A method and device for regulating black tea fermentation based on multi-enzyme synergistic effect

Abstract

The present application relates to the technical field of tea processing, in particular to a black tea fermentation control method and equipment based on multi-enzyme synergistic effect, comprising: S1. mixing polyphenol oxidase, peroxidase and other enzymes, adding trehalose, trisodium citrate, vitamin C, etc., and using phosphate buffer solution to make up the volume, and then activating at 30 DEG C water bath to obtain a composite enzyme solution; S2. wilting fresh leaves to a moisture content of 60-65%, and then gradient rolling after UV-C intermittent irradiation to obtain rolled leaves; S3. spraying the enzyme solution at 1.5-2.0% of the weight of the rolled leaves, and then ultrasonic assisted penetration; S4. three-stage temperature control fermentation, stirring and passing ozone air every 30 minutes, and adding a pulsed magnetic field every hour; S5. detecting the conversion rate of tea polyphenols, theaflavins, etc., and terminating the fermentation when the standard is met; S6. microwave fixation after hot air treatment; S7. segment drying to a moisture content of 5-6% to obtain finished products. The present method improves the conversion efficiency of tea polyphenols and the balance of theaflavins generation in black tea, retains aroma substances, and enhances functional activity; optimizes process stability, reduces energy consumption and pollution, and helps to improve the quality and industrial benefits of black tea.

Description

Technical Field

[0001] This invention relates to the field of tea processing technology, specifically to a method and equipment for regulating the fermentation of black tea based on the synergistic effect of multiple enzymes. Background Technology

[0002] As a fully fermented tea, the core quality of black tea depends on the efficiency of enzymatic oxidation of polyphenols during fermentation, as well as the balance of characteristic components such as theaflavins and thearubigins. Although the black tea industry has now formed a large-scale production system, traditional fermentation processes still face many technical bottlenecks, making it difficult to meet the demands of high-end black tea for quality stability and functional value.

[0003] Traditional black tea fermentation relies primarily on the natural catalysis of endogenous polyphenol oxidases in fresh leaves. However, the activity of these endogenous enzymes is easily affected by factors such as leaf variety, growing environment, and degree of withering, resulting in batch-to-batch fluctuations in enzyme activity of 30%-50%. This directly leads to unstable polyphenol conversion rates—some batches have conversion rates below 35%, with theaflavins content as low as 1.2%-1.5%, while over-fermented batches have excessively high thearubigin content, resulting in a sour and musty taste in the tea soup. Furthermore, traditional fermentation employs a single "constant temperature and humidity" environmental control mode, which cannot meet the phased requirements of multiple biochemical reactions, including polyphenol oxidation, cellulose degradation, and aroma compound generation. Higher temperatures are needed in the early stages of fermentation to accelerate enzymatic reactions; precise temperature control is required in the middle stages to prevent excessive theaflavin conversion; and humidity regulation is needed in the later stages to reduce aroma loss. This single environmental parameter results in poor synergy among the reactions, making it difficult to simultaneously optimize functional components and flavor compounds.

[0004] At the level of enzyme-catalyzed reaction regulation, traditional processes lack exogenous enzyme synergistic intervention methods, and relying solely on endogenous enzymes easily leads to problems such as "insufficient enzyme activity" or "enzymatic hydrolysis imbalance." On the one hand, the endogenous cellulase activity in fresh leaves is low, and the cell wall degradation is insufficient after cell disruption, limiting the contact area between the enzyme and polyphenolic substrates and resulting in low conversion efficiency. On the other hand, the activity of coenzymes such as peroxidase and laccase during fermentation is weak, failing to effectively remove free radicals generated in the reaction, leading to an accelerated oxidation rate of theaflavins, and the loss rate of characteristic flavor substances (such as linalool and geraniol) due to oxidative degradation exceeds 40%. In addition, traditional fermentation lacks targeted enzyme activity stabilization measures, and enzyme molecules are easily denatured and aggregated under high temperature and high humidity environments. The enzyme half-life is only 24 hours, requiring frequent replenishment of enzyme solution, increasing production costs and disrupting the continuity of fermentation.

[0005] At the equipment level, existing fermentation devices are functionally limited, mostly only providing temperature and humidity control, lacking modules for adjusting key parameters such as oxygen concentration and magnetic field assistance. Fluctuations in oxygen concentration directly affect the catalytic pathway of polyphenol oxidase; too low a concentration leads to incomplete fermentation, while too high a concentration accelerates the formation of theaflavins. Without physical field assistance, the conformation of enzyme molecules cannot be regulated through external energy, further limiting the improvement of conversion efficiency. Simultaneously, traditional equipment lacks real-time monitoring and intelligent control systems, requiring frequent manual sampling and testing. This not only lags behind the fermentation process but also easily introduces contamination from other microorganisms, with microbial contamination rates reaching 5%-8%, affecting product safety.

[0006] As consumers demand higher quality black tea and the industry seeks green and efficient production, the drawbacks of traditional fermentation processes—relying on experience, difficult to control, and resulting in poor quality—are becoming increasingly apparent. There is an urgent need to develop a systematic fermentation method that integrates multi-enzyme synergy, precise multi-parameter control, and intelligent equipment support to address issues such as unstable enzyme activity, poor reaction synergy, and insufficient equipment functionality, thereby achieving targeted control of black tea quality and industrial upgrading. Summary of the Invention

[0007] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method and equipment for regulating black tea fermentation based on the synergistic effect of multiple enzymes.

[0008] (II) Technical Solution A method for regulating the fermentation of black tea based on the synergistic effect of multiple enzymes includes the following steps: S1. Preparation of the composite enzyme solution: By weight, mix 0.08-0.12 parts of polyphenol oxidase, 0.04-0.06 parts of peroxidase, 0.02-0.03 parts of laccase, 0.01-0.02 parts of cellulase, and 0.005-0.01 parts of glucose oxidase; add 0.03-0.05 parts of trehalose, 0.01-0.02 parts of trisodium citrate, and 0.003-0.005 parts of vitamin C, then add 0.002-0.004 parts of copper theanine complex and 0.001-0.003 parts of propyl gallate-cyclodextrin inclusion complex; adjust the volume to 100 parts with phosphate buffer (pH 5.0-5.5), and activate in a 30°C water bath for 15 minutes, stirring every 5 minutes, to obtain the composite enzyme solution; the chemical structure of the copper theanine complex is:

[0009] S2. Fresh leaf pretreatment: Select one bud and two leaves of black tea fresh leaves and wither them in an environment of 25-28℃ until the moisture content of the fresh leaves is 60-65%; after withering, perform UV-C irradiation treatment using an intermittent irradiation mode; then perform gradient rolling to obtain rolled leaves; S3. Enzyme solution spraying: Spray compound enzyme solution at a ratio of 1.5-2.0% of the weight of the rolled leaves. Use a fan-shaped atomizing nozzle for spraying. After spraying, perform ultrasonic-assisted penetration treatment to ensure that the enzyme solution penetrates evenly into the leaf cells. S4. Segmented fermentation: Place the rubbed leaves sprayed with enzyme solution into the fermentation device and carry out three-stage temperature-controlled fermentation; stir once every 30 minutes during the fermentation process, and at the same time, introduce sterile air containing 0.01% ozone every 30 minutes. S5. Fermentation monitoring: Samples are taken every hour to test the quality indicators of the fermented leaves. Fermentation is terminated when the conversion rate of tea polyphenols reaches 40-45%, the content of theaflavins reaches 1.8-2.2%, and the ratio of thearubigins to theaflavins reaches 8-10. S6. Enzyme activation termination: Place the fermented leaves in a hot air treatment device and treat with hot air at 95-100℃ for 3-5 minutes; immediately after treatment, microwave fixation is performed to completely deactivate the compound enzymes, and the initial product of fermented black tea is obtained. S7. Drying and Shaping: First, dry the fermented black tea at 60-65℃ for 2 hours, then dry it at 75-80℃ for 1.5 hours. Turn it over every 30 minutes during the drying process, and finally dry it until the moisture content of the black tea is 5-6% to obtain the finished black tea.

[0010] Preferably, the method further includes a step for regulating the stability of the complex enzyme solution; the activated complex enzyme solution is stored in a refrigerated environment at 4°C, and 0.001-0.002 parts of xanthan gum are added before storage and stirred until completely dissolved.

[0011] Preferably, the method also includes a step of detecting the cell fragmentation degree of the twisted leaf; take 10g of twisted leaf sample, add 50ml of deionized water, stir magnetically for 3 minutes, filter with quantitative filter paper, and collect the filtrate; use an Abbe refractometer to detect the soluble solids content in the filtrate, and control the solids concentration at 6-8%.

[0012] Preferably, the theanine copper complex in S1 is prepared by the following method: theanine and copper sulfate are dissolved in deionized water at a molar ratio of 1:1 to prepare a solution with a concentration of 0.1 mol / L; the copper sulfate solution is slowly added dropwise to the theanine solution, and the reaction is carried out in a constant temperature water bath at 35°C for 2 hours with a stirring speed of 300 rpm; after the reaction is completed, the product is subjected to vacuum freeze drying at a drying temperature of -50°C, a vacuum degree of 10 Pa, and a drying time of 24 hours; the dried solid product is pulverized by an air jet mill to control the particle size to 50-100 μm to obtain the theanine copper complex.

[0013] Preferably, a pulsed magnetic field is added to the fermentation process in S4 to assist in the treatment; a pulsed magnetic field with an intensity of 0.2-0.3T is applied once per hour, the magnetic field lasts for 5 minutes, and the direction of the magnetic field changes every 30 seconds.

[0014] Preferably, when determining the theaflavins content in S5 using high performance liquid chromatography, a ZORBAX SB-C18 column is used, with a column temperature of 30℃, a mobile phase consisting of methanol and 0.1% phosphoric acid aqueous solution mixed at a volume ratio of 40:60, a flow rate of 1.0 ml / min, an injection volume of 10 μL, a detection wavelength of 280 nm, and the theaflavins content is calculated using the external standard method.

[0015] Preferably, in S6, the microwave blanching process adopts an intermittent processing mode with a microwave power of 600W. After processing for 10 seconds, the process is paused for 5 seconds, and the cycle is repeated 3 times.

[0016] Preferably, the withering process of fresh leaves in S2 adopts an intelligent ventilation system with a ventilation rate of 1.5-2.0 m / s, each ventilation lasts for 10 minutes, with an interval of 30 minutes; the withering environment is monitored in real time by temperature and humidity sensors, and ventilation is automatically started when the local temperature exceeds 28℃.

[0017] Preferably, the oxygen concentrations in the three fermentation stages of S4 are controlled as follows: 10-12% for the first stage, 8-10% for the second stage, and 12-14% for the third stage. The oxygen concentration in the fermentation chamber is monitored in real time by an oxygen sensor. When the concentration is lower than the set lower limit, the sterile air replenishment device is activated, and the replenishment rate is automatically adjusted according to the concentration difference.

[0018] Preferably, a black tea fermentation control device for any of the above methods includes a fermentation chamber; a multi-parameter sensor group is installed inside the chamber, with temperature detection accuracy ±0.1℃, humidity detection accuracy ±1%RH, and oxygen concentration detection accuracy ±0.5%; a pulse magnetic field generator is provided, with magnetic field strength adjustable from 0.1-0.5T and magnetic field direction automatically switchable; an ultrasonic spraying system is provided, with ultrasonic frequency adjustable from 20-40kHz and 6 atomizing nozzles evenly distributed in a ring; an ozone generating module is installed, with ozone concentration continuously adjustable from 0-0.05%, and ozone is mixed with sterile air and introduced into the chamber; an intelligent control system is configured, with a built-in touch screen display, which can preset a three-stage fermentation curve, automatically execute the adjustment of temperature, humidity, oxygen concentration, and magnetic field strength parameters, and supports remote data monitoring and operation via a wireless module.

[0019] (iii) Beneficial technical effects Compared with existing technologies, the beneficial effects of this invention are: 1. By synergistically combining multiple enzymes such as polyphenol oxidase, peroxidase, and laccase in the compound enzyme solution, along with the stabilizing effect of theanine copper complex on enzyme activity, the problem of large enzyme activity fluctuations in traditional processes is effectively solved. This ensures the targeted conversion of tea polyphenols into theaflavins while inhibiting excessive theabrownin production, resulting in a bright tea liquor with a mellow and rich flavor. The addition of propyl gallate-cyclodextrin inclusion complex reduces the oxidative degradation of aroma substances, retaining more characteristic aroma components such as linalool and geraniol, thus enhancing the richness and persistence of the black tea aroma. Furthermore, the combination of segmented fermentation and pulsed magnetic field assistance further balances the ratio of theaflavins to thearubigins, avoiding quality defects caused by "over-fermentation" or "under-fermentation" in traditional processes, thereby simultaneously improving the sensory experience and functional value of the finished black tea.

[0020] 2. The addition of xanthan gum to the compound enzyme solution prolongs the enzyme's half-life, reduces the frequency of enzyme replenishment, and lowers production costs. The combination of intermittent UV-C irradiation and gradient kneading ensures cell breakage while preventing excessive oxidation of tea polyphenols, laying a good foundation for subsequent enzymatic reactions. Precise regulation of temperature, humidity, and oxygen concentration during segmented fermentation, along with the application of auxiliary methods such as pulsed magnetic fields and ozone ventilation, enables the synergistic advancement of multiple biochemical reactions, significantly shortening the fermentation cycle while reducing energy consumption. The introduction of a real-time monitoring system allows for timely detection of quality changes during fermentation, avoiding the lag and contamination risks of manual testing, and ensuring consistent product quality across each batch.

[0021] 3. The significant improvement in the quality of finished black tea gives it a higher market premium, meeting the demands of high-end consumers and helping enterprises improve their economic benefits. The application of intelligent fermentation equipment can replace traditional manual operations, reducing reliance on experienced workers, while also reducing contamination by miscellaneous bacteria, improving product safety, and meeting the requirements of green and safe production in the food industry. The combination of enzyme activity stabilization technology and multi-parameter control in the process can be adapted to different varieties and grades of fresh leaf raw materials, improving raw material utilization, reducing resource waste, providing a reliable technological paradigm for the large-scale and standardized production of black tea, and promoting the industry's transformation from "extensive processing" to "precision manufacturing." Attached Figure Description

[0022] Figure 1 This is a flowchart of a method for regulating black tea fermentation based on the synergistic effect of multiple enzymes, as disclosed in this invention. Figure 2 The above are line graphs comparing the content of theaflavins and thearubigins to theaflavins in the examples and comparative examples. Figure 3 This is a bar chart comparing the conversion rate of tea polyphenols and the retention rate of aroma substances between the examples and the comparative examples; Figure 4 This is a radar comparison chart created by comparing the quality indicators of the examples and comparative examples with the sensory indicators of the finished black tea after unifying the dimensions. Detailed Implementation

[0023] according to Figures 1 to 4 The specific embodiments of the present invention are as follows: Example

[0024] Preparation of raw materials and reagents Fresh leaves of Qimen black tea (one bud and two leaves), intact and free from pests and diseases, with a moisture content of 75%, were selected. The polyphenol oxidase activity was 6000 U / g, peroxidase activity 4000 U / g, laccase activity 2500 U / g, cellulase activity 1500 U / g, and glucose oxidase activity 1200 U / g, all of which were food grade. The theanine copper complex was prepared by the following method: theanine and copper sulfate were dissolved separately in deionized water at a 1:1 molar ratio to prepare a 0.1 mol / L solution; the copper sulfate solution was slowly added dropwise to the theanine solution, and the reaction was carried out in a constant temperature water bath at 35℃ with stirring at a speed of 300 rpm for 2 hours; after the reaction, the mixture was freeze-dried under vacuum at -50℃ and a vacuum of 10 Pa for 24 hours; the dried solid product was pulverized using an air jet mill to control the particle size at 80 μm, yielding the theanine copper complex. The inclusion molar ratio of propyl gallate-cyclodextrin inclusion complex was 1:2. Phosphate buffer pH 5.2. Trehalose, trisodium citrate, vitamin C, and xanthan gum are all food grade.

[0025] Preparation steps S1. Preparation of composite enzyme solution Weigh out 0.1 parts by weight of polyphenol oxidase, 0.05 parts of peroxidase, 0.025 parts of laccase, 0.015 parts of cellulase, and 0.008 parts of glucose oxidase and mix them. Add 0.04 parts of trehalose, 0.015 parts of trisodium citrate, 0.004 parts of vitamin C, 0.003 parts of copper theanine complex, and 0.002 parts of propyl gallate-cyclodextrin inclusion complex. Adjust the volume to 100 parts with pH 5.2 phosphate buffer. Activate in a 30°C water bath for 15 minutes, stirring every 5 minutes at 300 rpm. After activation, add 0.0015 parts of xanthan gum and refrigerate at 4°C for later use.

[0026] S2. Fresh Leaf Pretreatment Fresh leaves were spread out to a thickness of 4 cm and wilted at 26℃ with a controlled relative humidity of 70%. An intelligent ventilation system was used, with a ventilation rate of 1.8 m / s, 10 minutes per ventilation, and a 30-minute interval between ventilations. Wilting continued until the fresh leaves reached a moisture content of 62%. UV-C irradiation was then applied at a wavelength of 254 nm, a power of 15 W, and an irradiation distance of 30 cm. An intermittent irradiation mode was used, with 30 seconds of irradiation followed by a 30-second pause, repeated four times. This was followed by gradient rolling with an initial pressure of 0.3 kg / cm². 2 Maintain for 5 minutes, gradually increase to 0.5 kg / cm² and maintain for 10 minutes, finally decrease to 0.3 kg / cm². 2Maintain for 5 minutes. Sampling and cell disruption determination: Take 10g of shredded leaves, add 50ml of deionized water, stir for 3 minutes, filter with quantitative filter paper, and collect the filtrate. The soluble solids content in the filtrate was determined to be 7% and the cell disruption degree to be 72% using an Abbe refractometer.

[0027] S3. Enzyme solution spraying Apply the compound enzyme solution at a ratio of 1.8% of the weight of the rolled leaves. Use a fan-shaped atomizing nozzle, 30 cm away from the surface of the rolled leaves, with an atomized particle size of 65 μm. After spraying, perform ultrasonic-assisted penetration treatment with an ultrasonic power of 200 W, a frequency of 25 kHz, and a treatment time of 3 minutes.

[0028] S4. Segmented fermentation The rolled tea leaves sprayed with enzyme solution were placed in a black tea fermentation control device and subjected to a three-stage temperature-controlled fermentation. The first stage maintained a temperature of 29℃, relative humidity of 88%, fermentation time of 2 hours, and an oxygen concentration of 11%. The second stage maintained a temperature of 33℃, relative humidity of 82%, fermentation time of 1.5 hours, and an oxygen concentration of 9%. The third stage maintained a temperature of 27℃, relative humidity of 78%, fermentation time of 1 hour, and an oxygen concentration of 13%. During fermentation, the tea was stirred every 30 minutes at a speed of 12 rpm for 30 seconds. Simultaneously, sterile air containing 0.01% ozone was introduced every 30 minutes at a rate of 1.5 L / min for 30 seconds. A pulsed magnetic field with an intensity of 0.25T was applied every hour for 5 minutes, with the magnetic field direction alternating every 30 seconds.

[0029] S5. Fermentation Monitoring Fermented leaf quality indicators were tested every hour. The Folin-Ciocalteu method was used to determine the conversion rate of tea polyphenols, high-performance liquid chromatography (HPLC) was used to determine the theaflavins content, and spectrophotometry was used to determine the absorbance of thearubigins at 460 nm. The results after 1 hour showed a tea polyphenol conversion rate of 32%, theaflavins of 1.5%, and a thearubigin / theaflavins ratio of 7.2. After 2 hours, the results showed a tea polyphenol conversion rate of 38%, theaflavins of 1.9%, and a ratio of 8.5. After 3 hours, the results showed a tea polyphenol conversion rate of 42%, theaflavins of 2.0%, and a ratio of 9.0, reaching the preset targets, and fermentation was terminated.

[0030] S6. Enzyme activity termination The fermented leaves were placed in a hot air treatment device and treated with 98℃ hot air for 4 minutes at a wind speed of 2m / s. Immediately after treatment, microwave blanching was performed with a microwave power of 600W in an intermittent mode, with a 10-second treatment followed by a 5-second pause, repeated 3 times.

[0031] S7. Drying and Shaping The initial fermented black tea was first dried at 62℃ for 2 hours, and then at 78℃ for 1.5 hours. During the drying process, the tea was turned over every 30 minutes at a turning speed of 6 rpm for 1 minute. Finally, the tea was dried until the moisture content was 5.5%, yielding the finished black tea.

[0032] Example 2

[0033] Preparation of raw materials and reagents Fresh leaves of the Zhengshan Xiaozhong variety, consisting of one bud and two leaves, were selected. The leaves were intact and free from pests and diseases, with a moisture content of 74%. The polyphenol oxidase activity was 5000 U / g, peroxidase activity 3500 U / g, laccase activity 2000 U / g, cellulase activity 1000 U / g, and glucose oxidase activity 800 U / g, all of which were food grade. The theanine copper complex was prepared as follows: theanine and copper sulfate were dissolved separately in deionized water at a 1:1 molar ratio to prepare a 0.1 mol / L solution. The copper sulfate solution was slowly added dropwise to the theanine solution, and the reaction was carried out in a constant temperature water bath at 35℃ with stirring at a speed of 300 rpm for 2 hours. After the reaction, the mixture was freeze-dried under vacuum at -50℃ and a vacuum of 10 Pa for 24 hours. The dried solid product was pulverized using an air jet mill to control the particle size to 50 μm, yielding the theanine copper complex. The propyl gallate-cyclodextrin inclusion complex had an inclusion molar ratio of 1:2. Phosphate buffer pH 5.0. Trehalose, trisodium citrate, vitamin C, and xanthan gum are all food grade.

[0034] Preparation steps S1. Preparation of composite enzyme solution Weigh out 0.08 parts by weight of polyphenol oxidase, 0.04 parts by weight of peroxidase, 0.02 parts by weight of laccase, 0.01 parts by weight of cellulase, and 0.005 parts by weight of glucose oxidase and mix them. Add 0.03 parts by weight of trehalose, 0.01 parts by weight of trisodium citrate, 0.003 parts by weight of vitamin C, 0.002 parts by weight of copper theanine complex, and 0.001 parts by weight of propyl gallate-cyclodextrin inclusion complex. Adjust the volume to 100 parts by weight with pH 5.0 phosphate buffer. Activate in a 30°C water bath for 15 minutes, stirring every 5 minutes at a stirring speed of 300 rpm. After activation, add 0.001 parts by weight of xanthan gum and refrigerate at 4°C for later use.

[0035] S2. Fresh Leaf Pretreatment Fresh leaves were spread out to a thickness of 3 cm and wilted at 25℃ with a relative humidity of 65%. An intelligent ventilation system was used, with a ventilation rate of 1.5 m / s, 10 minutes per ventilation, and a 30-minute interval between ventilations. Wilting continued until the fresh leaves reached a moisture content of 60%. UV-C irradiation was then applied at a wavelength of 254 nm, a power of 15 W, and an irradiation distance of 30 cm. An intermittent irradiation mode was used, with 30 seconds of irradiation followed by a 30-second pause, repeated four times. This was followed by gradient rolling with an initial pressure of 0.3 kg / cm². 2Maintain for 5 minutes, then gradually increase to 0.5 kg / cm². 2 Maintain for 10 minutes, eventually dropping to 0.3 kg / cm². 2 Maintain for 5 minutes. Sampling and cell disruption determination: Take 10g of shredded leaves, add 50ml of deionized water, stir for 3 minutes, filter with quantitative filter paper, and collect the filtrate. The soluble solids content in the filtrate was determined to be 6% and the cell disruption degree to be 70% using an Abbe refractometer.

[0036] S3. Enzyme solution spraying Apply the compound enzyme solution at a ratio of 1.5% of the weight of the rolled leaves. Use a fan-shaped atomizing nozzle, 30 cm away from the surface of the rolled leaves, with atomized particles of 50 μm. After spraying, perform ultrasonic-assisted penetration treatment at a power of 200 W, a frequency of 25 kHz, and a treatment time of 3 minutes.

[0037] S4. Segmented fermentation The rolled tea leaves sprayed with enzyme solution were placed in a black tea fermentation control device for three-stage temperature-controlled fermentation. The first stage maintained a temperature of 28℃, relative humidity of 85%, fermentation time of 2 hours, and an oxygen concentration of 10%. The second stage maintained a temperature of 32℃, relative humidity of 80%, fermentation time of 1.5 hours, and an oxygen concentration of 8%. The third stage maintained a temperature of 26℃, relative humidity of 75%, fermentation time of 1 hour, and an oxygen concentration of 12%. During fermentation, the tea was stirred every 30 minutes at a speed of 12 rpm for 30 seconds. Simultaneously, sterile air containing 0.01% ozone was introduced every 30 minutes at a rate of 1.5 L / min for 30 seconds. A pulsed magnetic field with an intensity of 0.2T was applied every hour for 5 minutes, with the magnetic field direction alternating every 30 seconds.

[0038] S5. Fermentation Monitoring Fermented leaf quality indicators were tested every hour. The Folin-Ciocalteu method was used to determine the conversion rate of tea polyphenols, high-performance liquid chromatography (HPLC) was used to determine the theaflavins content, and spectrophotometry was used to determine the absorbance of thearubigins at 460 nm. After 3 hours, the results showed a tea polyphenol conversion rate of 40%, theaflavins content of 1.8%, and a thearubigin / theaflavins ratio of 8.0, meeting the preset targets, and fermentation was terminated.

[0039] S6. Enzyme activity termination The fermented leaves were placed in a hot air treatment device and treated with 95℃ hot air for 3 minutes at a wind speed of 2m / s. Immediately after treatment, microwave blanching was performed with a microwave power of 600W in an intermittent mode, with a 10-second treatment followed by a 5-second pause, repeated 3 times.

[0040] S7. Drying and Shaping The initial fermented black tea was first dried at 60℃ for 2 hours, and then at 75℃ for 1.5 hours. During the drying process, the tea was turned over every 30 minutes at a turning speed of 6 rpm for 1 minute. Finally, the tea was dried until the moisture content was 5%, yielding the finished black tea.

[0041] Example 3

[0042] Preparation of raw materials and reagents Fresh leaves of Dianhong (Yunnan black tea) with one bud and two leaves, intact and free from pests and diseases, with a moisture content of 76%, were selected. The polyphenol oxidase activity was 8000 U / g, peroxidase activity 5000 U / g, laccase activity 3000 U / g, cellulase activity 2000 U / g, and glucose oxidase activity 1500 U / g, all of which were food grade. The theanine copper complex was prepared by the following method: theanine and copper sulfate were dissolved separately in deionized water at a 1:1 molar ratio to prepare a 0.1 mol / L solution; the copper sulfate solution was slowly added dropwise to the theanine solution, and the reaction was carried out in a constant temperature water bath at 35℃ with stirring at a speed of 300 rpm for 2 hours; after the reaction, the mixture was freeze-dried under vacuum at -50℃ and a vacuum of 10 Pa for 24 hours; the dried solid product was pulverized using an air jet mill to control the particle size to 100 μm, yielding the theanine copper complex. The inclusion molar ratio of propyl gallate-cyclodextrin inclusion complex was 1:2. Phosphate buffer pH 5.5. Trehalose, trisodium citrate, vitamin C, and xanthan gum are all food grade.

[0043] Preparation steps S1. Preparation of composite enzyme solution Weigh out 0.12 parts by weight of polyphenol oxidase, 0.06 parts of peroxidase, 0.03 parts of laccase, 0.02 parts of cellulase, and 0.01 parts of glucose oxidase and mix them. Add 0.05 parts of trehalose, 0.02 parts of trisodium citrate, 0.005 parts of vitamin C, 0.004 parts of copper theanine complex, and 0.003 parts of propyl gallate-cyclodextrin inclusion complex. Adjust the volume to 100 parts with pH 5.5 phosphate buffer. Activate in a 30°C water bath for 15 minutes, stirring every 5 minutes at 300 rpm. After activation, add 0.002 parts of xanthan gum and refrigerate at 4°C for later use.

[0044] S2. Fresh Leaf Pretreatment Fresh leaves were spread out to a thickness of 5 cm and wilted at 28℃ with a relative humidity of 75%. An intelligent ventilation system was used, with a ventilation rate of 2.0 m / s, 10 minutes per ventilation, and a 30-minute interval between ventilations. Wilting continued until the fresh leaves reached a moisture content of 65%. UV-C irradiation was then applied at a wavelength of 254 nm, a power of 15 W, and an irradiation distance of 30 cm. An intermittent irradiation mode was used, with 30 seconds of irradiation followed by a 30-second pause, repeated four times. This was followed by gradient rolling with an initial pressure of 0.3 kg / cm². 2Maintain for 5 minutes, then gradually increase to 0.5 kg / cm². 2 Maintain for 10 minutes, eventually dropping to 0.3 kg / cm². 2 Maintain for 5 minutes. Sampling and cell disruption determination: Take 10g of shredded leaves, add 50ml of deionized water, stir for 3 minutes, filter with quantitative filter paper, and collect the filtrate. The soluble solids content in the filtrate was determined to be 8% and the cell disruption degree to be 75% using an Abbe refractometer.

[0045] S3. Enzyme solution spraying Apply the compound enzyme solution at a ratio of 2.0% of the weight of the rolled leaves. Use a fan-shaped atomizing nozzle, 30 cm away from the surface of the rolled leaves, with an atomized particle size of 80 μm. After spraying, perform ultrasonic-assisted penetration treatment with an ultrasonic power of 200 W, a frequency of 25 kHz, and a treatment time of 3 minutes.

[0046] S4. Segmented fermentation The rolled tea leaves sprayed with enzyme solution were placed in a black tea fermentation control device for three-stage temperature-controlled fermentation. The first stage maintained a temperature of 30℃, relative humidity of 90%, fermentation time of 2 hours, and an oxygen concentration of 12%. The second stage maintained a temperature of 34℃, relative humidity of 85%, fermentation time of 1.5 hours, and an oxygen concentration of 10%. The third stage maintained a temperature of 28℃, relative humidity of 80%, fermentation time of 1 hour, and an oxygen concentration of 14%. During fermentation, the tea was stirred every 30 minutes at a speed of 12 rpm for 30 seconds. Simultaneously, sterile air containing 0.01% ozone was introduced every 30 minutes at a rate of 1.5 L / min for 30 seconds. A pulsed magnetic field with an intensity of 0.3T was applied every hour for 5 minutes, with the magnetic field direction alternating every 30 seconds.

[0047] S5. Fermentation Monitoring Fermented leaf quality indicators were tested every hour. The Folin-Ciocalteu method was used to determine the conversion rate of tea polyphenols, high-performance liquid chromatography (HPLC) was used to determine the theaflavins content, and spectrophotometry was used to determine the absorbance of thearubigins at 460 nm. After 3 hours, the results showed a tea polyphenol conversion rate of 45%, theaflavins of 2.2%, and a thearubigin / theaflavins ratio of 10.0, meeting the preset targets, and fermentation was terminated.

[0048] S6. Enzyme activity termination The fermented leaves were placed in a hot air treatment device and treated with 100℃ hot air for 5 minutes at a wind speed of 2m / s. Immediately after treatment, microwave blanching was performed with a microwave power of 600W in an intermittent mode, with a 10-second treatment followed by a 5-second pause, repeated 3 times.

[0049] S7. Drying and Shaping The initial fermented black tea was first dried at 65℃ for 2 hours, and then at 80℃ for 1.5 hours. During the drying process, the tea was turned over every 30 minutes at a turning speed of 6 rpm for 1 minute. Finally, the tea was dried until the moisture content was 6%, yielding the finished black tea.

[0050] Comparative Example Raw materials and methods The same fresh Qimen black tea leaves as in Example 1 were selected and traditional fermentation process was used.

[0051] S1. No compound enzyme solution is prepared; it relies solely on endogenous enzymes from fresh leaves.

[0052] S2. Fresh leaves are naturally withered at an ambient temperature of 25-30℃ without humidity control until the moisture content reaches 62%. They are then directly rolled using a constant pressure of 0.4 kg / cm² for 20 minutes.

[0053] S3. Enzyme-free solution spraying procedure.

[0054] S4. Fermentation is carried out in an environment with a constant temperature of 30℃ and a relative humidity of 85% for 4.5 hours, without stirring or ozone aeration during the fermentation process.

[0055] S5. There is no real-time monitoring step; the fermentation endpoint is determined based on experience.

[0056] S6.95℃ hot air treatment for 10 minutes to terminate enzyme activity.

[0057] S7.70℃ constant temperature drying for 3 hours to a moisture content of 5-6% to obtain finished black tea.

[0058] The quality indicators of the examples and comparative examples are compared in the table below: Table 1

[0059] The sensory and functional characteristics of the finished black tea products from the examples and comparative examples are compared in the table below: Table 2

[0060] As can be seen from the two comparison tables, the polyphenol conversion rate and theaflavin content of the embodiment are significantly higher than those of the comparative example, and the thearubigin to theaflavin ratio is more reasonable, effectively avoiding the over-fermentation problem of traditional processes. Simultaneously, the aroma retention rate is significantly improved, and the microbial contamination rate is far lower than that of the comparative example. In terms of sensory and functional characteristics, the tea liquor of the embodiment has a brighter color and a higher taste score. The water extract content and free radical scavenging rate are also superior to those of the comparative example, and the quality degradation rate after 3 months of storage is only about one-third of that of the comparative example. Overall, the data fully demonstrate that this invention, through multi-enzyme synergy, segmented regulation, and intelligent equipment assistance, effectively solves the problems of unstable quality, flavor loss, and high contamination risk in traditional black tea fermentation, significantly improving the overall quality and market value of black tea.

Claims

1. A method for regulating the fermentation of black tea based on the synergistic effect of multiple enzymes, characterized in that, Includes the following steps: S1. Preparation of the composite enzyme solution: By weight, mix 0.08-0.12 parts of polyphenol oxidase, 0.04-0.06 parts of peroxidase, 0.02-0.03 parts of laccase, 0.01-0.02 parts of cellulase, and 0.005-0.01 parts of glucose oxidase; add 0.03-0.05 parts of trehalose, 0.01-0.02 parts of trisodium citrate, and 0.003-0.005 parts of vitamin C, then add 0.002-0.004 parts of copper theanine complex and 0.001-0.003 parts of propyl gallate-cyclodextrin inclusion complex; adjust the volume to 100 parts with phosphate buffer (pH 5.0-5.5), and activate in a 30°C water bath for 15 minutes, stirring every 5 minutes, to obtain the composite enzyme solution; the chemical structure of the copper theanine complex is: ； S2. Fresh leaf pretreatment: Select one bud and two leaves of black tea fresh leaves and wither them in an environment of 25-28℃ until the moisture content of the fresh leaves is 60-65%; after withering, perform UV-C irradiation treatment using an intermittent irradiation mode; then perform gradient rolling to obtain rolled leaves; S3. Enzyme solution spraying: Spray compound enzyme solution at a ratio of 1.5-2.0% of the weight of the rolled leaves. Use a fan-shaped atomizing nozzle for spraying. After spraying, perform ultrasonic-assisted penetration treatment to ensure that the enzyme solution penetrates evenly into the leaf cells. S4. Segmented fermentation: Place the rubbed leaves sprayed with enzyme solution into the fermentation device and carry out three-stage temperature-controlled fermentation; stir once every 30 minutes during the fermentation process, and at the same time, introduce sterile air containing 0.01% ozone every 30 minutes. S5. Fermentation monitoring: Samples are taken every hour to test the quality indicators of the fermented leaves. Fermentation is terminated when the conversion rate of tea polyphenols reaches 40-45%, the content of theaflavins reaches 1.8-2.2%, and the ratio of thearubigins to theaflavins reaches 8-10. S6. Enzyme activation termination: Place the fermented leaves in a hot air treatment device and treat with hot air at 95-100℃ for 3-5 minutes; immediately after treatment, microwave fixation is performed to completely deactivate the compound enzymes, and the initial product of fermented black tea is obtained. S7. Drying and Shaping: First, dry the fermented black tea at 60-65℃ for 2 hours, then dry it at 75-80℃ for 1.5 hours. Turn it over every 30 minutes during the drying process, and finally dry it until the moisture content of the black tea is 5-6% to obtain the finished black tea. Stability control steps for the compound enzyme solution: Store the activated compound enzyme solution in a refrigerated environment at 4°C. Before storage, add 0.001-0.002 parts of xanthan gum and stir until completely dissolved. Procedure for detecting cell fragmentation in twisted leaves: Take 10g of twisted leaf sample, add 50ml of deionized water, stir magnetically for 3 minutes, filter with quantitative filter paper, and collect the filtrate; use an Abbe refractometer to detect the soluble solids content in the filtrate, and control the solids concentration at 6-8%.

2. The method for regulating black tea fermentation based on multi-enzyme synergy according to claim 1, characterized in that, The theanine copper complex in S1 was prepared as follows: theanine and copper sulfate were dissolved in deionized water at a molar ratio of 1:1 to prepare a solution with a concentration of 0.1 mol / L; the copper sulfate solution was slowly added dropwise to the theanine solution, and the mixture was stirred in a constant temperature water bath at 35°C for 2 hours at a stirring speed of 300 rpm; after the reaction was completed, the mixture was freeze-dried under vacuum at a temperature of -50°C and a vacuum degree of 10 Pa for 24 hours; the dried solid product was pulverized using an air jet mill to control the particle size to 50-100 μm, thus obtaining the theanine copper complex.

3. The method for regulating black tea fermentation based on multi-enzyme synergy according to claim 1, characterized in that, In S4, a pulsed magnetic field is added to assist the fermentation process; a pulsed magnetic field with an intensity of 0.2-0.3T is applied once per hour, the magnetic field lasts for 5 minutes, and the direction of the magnetic field changes every 30 seconds.

4. The method for regulating black tea fermentation based on multi-enzyme synergy according to claim 1, characterized in that, When determining the theaflavins content in S5 using high performance liquid chromatography, a ZORBAX SB-C18 column was used at a column temperature of 30℃. The mobile phase consisted of a mixture of methanol and 0.1% phosphoric acid aqueous solution at a volume ratio of 40:

60. The flow rate was 1.0 ml / min, the injection volume was 10 μL, and the detection wavelength was 280 nm. The theaflavins content was calculated using the external standard method.

5. The method for regulating black tea fermentation based on multi-enzyme synergy according to claim 1, characterized in that, The S6 uses an intermittent microwave blanching mode with a microwave power of 600W. After processing for 10 seconds, it pauses for 5 seconds, and repeats this cycle 3 times.

6. The method for regulating black tea fermentation based on multi-enzyme synergy according to claim 1, characterized in that, The S2 uses an intelligent ventilation system for the fresh leaf wilting process, with a ventilation rate of 1.5-2.0 m / s, each ventilation lasting 10 minutes, with a 30-minute interval; the wilting environment is monitored in real time by temperature and humidity sensors, and ventilation is automatically activated when the local temperature exceeds 28℃.

7. The method for regulating black tea fermentation based on multi-enzyme synergistic effect according to claim 1, characterized in that, In S4, the oxygen concentration in the three fermentation stages is controlled as follows: 10-12% in the first stage, 8-10% in the second stage, and 12-14% in the third stage. The oxygen concentration in the fermentation chamber is monitored in real time by an oxygen sensor. When the concentration is lower than the set lower limit, the sterile air replenishment device is activated, and the replenishment rate is automatically adjusted according to the concentration difference.

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