Green tea extraction process

Abstract

According to the green tea extraction process provided by the invention, the extraction efficiency and quality of active ingredients of tea leaves are remarkably improved through combination of pulsed electric field pretreatment and a two-stage enzymolysis technology. The pulsed electric field enhances the cell permeability; cellulase and pectinase degrade cell walls in stages and release intracellular substances; as a natural activator, saccharicterpenin stabilizes chitinase activity and enhances antioxidant protection. And in the enzymolysis process, near infrared spectrum is combined to monitor the dissolution dynamic state of tea polyphenol in real time, so that the enzymolysis end point is accurately controlled, the obtained extract tea is high in active ingredient and excellent in oxidation resistance, and the batch stability is ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of green tea extraction, and particularly relates to a green tea extraction process. Background Art

[0002] In today's society, increasing public health concerns and increasing demands for food safety have fueled research into extracting natural active ingredients from tea. In particular, tea polyphenols, as key active ingredients in tea, have garnered widespread attention from both academia and industry. Tea polyphenols in green tea possess diverse biological activities, including antioxidant and anti-tumor properties, offering numerous potential health benefits. Therefore, developing efficient extraction processes to maximize the yield of active ingredients like tea polyphenols is crucial.

[0003] Traditional solvent extraction is widely used due to its simplicity and low cost, but it also suffers from low efficiency and insufficient purity. Emerging technologies such as ultrasonic extraction, pulsed electric field extraction, microwave extraction, aqueous two-phase extraction, enzyme extraction, and deep eutectic solvent extraction not only improve extraction yields and reduce energy consumption and time, but also minimize resource waste and environmental pollution. However, their high cost limits their industrial application. In the future, researchers should strive to integrate new technologies with traditional processes to reduce costs, ensure product quality, and balance energy conservation and environmental protection.

[0004] Pulsed electric field (PEF) technology uses short, high-voltage pulses to act on plant cell membranes, creating irreversible permeability through electroporation and promoting the release of intracellular active substances. Compared to traditional thermal extraction, PEF offers key advantages such as non-thermal processing, low energy consumption, shortened extraction time, and high selectivity. Enzymatic hydrolysis, on the other hand, utilizes enzymes such as cellulase and pectinase to specifically hydrolyze the polysaccharides that form the green tea cell wall, disrupting the cell wall barrier and significantly increasing the dissolution rate of active substances. Its advantages lie in its mild reaction conditions, strong substrate specificity, and environmental friendliness.

[0005] Existing research has confirmed that PEF pretreatment can enhance enzymatic hydrolysis efficiency, owing to the increased enzyme-substrate contact area achieved through electroporation. However, this combined process still faces two major bottlenecks: First, the direct addition of the enzyme complex after PEF treatment releases intracellular polyphenol oxidase, which catalyzes the oxidative polymerization of catechins and other compounds, leading to browning of the extract; second, proteases compete with polysaccharide hydrolases for binding sites, reducing the activity of the target enzyme. Therefore, ensuring both the extraction yield and enzymatic hydrolysis efficiency in this process is a key focus of this study.

[0006] There are differences in the components of green tea of ​​different varieties and batches at different times, which means that the optimal PEF-enzymatic hydrolysis parameters, such as electric field strength, enzyme ratio, and time, need to be repeatedly optimized. Traditional endpoint detection methods, such as high-performance liquid chromatography, lag and destroy samples, and cannot control the process in real time.

[0007] To address these issues, there is an urgent need to develop online monitoring methods to achieve precise control of the enzymatic hydrolysis process. Near-infrared spectroscopy (NIRS) can non-destructively and in real time measure the dynamic content of key components in the reaction system by observing changes in the absorption peaks of molecular bond vibrations.

[0008] Therefore, how to combine the PEF-enzymatic hydrolysis process with NIRS process analysis technology to achieve precise, intelligent and standardized extraction of green tea active substances, solve the problems of fuzzy reaction endpoints and uneven quality in traditional combined technologies, and provide technical support for the large-scale production of high-value-added green tea extracts is the key research direction of this invention. Summary of the Invention

[0009] The present invention discloses a green tea extraction process to solve the above-mentioned and potential problems in the prior art. In order to solve the above-mentioned technical problems, the process is as follows:

[0010] Raw material pretreatment: Wash the fresh tea leaves to remove impurities, dry them at a temperature below 40°C to retain heat-sensitive components, grind them and pass them through a 40-60 mesh sieve to obtain green tea powder;

[0011] Pulsed electric field treatment: pure water is added to the green tea powder at a solid-liquid ratio of 1:15-1:20, and pulsed electric field treatment is performed under the conditions of electric field intensity of 15-25 kV / cm, pulse number of 50-200, pulse width of 10-50 μs, frequency of 50-200 Hz, treatment time of 100-500 μs, and temperature ≤40°C;

[0012] The first stage of enzymatic hydrolysis: the mixed solution after pulsed electric field treatment was added with acetic acid solution to adjust the pH to 4.5, and 1.0% by weight of cellulase and 0.8% by weight of pectinase were added, and the mixture was reacted at 50°C and 120 rpm for 30 minutes;

[0013] The second stage of enzymatic hydrolysis: add 0.4-0.8% by weight of chitinase complex solution to continue enzymatic hydrolysis, and monitor by near-infrared spectroscopy until the enzymatic hydrolysis is completed. The total enzymatic hydrolysis time is controlled at 60-80 minutes;

[0014] Elution: After enzymatic hydrolysis is completed, the temperature is rapidly raised to 90°C and maintained for 10 minutes. Then, the mixture is cooled in an ice-water bath to below 25°C. Pure water is added and stirred for 10 minutes, and the mixture is centrifuged at 8000 rpm for 15 minutes. Acetic acid solution is added to the supernatant to adjust the pH to 3.0-3.5. The pH-adjusted supernatant is pumped into a pretreated AB-8 resin column at a flow rate of 2 BV / h. The resin column is rinsed with 5 BV of pure water to remove impurities. The target component is then eluted with 70% ethanol at a flow rate of 1 BV / h.

[0015] Freeze drying: the eluate was concentrated under reduced pressure at 40°C until there was no residual ethanol <0.5%, and freeze dried at -40°C and an air pressure of 1.5-10 Pa for 4 hours, and then the temperature was increased to 15-35°C at a rate of 1°C / min and further dried to a water content of ≤5% to obtain the green tea extract.

[0016] The chitinase complex solution is as follows: a 0.25-0.5% saccharide terpenoid solution is prepared, preheated at 45°C for 5 minutes, adjusted to pH 5.0 with acetic acid solution, chitinase is added to a content of 0.4-0.8%, magnetically stirred at 50 rpm for 5 minutes, allowed to stand at 40°C for 10 minutes, and then raised to 45°C at 1°C / min and maintained for 5 minutes to obtain a pre-activated chitinase complex solution.

[0017] The near-infrared spectroscopy monitoring includes: using a near-infrared spectroscopy fiber probe to monitor the absorbance changes of a characteristic peak in the wavelength range of 1000-2500 nm in real time, and automatically terminating the enzymatic hydrolysis when the rate of change is less than 0.1% / min. Preferably, the wavelength is 1690 nm.

[0018] The aforementioned near-infrared spectroscopy monitoring includes:

[0019] (1) Instrument configuration: Fourier transform near-infrared spectrometer equipped with an immersed fiber optic probe, an InGaAs detector, a wavelength range of 1000-2500 nm, a resolution of 8 cm⁻¹, and 64 scans per sample;

[0020] (2) Characteristic peak selection: 1690 nm was used as the main characteristic peak, supplemented by 1450 nm (OH bond secondary harmonic frequency) and 1940 nm (water molecule combination frequency) as reference calibration peaks;

[0021] (3) Real-time monitoring: Spectral data is collected every 30 seconds, and the end point of enzymatic hydrolysis is determined by the following algorithm:

[0022] a. Calculate the absorbance change rate at 1690nm ΔA / Δt=(A n -A n-1 ) / Δt, where Δt=30s;

[0023] b. Use the moving average method to smooth 5 consecutive data points;

[0024] c. When ΔA / Δt < 0.1% / min (relative rate of change) for three consecutive times, it is determined that the dissolution of tea polyphenols has reached equilibrium and the termination signal is triggered;

[0025] (4) Quality control and verification: Random samples are taken from each batch, and the EGCG content is tested by HPLC and compared with the NIR predicted value. If the deviation is >5%, the system will automatically alarm and review the spectral data.

[0026] Among them, the PLS regression model is used to predict the concentration of tea polyphenols in real time, and the model satisfies:

[0027] The optimal number of principal components is 6;

[0028] Calibration set R²≥0.982, cross-validation R²≥0.953;

[0029] Root mean square error of prediction RMSEP≤0.15%;

[0030] The relative analytical error RPD is ≥8.7.

[0031] The mathematical expression of the PLS regression model is: ;

[0032] is the predicted value of tea polyphenols concentration;

[0033] is the pre-processed spectrum matrix at 1690 nm;

[0034] is the regression coefficient matrix, optimized by minimizing the predicted residual sum of squares (PRESS):

[0035]

[0036] is the residual matrix.

[0037] represents the actual measurement value of the i-th sample;

[0038] represents the model prediction value of the i-th sample.

[0039] Model parameters (based on PLS regression modeling):

[0040] index Tea polyphenols model EGCG model Optimal number of principal components 6 8 Calibration set R² 0.982 0.961 Cross-validation R² 0.953 0.932 RMSEP(%) 0.15 0.21 RPD (Relative Analytical Deviation) 8.7 6.2

[0041] (5) Quality grading: NIR prediction value grading standard:

[0042] NIR predicted value (tea polyphenols) Quality grade Key indicator requirements >24.5% Special EGCG ≥ 12%, caffeine ≤ 5% 22.0–24.5% Level 1 EGCG 10-12%, caffeine 2.5-5.0% <22.0% Level 2 Triggering the re-inspection mechanism

[0043] (6) Data feedback optimization:

[0044] Second-level products automatically trigger re-inspection to avoid losses from misjudgment. Long-term accumulated data can be used to optimize parameters such as PEF strength and enzyme ratio.

[0045] The advantages and beneficial effects of the present invention are:

[0046] 1. This process combines pulsed electric field cell wall disruption, step-by-step enzymatic hydrolysis, and near-infrared spectroscopy monitoring technologies to achieve efficient and green extraction of tea's active ingredients. Pulsed electric field non-thermal treatment protects heat-sensitive components while improving cell membrane permeability; two-stage enzymatic hydrolysis optimizes substrate contact efficiency. A near-infrared dynamic monitoring system accurately determines the enzymatic hydrolysis endpoint, combining automated control with data feedback to form a digital production closed loop. This extraction process combines high extraction rates of tea polyphenols and EGCG with strong free radical scavenging capabilities. The entire process is precisely controllable, providing an intelligent solution for traditional tea processing.

[0047] 2. By integrating pulsed electric field pretreatment with multi-stage enzymatic hydrolysis technology, the extraction efficiency and quality of tea's active ingredients are significantly improved. During the initial treatment phase, the pulsed electric field instantly penetrates the cell membrane in a non-thermal manner, effectively enhancing cell membrane permeability while preventing the damage to heat-sensitive components caused by traditional thermal or mechanical methods. This physical treatment preemptively opens the exudation channels for water-soluble substances such as tea polyphenols and amino acids, creating favorable conditions for subsequent enzymatic hydrolysis and improving initial extraction yields.

[0048] 3. In the biological fermentation process, a two-stage strategy is adopted to achieve a step-by-step decomposition of the cell wall. In the first stage, the physical barrier of the plant cell is precisely broken down through the synergistic action of cellulase and pectinase: cellulase targets the degradation of the cellulose skeleton of the cell wall, while pectinase specifically decomposes the pectin in the intercellular layer. The step-by-step treatment not only releases most of the intracellular substances, but also provides a pure substrate environment for the efficient action of chitinase in the second stage by eliminating the enzyme competition effect. The natural activator saccharide terpenoids is introduced to pre-activate the chitinase, forming a dual protection mechanism. The triterpenoid saponins it contains stabilize the enzyme active center through hydrophobic interactions, and the carbohydrate components construct a hydrogen bond network to maintain the enzyme molecular conformation, so that the chitinase can still maintain a high relative activity in complex extraction systems. Saccharide terpenoids simultaneously enhance the chitinase activity and product antioxidant properties by stabilizing the enzyme structure and scavenging free radicals.

[0049] 4. This in-situ monitoring technology forms a dynamic closed-loop coupling with a two-stage enzymatic hydrolysis process. After pulsed electric field pretreatment, cellulase and pectinase efficiently decompose the cell wall skeleton in the first stage, resulting in a regular exponential growth in the tea polyphenol dissolution curve. During the second stage, chitinase, pre-activated by saccharoterpenoids, targets the tea polyphenols, while the near-infrared system precisely captures the inflection point in the dissolution rate, preventing catechin oxidation loss caused by excessive enzymatic hydrolysis. The two processes work together to stabilize the tea polyphenol extraction rate while reducing the endpoint response delay to less than 10 seconds. This significantly improves efficiency and reduces the risk of browning compared to traditional processes, achieving digital intelligent control.

[0050] 5. The near-infrared spectroscopy monitoring system ensures accurate endpoint control of enzymatic hydrolysis through the deep synergy of high-precision hardware and intelligent algorithms. The system utilizes a Fourier transform spectrometer with an immersed fiber optic probe to collect characteristic absorbance data of phenolic hydroxyl groups at a wavelength of 1690 nm every 30 seconds. After Savitzky-Golay smoothing and standard normal transformation preprocessing, the data is input into a quantitative model based on partial least squares regression. This model calculates the rate of change of tea polyphenol concentration in real time with a cross-validated R² ≥ 0.953 and a root mean square error of prediction ≤ 0.15%. The model automatically terminates when the rate of change after three consecutive moving averages falls below 0.1% / minute. Its relative analytical error (RPD) of ≥ 8.7 overcomes the hysteresis associated with traditional offline testing. This process tracks changes in phenolic hydroxyl concentration in real time using the characteristic 1690 nm wavelength. When the dissolution rate of tea polyphenols reaches equilibrium, the system automatically terminates the process, transitioning from enzymatic inactivation to centrifugal purification. This in-situ monitoring technology significantly reduces quality inspection time and improves prediction accuracy compared to traditional testing methods. The active substances such as tea polyphenols and EGCG in the final product show excellent biological activity, and their DPPH free radical scavenging rate is improved compared with conventional extracts.

[0051] 6. This system establishes a complete digital process from pretreatment and enzymatic hydrolysis optimization to quality control. The automated re-inspection mechanism for secondary products and the production data accumulation system mutually support each other, preventing product degradation and providing continuous data support for process parameter optimization. This technology not only achieves efficient extraction of tea's active ingredients but also, through the deep integration of physical, biological, and information technologies, enables precise processing of green tea products. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below with reference to the examples. This application uses fresh tea leaves from Wuyuan as the subject of extraction. The near-infrared spectrometer used below is a Bruker Micro NIRPAT-L, purchased from Beijing Gezhi Tongde Technology Co., Ltd., and the optical fiber probe is a Series 650 transflective probe, purchased from Thermo Fisher Scientific .

[0053] Example 1

[0054] Raw material pretreatment: 50g of fresh tea leaves were washed to remove impurities, dried at a temperature below 40°C to retain heat-sensitive components, crushed, and passed through a 50-mesh sieve to obtain green tea powder;

[0055] Pulsed electric field treatment: Pure water was added to the green tea powder at a solid-liquid ratio of 1:15-1:20, and pulsed electric field treatment was performed under the conditions of an electric field intensity of 20 kV / cm, 120 pulses, a pulse width of 40 μs, a frequency of 120 Hz, a treatment time of 300 μs, and a temperature of ≤40°C.

[0056] The first stage of enzymatic hydrolysis: the mixed solution after pulsed electric field treatment was added with acetic acid solution to adjust the pH to 4.5, and 1.0% by weight of cellulase and 0.8% by weight of pectinase were added, and the mixture was reacted at 50°C and 120 rpm for 30 minutes;

[0057] The chitinase complex solution was prepared as follows: a 0.38% saccharide terpenoid solution was prepared, preheated at 45°C for 5 minutes, adjusted to pH 5.0 with acetic acid solution, chitinase was added to a content of 0.6%, magnetically stirred at 50 rpm for 5 minutes, allowed to stand at 40°C for 10 minutes, and then raised to 45°C at a rate of 1°C / min and maintained for 5 minutes to obtain a pre-activated chitinase complex solution.

[0058] The second stage of enzymatic hydrolysis: 0.6% by weight of chitinase complex solution was added to continue enzymatic hydrolysis. The absorbance change of the characteristic peak in the wavelength range of 1000-2500 nm was monitored in real time by near-infrared spectroscopy. The enzymatic hydrolysis was terminated when the change rate was <0.1% / min. The total enzymatic hydrolysis time was 78 min.

[0059] Elution: After enzymatic hydrolysis is completed, the temperature is rapidly raised to 90°C and maintained for 10 minutes. Then, the mixture is cooled in an ice-water bath to below 25°C. Pure water is added and stirred for 10 minutes, and the mixture is centrifuged at 8000 rpm for 15 minutes. Acetic acid solution is added to the supernatant to adjust the pH to 3.2. The pH-adjusted supernatant is pumped into a pretreated AB-8 resin column at a flow rate of 2 BV / h. The resin column is rinsed with 5 BV of pure water to remove impurities. The target component is then eluted with 70% ethanol at a flow rate of 1 BV / h.

[0060] Freeze drying: the eluate was concentrated under reduced pressure at 40°C until there was no residual ethanol <0.5%, and freeze dried at -40°C and an air pressure of 6 Pa for 4 h. The temperature was then increased to 25°C at a rate of 1°C / min and further dried to a water content of 3% to obtain the green tea extract.

[0061] Example 2

[0062] Raw material pretreatment: 50g of fresh tea leaves were washed to remove impurities, dried at a temperature below 40°C to retain heat-sensitive components, crushed, and passed through a 60-mesh sieve to obtain green tea powder;

[0063] Pulsed electric field treatment: Pure water was added to the green tea powder at a solid-liquid ratio of 1:15, and pulsed electric field treatment was performed under the conditions of an electric field intensity of 25 kV / cm, 50 pulses, a pulse width of 50 μs, a frequency of 50 Hz, a treatment time of 500 μs, and a temperature of ≤40°C.

[0064] The first stage of enzymatic hydrolysis: the mixed solution after pulsed electric field treatment was added with acetic acid solution to adjust the pH to 4.5, and 1.0% by weight of cellulase and 0.8% by weight of pectinase were added, and the mixture was reacted at 50°C and 120 rpm for 30 minutes;

[0065] The chitinase complex solution is as follows: a 0.25% saccharide terpenoid solution is prepared, preheated at 45°C for 5 minutes, adjusted to pH 5.0 with acetic acid solution, chitinase is added to a content of 0.8%, magnetic stirring is performed at 50 rpm for 5 minutes, and the solution is first allowed to stand at 40°C for 10 minutes, then raised to 45°C at a rate of 1°C / min and maintained for 5 minutes to obtain a pre-activated chitinase complex solution.

[0066] The second stage of enzymatic hydrolysis: 0.4% by weight of chitinase complex solution was added to continue enzymatic hydrolysis. The absorbance change of the characteristic peak in the wavelength range of 1000-2500 nm was monitored in real time by near-infrared spectroscopy. The enzymatic hydrolysis was terminated when the change rate was <0.1% / min. The total enzymatic hydrolysis time was 60 min.

[0067] Elution: After enzymatic hydrolysis is completed, the temperature is rapidly raised to 90°C and maintained for 10 minutes. Then, the mixture is cooled in an ice-water bath to below 25°C. Pure water is added and stirred for 10 minutes, and the mixture is centrifuged at 8000 rpm for 15 minutes. Acetic acid solution is added to the supernatant to adjust the pH to 3.0. The pH-adjusted supernatant is pumped into a pretreated AB-8 resin column at a flow rate of 2 BV / h. The resin column is rinsed with 5 BV of pure water to remove impurities. The target component is then eluted with 70% ethanol at a flow rate of 1 BV / h.

[0068] Freeze drying: the eluate was concentrated under reduced pressure at 40°C until there was no residual ethanol <0.5%, and freeze dried at -40°C and an air pressure of 10 Pa for 4 h. The temperature was then increased to 15°C at a rate of 1°C / min and further dried to a water content of 5% to obtain the green tea extract.

[0069] Example 3

[0070] Raw material pretreatment: 50g of fresh tea leaves were washed to remove impurities, dried at a temperature below 40°C to retain heat-sensitive components, crushed, and passed through a 40-mesh sieve to obtain green tea powder;

[0071] Pulsed electric field treatment: Pure water was added to the green tea powder at a solid-liquid ratio of 1:20, and pulsed electric field treatment was performed under the conditions of an electric field intensity of 15 kV / cm, 200 pulses, a pulse width of 10 μs, a frequency of 200 Hz, a treatment time of 100 μs, and a temperature of ≤40°C.

[0072] The first stage of enzymatic hydrolysis: the mixed solution after pulsed electric field treatment was added with acetic acid solution to adjust the pH to 4.5, and 1.0% by weight of cellulase and 0.8% by weight of pectinase were added, and the mixture was reacted at 50°C and 120 rpm for 30 minutes;

[0073] The chitinase complex solution is as follows: prepare a 0.5% saccharide terpenoid solution by mass, preheat at 45°C for 5 minutes, adjust the pH to 5.0 with acetic acid solution, add chitinase to make its content 0.4%, stir magnetically at 50 rpm for 5 minutes, let stand at 40°C for 10 minutes, then increase the temperature to 45°C at 1°C / min and maintain for 5 minutes to obtain a pre-activated chitinase complex solution.

[0074] The second stage of enzymatic hydrolysis: 0.8% by weight of chitinase complex solution was added to continue enzymatic hydrolysis. The absorbance change of the characteristic peak in the wavelength range of 1000-2500 nm was monitored in real time by near-infrared spectroscopy. The enzymatic hydrolysis was terminated when the change rate was <0.1% / min. The total enzymatic hydrolysis time was 80 min.

[0075] Elution: After enzymatic hydrolysis is completed, the temperature is rapidly raised to 90°C and maintained for 10 minutes. Then, the mixture is cooled in an ice-water bath to below 25°C. Pure water is added and stirred for 10 minutes, and the mixture is centrifuged at 8000 rpm for 15 minutes. Acetic acid solution is added to the supernatant to adjust the pH to 3.5. The pH-adjusted supernatant is pumped into a pretreated AB-8 resin column at a flow rate of 2 BV / h. The resin column is rinsed with 5 BV of pure water to remove impurities. The target component is then eluted with 70% ethanol at a flow rate of 1 BV / h.

[0076] Freeze drying: the eluate was concentrated under reduced pressure at 40°C until there was no residual ethanol <0.5%, and freeze dried at -40°C and an air pressure of 1.5 Pa for 4 h. The temperature was then increased to 35°C at a rate of 1°C / min and further dried to a water content of 4% to obtain the green tea extract.

[0077] Comparative Example 1

[0078] The difference between this comparative example and Example 1 is that the process of this comparative example is as follows:

[0079] Raw material pretreatment: 50g of fresh tea leaves were washed to remove impurities, dried at a temperature below 40°C to retain heat-sensitive components, crushed, and passed through a 50-mesh sieve to obtain green tea powder;

[0080] Pulsed electric field treatment: Pure water was added to the green tea powder at a solid-liquid ratio of 1:15-1:20, and pulsed electric field treatment was performed under the conditions of an electric field intensity of 20 kV / cm, 120 pulses, a pulse width of 40 μs, a frequency of 120 Hz, a treatment time of 300 μs, and a temperature of ≤40°C.

[0081] Enzyme hydrolysis: The mixed solution after pulsed electric field treatment was added with acetic acid solution to adjust the pH to 4.5, and 1.0% by weight of cellulase, 0.8% by weight of pectinase, and 0.6% by weight of chitinase were added. The reaction was stirred at 50°C and 120 rpm. The absorbance change of the characteristic peak in the wavelength range of 1000-2500 nm was monitored in real time by near-infrared spectroscopy. The enzymatic hydrolysis was terminated when the rate of change was <0.1% / min. The total enzymatic hydrolysis time was 78 min.

[0082] Elution: After enzymatic hydrolysis is completed, the temperature is rapidly raised to 90°C and maintained for 10 minutes. Then, the mixture is cooled in an ice-water bath to below 25°C. Pure water is added and stirred for 10 minutes, and the mixture is centrifuged at 8000 rpm for 15 minutes. Acetic acid solution is added to the supernatant to adjust the pH to 3.2. The pH-adjusted supernatant is pumped into a pretreated AB-8 resin column at a flow rate of 2 BV / h. The resin column is rinsed with 5 BV of pure water to remove impurities. The target component is then eluted with 70% ethanol at a flow rate of 1 BV / h.

[0083] Freeze drying: the eluate was concentrated under reduced pressure at 40°C until there was no residual ethanol <0.5%, and freeze dried at -40°C and an air pressure of 6 Pa for 4 h. The temperature was then increased to 25°C at a rate of 1°C / min and further dried to a water content of 3% to obtain the green tea extract.

[0084] Comparative Example 2

[0085] The difference between this comparative example and Example 1 is that the specific process of this comparative example is as follows:

[0086] Raw material pretreatment: 50g of fresh tea leaves were washed to remove impurities, dried at a temperature below 40°C to retain heat-sensitive components, crushed, and passed through a 50-mesh sieve to obtain green tea powder;

[0087] Pulsed electric field treatment: Pure water was added to the green tea powder at a solid-liquid ratio of 1:15-1:20, and pulsed electric field treatment was performed under the conditions of an electric field intensity of 20 kV / cm, 120 pulses, a pulse width of 40 μs, a frequency of 120 Hz, a treatment time of 300 μs, and a temperature of ≤40°C.

[0088] The chitinase complex solution was prepared as follows: a 0.38% saccharide terpenoid solution was prepared, preheated at 45°C for 5 minutes, adjusted to pH 5.0 with acetic acid solution, chitinase was added to a content of 0.6%, magnetically stirred at 50 rpm for 5 minutes, allowed to stand at 40°C for 10 minutes, and then raised to 45°C at a rate of 1°C / min and maintained for 5 minutes to obtain a pre-activated chitinase complex solution.

[0089] Enzyme hydrolysis: The mixed solution after pulsed electric field treatment was added with acetic acid solution to adjust the pH to 4.5, and 1.0% by weight of cellulase, 0.8% by weight of pectinase, and 0.6% by weight of chitinase complex were added. The reaction was stirred at 50°C and 120 rpm. The absorbance change of the characteristic peak in the wavelength range of 1000-2500 nm was monitored in real time by near-infrared spectroscopy. The enzymatic hydrolysis was terminated when the rate of change was <0.1% / min. The total enzymatic hydrolysis time was 76 min.

[0090] Elution: After enzymatic hydrolysis is completed, the temperature is rapidly raised to 90°C and maintained for 10 minutes. Then, the mixture is cooled in an ice-water bath to below 25°C. Pure water is added and stirred for 10 minutes, and the mixture is centrifuged at 8000 rpm for 15 minutes. Acetic acid solution is added to the supernatant to adjust the pH to 3.2. The pH-adjusted supernatant is pumped into a pretreated AB-8 resin column at a flow rate of 2 BV / h. The resin column is rinsed with 5 BV of pure water to remove impurities. The target component is then eluted with 70% ethanol at a flow rate of 1 BV / h.

[0091] Freeze drying: the eluate was concentrated under reduced pressure at 40°C until there was no residual ethanol <0.5%, and freeze dried at -40°C and an air pressure of 6 Pa for 4 h. The temperature was then increased to 25°C at a rate of 1°C / min and further dried to a water content of 3% to obtain the green tea extract.

[0092] Comparative Example 3

[0093] The difference between this comparative example and Example 1 is that in this comparative example, saccharitrenin is replaced by Tween 80; the rest is the same as Example 1.

[0094] Comparative Example 4

[0095] The difference between this comparative example and Example 1 is that in this comparative example, the chitinase complex solution is prepared as follows: a 1.0% saccharide terpenoid solution is prepared, preheated at 45°C for 5 minutes, adjusted to pH 5.0 with acetic acid solution, chitinase is added to a content of 0.6%, magnetic stirring is performed at 50 rpm for 5 minutes, the temperature is first allowed to stand at 40°C for 10 minutes, and then raised to 45°C at 1°C / min and maintained for 5 minutes to obtain a pre-activated chitinase complex solution; the rest is the same as in Example 1.

[0096] Comparative Example 5

[0097] The difference between this comparative example and Example 1 is that in this comparative example, the chitinase complex solution is prepared as follows: a 0.1% saccharide terpenoid solution is prepared, preheated at 45°C for 5 minutes, adjusted to pH 5.0 with acetic acid solution, chitinase is added to a content of 0.6%, magnetic stirring is performed at 50 rpm for 5 minutes, the temperature is first allowed to stand at 40°C for 10 minutes, and then raised to 45°C at 1°C / min and maintained for 5 minutes to obtain a pre-activated chitinase complex solution; the rest is the same as in Example 1.

[0098] Test 1: Active ingredient detection and verification

[0099] With reference to GB / T8313-2002, the tea polyphenol content was determined by ultraviolet spectrophotometry. The enzymatically hydrolyzed extract was used for determination. The determination principle is: tea polyphenols and ferrous ions form a blue-purple complex in the solution. The absorbance of the complex is used to determine the tea polyphenol content and calculate the extraction rate. The extraction rate formula for tea polyphenols is:

[0100] TP(%)=Tea polyphenols product quality / tea quality

[0101] EGCG content detection: According to the HPLC method in GB / T22244-2008 standard, the chromatographic conditions are: C18 column, mobile phase of methanol-water-phosphoric acid (30:70:0.1), and detection wavelength of 280 nm.

[0102] Caffeine content: According to GB / T8312-2013, HPLC method, conditions: C18 column, mobile phase methanol-water (3:7), detection wavelength 274nm.

[0103] The results are shown in Table 1 below:

[0104] Table 1

[0105] Group Tea polyphenols content (mg / g) EGCG content (mg / g) Caffeine content (mg / g) Example 1 308.47 151.20 45.78 Example 2 305.13 147.52 43.61 Example 3 294.84 145.69 42.80 Comparative Example 1 221.56 87.45 21.47 Comparative Example 2 247.92 121.08 32.49 Comparative Example 3 234.49 113.42 25.87 Comparative Example 4 293.07 141.59 41.08 Comparative Example 5 267.15 130.83 36.54

[0106] Test 2: Determination of antioxidant activity

[0107] (1) Determination of DPPH free radical scavenging ability:

[0108] The specific operation is as follows: use anhydrous ethanol to prepare a concentration of 1.0x10 -4 mol DPPH solution, take the green tea extracts of Examples 1-3 and Comparative Examples 1-5, prepare a solution with a concentration of 50 μg / mL, take 100 μL of each and mix with an equal volume of DPPH solution, incubate in the dark at 25°C for 30 minutes, and measure the absorbance at 517 nm. Anhydrous ethanol is used as a blank control. The DPPH inhibition rate (%) is calculated using the following formula:

[0109] I DPPH (%) = [(A control -A sample ) / A control ]*100

[0110] In the formula: I DPPH Refers to the DPPH free radical scavenging rate (%); A control Refers to the absorbance of the blank control group; A sample Refers to the absorbance of the sample solution.

[0111] (2) Determination of ABTS free radical scavenging ability

[0112] First, prepare the ABTS working solution as follows: prepare 7.0mM ABTS solution and 2.45mM potassium persulfate solution with ultrapure water, mix the two in a 1:1 volume ratio, and store the reagent in the dark. The solution is then diluted 50 times with ethanol to obtain an ABTS working solution with an absorbance of 0.70. Take 20μL of 50μg / mL green tea extract solution and add it to 180μL of ABTS working solution, incubate in the dark at 25°C for 30min, and detect the absorbance at 734nm. Use an equal amount of anhydrous ethanol (20μL) as a blank control, and calculate the inhibition rate (%) of ABTS using the formula:

[0113] I ABTS (%) = [(A control -A sample ) / A control ]*100

[0114] In the formula: I ABTS Refers to the ABTS free radical scavenging rate (%); A control Refers to the absorbance of the blank control group; A sample Refers to the absorbance of the sample solution.

[0115] The experimental data are all based on the average value of three replicates of the parallel experiment to represent the final experimental results. The results are shown in Table 2 below.

[0116] Table 2

[0117] Group DPPH clearance rate / % ABTS clearance / % Example 1 79.48 92.81 Example 2 78.41 91.47 Example 3 76.25 89.06 Comparative Example 1 50.24 61.73 Comparative Example 2 64.31 75.38 Comparative Example 3 58.27 71.94 Comparative Example 4 75.09 88.76 Comparative Example 5 71.42 82.52

Claims

1. A green tea extraction process, characterized in that: The process is: Raw material pretreatment: Wash the fresh tea leaves to remove impurities, dry them at a temperature below 40°C to retain heat-sensitive components, grind them and pass them through a 40-60 mesh sieve to obtain green tea powder; Pulsed electric field treatment: pure water is added to the green tea powder at a solid-liquid ratio of 1:15-1:20, and pulsed electric field treatment is performed under the conditions of electric field intensity of 15-25 kV / cm, pulse number of 50-200, pulse width of 10-50 μs, frequency of 50-200 Hz, treatment time of 100-500 μs, and temperature ≤40°C; The first stage of enzymatic hydrolysis: the mixed solution after pulsed electric field treatment was added with acetic acid solution to adjust the pH to 4.5, and 1.0% by weight of cellulase and 0.8% by weight of pectinase were added, and the mixture was reacted at 50°C and 120 rpm for 30 minutes; The second stage of enzymatic hydrolysis: add 0.4-0.8% by weight of chitinase complex solution to continue enzymatic hydrolysis, and monitor by near-infrared spectroscopy until the enzymatic hydrolysis is completed. The total enzymatic hydrolysis time is controlled at 60-80 minutes; Elution: After enzymatic hydrolysis is completed, the temperature is rapidly raised to 90°C and maintained for 10 minutes. Then, the mixture is cooled in an ice-water bath to below 25°C. Pure water is added and stirred for 10 minutes, and the mixture is centrifuged at 8000 rpm for 15 minutes. Acetic acid solution is added to the supernatant to adjust the pH to 3.0-3.

5. The pH-adjusted supernatant is pumped into a pretreated AB-8 resin column at a flow rate of 2 BV / h. The resin column is rinsed with 5 BV of pure water to remove impurities. The target component is then eluted with 70% ethanol at a flow rate of 1 BV / h. Freeze drying: the eluate was concentrated under reduced pressure at 40°C until there was no residual ethanol <0.5%, and freeze dried at -40°C and an air pressure of 1.5-10 Pa for 4 hours, and then the temperature was increased to 15-35°C at a rate of 1°C / min and further dried to a water content of ≤5% to obtain the green tea extract.

2. The process according to claim 1, characterized in that: The chitinase complex solution is prepared by preparing a 0.25-0.5% saccharide terpenoid solution by mass, preheating at 45° C. for 5 minutes, adjusting the pH to 5.0 with acetic acid solution, adding chitinase to a content of 0.4-0.8%, magnetically stirring at 50 rpm for 5 minutes, first standing at 40° C. for 10 minutes, then heating to 45° C. at 1° C. / min and maintaining for 5 minutes to obtain a preactivated chitinase complex solution.

3. The process according to claim 1, characterized in that The near-infrared spectroscopy monitoring is to terminate the enzymatic hydrolysis by monitoring the wavelength absorbance change in real time through a near-infrared spectroscopy optical fiber probe.

4. The process according to claim 3, characterized in that The changes in the absorbance of the characteristic peak in the wavelength range of 1000-2500nm are monitored in real time by a near-infrared spectroscopy fiber optic probe.

5. The process according to claim 4, characterized in that in, The wavelength is 1690nm.

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