the linear fitting coefficient.

7. The method for determining the content of tea polyphenols in tea leaves according to claim 6, characterized in that: The final content of tea polyphenols in tea leaves is calculated according to the concentration of tea polyphenols in the diluted sample combined with the optical analysis sample dilution multiple and extraction volume. The specific steps are: According to the absorbance of the diluted sample, the dilution multiple, the volume of the diluted sample, the volume of the extract D and the mass of the initial tea sample, the final content of tea polyphenols in the tea sample is calculated as follows: ； in, Indicates the final content of tea polyphenols in tea samples. represents the volume of tea polyphenol solution D after dilution, Indicates the mass of the initial tea sample.

8. A system for determining the content of tea polyphenols in tea leaves, based on the method for determining the content of tea polyphenols in tea leaves according to any one of claims 1 to 7, characterized in that: Including, sample processing module, solvent separation module, optical analysis module, efficiency evaluation module and tea polyphenol content module; The sample processing module uses cellulase and pectinase to destroy the cell wall structure of tea leaves, combines hot water extraction to increase the dissolution rate of tea polyphenols, and filters to remove insoluble impurities to obtain a primary extract A; The solvent separation module uses a solvent to perform multi-stage separation on the primary extract B to remove water-soluble and fat-soluble impurities; The optical analysis module dilutes the tea polyphenol solution D according to the optical detection range, measures the absorbance of the diluted sample in an ultraviolet spectrophotometer, extracts the absorbance value A at a specific wavelength, and calculates the concentration of tea polyphenols in the diluted sample using a standard curve fitting coefficient; The efficiency evaluation module is used to evaluate the tea polyphenols extraction efficiency, filtration efficiency, solvent separation efficiency and dilution retention efficiency in each step, and optimize the extraction and analysis conditions; The tea polyphenol content module is used to comprehensively calculate the tea polyphenol content in the tea sample by combining the concentration of the diluted sample, the dilution multiple, the volume of the tea polyphenol solution D, and the mass of the initial tea sample.

9. A computer device comprising a memory and a processor, wherein the memory stores a computer program, wherein: When the processor executes the computer program, the steps of the method for determining the content of tea polyphenols in tea leaves according to any one of claims 1 to 7 are implemented.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed by a processor, the steps of the method for determining the content of tea polyphenols in tea leaves according to any one of claims 1 to 7 are implemented.

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