Bidirectional liquid fermentation method for preparing tea extract and application of bidirectional liquid fermentation method

Abstract

The invention relates to the technical field of fermentation, and particularly discloses a two-way liquid fermentation method for preparing a tea extract and application of the two-way liquid fermentation method. According to the method, firstly, the tea leaves are subjected to plasma pretreatment, the bright green color and nutritional ingredients of the green tea are reserved, and the content of EGCG in the tea leaf extract can be increased; furthermore, the dried tea leaves are subjected to gradient crushing and grading treatment, so that the initial dissolution rate of EGCG and other core components in the tea leaf extract is increased by 30% or above; in addition, the mixed material is subjected to synergistic fermentation by adopting a composite strain of lactic acid bacteria and saccharomycetes, the synergistic effect can deeply convert the components of the tea leaf extract, the tea leaf extract can be induced to generate more novel active substances, novel effects are exerted, and meanwhile, the extraction rate of EGCG is remarkably increased. By adopting the two-way liquid state fermentation method, the content of EGCG in the tea leaf extract can be increased, and the extraction rate and purity of EGCG are remarkably improved.

Description

Technical Field

[0001] This application relates to the field of fermentation technology, and in particular to a two-way liquid fermentation method for preparing tea extract and its application. Background Technology

[0002] Different types of tea will exhibit different aromas. The aroma components of tea mainly include alcohols, aldehydes, esters, ketones, heterocyclic compounds, etc., and the main aroma types are sweet, floral, fruity, honey, roasted, etc. However, the compounds that present these aromas are usually very unstable and are easily affected by environmental factors such as oxygen, light, and heat, resulting in oxidation, polymerization, condensation and other reactions, which leads to unnatural aroma and loss of reducing power in tea extracts.

[0003] Tea extract, with its rich content of active ingredients such as tea polyphenols and catechins, is widely used in skin care products, personal care products and cosmetics. It mainly plays a role in anti-oxidation, anti-inflammation, whitening and anti-aging, and is one of the important raw materials of natural plant extracts in the daily chemical industry.

[0004] The extraction technology of tea extracts has been continuously evolving, presenting a diversified landscape. From liquid-liquid extraction based on simple solubility differences, to column chromatography and low-to-medium pressure column chromatography utilizing the principle of substance distribution between the stationary and mobile phases, to high-pressure preparative liquid chromatography that achieves efficient separation and purification using high pressure, and high-speed countercurrent chromatography that uses centrifugal force to form a phase system for separation, each technology has been continuously optimized and developed in specific aspects, providing multiple options for the extraction of tea extracts and promoting technological progress and application expansion in the field of tea extract extraction.

[0005] While the above extraction methods have the advantages mentioned above, they also have the following drawbacks: liquid-liquid extraction is simple to operate, but requires a large amount of extractant, involves multiple extraction operations which are cumbersome and result in significant losses, and consumes a lot of energy for organic solvent recovery; column chromatography has low extraction efficiency and poor extraction effect on low-content components; medium and low-pressure column chromatography has limited ability to handle complex systems; high-pressure preparative liquid chromatography equipment and operating costs are high; high-speed countercurrent chromatography has a low mobile phase flow rate, high time cost, and complex instruments, making it difficult to operate. Summary of the Invention

[0006] The purpose of this application is to overcome the shortcomings of the prior art and provide a two-way liquid fermentation method for preparing tea extract and its application. Compared with traditional methods, this application uses a two-way liquid fermentation method to prepare tea extract, which can increase the EGCG content in the tea extract and significantly improve the extraction rate and purity of EGCG.

[0007] To achieve the above objectives, the technical solution adopted in this application is as follows: This application provides a two-way liquid fermentation method for preparing tea extract, comprising the following steps: S1. The tea leaves are pretreated with plasma, then dried, and subjected to gradient pulverization and grading to obtain tea powder; then the tea powder is mixed with deionized water to obtain a mixture. S2. Inoculate the bacterial culture of the compound strain of lactic acid bacteria and yeast into the mixture in step S1 for fermentation to obtain fermentation broth; S3. Centrifuge the fermentation broth from step S2, collect the supernatant and ultrafilter it to obtain a concentrated liquid; add an extractant to the concentrated liquid and rotary evaporate it to obtain crude tea extract, then purify it to obtain tea extract.

[0008] In this application, the tea leaves are first subjected to a combination of plasma pretreatment and gradient pulverization and grading. The plasma pretreatment kills microorganisms (such as bacteria and fungi) on the surface of the tea leaves through the oxygen / nitrogen free radicals and ions generated by the plasma, while degrading any pesticide residues that may be present, reducing allergens. The low temperature characteristic avoids the damage of heat-sensitive components such as chlorophyll and tea polyphenols caused by high temperatures, thus preserving the bright green color and nutritional components of green tea. At the same time, the gradient pulverization and grading process allows the core components such as EGCG in the tea extract to dissolve better, which helps to increase the content of epigallocatechin gallate (EGCG) in the tea extract.

[0009] This application employs a composite strain of lactic acid bacteria and yeast for the synergistic fermentation of mixed materials. This synergistic fermentation utilizes a multi-probiotic combination system, forming a metabolic cascade between different strains. The metabolites of one strain become nutrient substrates for another. Lactic acid bacteria metabolites (such as glucose and amino acids) are key nutrient substrates for yeast, supporting its enzyme secretion and growth; yeast metabolites (such as enzymes and ethanol) act as environmental regulators and substrate precursors for lactic acid bacteria, promoting acid production and EGCG protection. The two strains establish a mutually dependent and highly efficient metabolic model. This synergistic effect can deeply transform the components of tea extract, inducing the production of more novel active substances and exerting novel efficacy, while significantly improving the extraction rate of EGCG. Using the two-way liquid fermentation method of this application, the EGCG content in tea extract can be increased, significantly improving the extraction rate and purity of EGCG.

[0010] Compared with traditional methods, the two-phase liquid fermentation method provided in this application can improve fermentation efficiency and product yield; at the same time, it protects heat-sensitive active ingredients (such as EGCG) from degradation, improving product yield and stability; and during the two-phase liquid fermentation process, the plant obtains nutrients such as polysaccharides, proteins, and vitamins through fermentation, which can provide nutrition for the growth and metabolism of fermenting bacteria. Simultaneously, the fermenting bacteria can help the plant produce more active substances through microbial cell biotransformation, producing a "two-way" synergistic effect. This application uses the two-phase liquid fermentation method to prepare tea extract, and then the tea extract can be purified to obtain EGCG.

[0011] In a preferred embodiment of the two-way liquid fermentation method for preparing tea extract described in this application, the plasma pretreatment in step S1 includes: The tea leaves were pretreated under conditions of 12~13.6 kV voltage, 20~30℃ temperature, and 40~60% humidity for 10~20 minutes.

[0012] This application involves low-temperature plasma pretreatment of tea leaves, which can kill microorganisms on the surface of the tea leaves, degrade potential pesticide residues, reduce allergens, better preserve the bright green color and nutrients of the tea leaves, and increase the EGCG content in the tea extract.

[0013] Preferably, the drying temperature is 40~50℃, the drying time is 2~3 hours, and the moisture content of the dried tea is 3~5%.

[0014] In a preferred embodiment of the two-way liquid fermentation method for preparing tea extract described in this application, step S1, the gradient pulverization and classification process includes: The dried tea leaves are crushed in a jaw crusher at a speed of 500-800 r / min for 5-8 minutes until they are crushed to 20-40 mesh, thus obtaining the first grade of tea. The first-grade tea leaves are ultra-finely pulverized using an airflow mill at an inlet pressure of 0.6~0.8 MPa and a pulverizing pressure of 0.3~0.5 MPa, until the tea leaves are pulverized to 70~120 mesh, thus obtaining the second-grade tea leaves. The second-grade tea leaves are placed in a cyclone grading machine with an air intake of 30-50m³. 3 The tea leaves are screened at a grading frequency of 20-30Hz to obtain uniform particles with a mesh size of 80-140 mesh, thus obtaining the third-grade tea.

[0015] The two-way liquid fermentation method of this application abandons the single crushing mode and adopts a gradient process of "coarse crushing-ultrafine crushing and grading". First, the crushed tea leaves are crushed to 20-40 mesh by a jaw crusher to destroy the surface structure of the tea leaves. Then, an air jet mill is used for ultrafine crushing to 70-120 mesh, controlling the particle size distribution of the first-grade tea leaves to 1000μm, so that the cell wall breakage rate is increased to more than 85%. Finally, the particles are graded by a cyclone classifier to uniformly grade the tea leaves to a mesh size of 80-140 mesh, avoiding the problem of uneven mixing caused by fine powder agglomeration.

[0016] In a preferred embodiment of the two-way liquid fermentation method for preparing tea extract described in this application, in step S1, the mass ratio of deionized water to tea powder is 1:(1.5~5).

[0017] In this application, the above-mentioned mass ratio of deionized water and tea powder provides a favorable moisture environment for microorganisms, ensuring the dissolution and transport of nutrients, which is beneficial to their growth and metabolism. Simultaneously, a suitable mass ratio ensures good fluidity and mass transfer properties in the fermentation system, promoting sufficient contact between microorganisms and the substrate and improving fermentation efficiency. If the mass ratio is too low, the fermentation system becomes too viscous, making nutrient transport difficult, limiting microbial growth, and reducing the amount of fermentation products produced. Furthermore, a viscous system hinders oxygen dissolution and transfer, further affecting the metabolic activities of aerobic microorganisms. In addition, a low mass ratio may lead to difficulty in heat dissipation during fermentation, causing localized temperature increases and affecting microbial activity. If the mass ratio is too high, nutrient dilution and osmotic pressure imbalance will affect microbial growth, reduce product concentration, increase impurities affecting quality, prolong the fermentation cycle, increase energy consumption and reduce efficiency, and also increase the difficulty of subsequent separation and purification and the burden of wastewater treatment.

[0018] In a preferred embodiment of the two-way liquid fermentation method for preparing tea extract described in this application, the mass ratio of lactic acid bacteria to yeast in step S2 is 1:(1~2).

[0019] This application employs synergistic fermentation of lactic acid bacteria and yeast within the aforementioned mass ratio range. This creates a metabolic cascade between different bacterial species, where the metabolites of one species become nutrient substrates for another, constructing a mutually dependent and highly efficient metabolic model. This synergistic effect can deeply transform the components of tea extract, inducing the production of more novel active substances and exerting new therapeutic effects, while significantly improving the extraction rate of EGCG.

[0020] In a preferred embodiment of the two-way liquid fermentation method for preparing tea extract described in this application, the inoculation mass ratio of the bacterial solution in step S2 is 2-5%.

[0021] Using bacterial solutions within the above-mentioned inoculation range can significantly improve the extraction rate and purity of EGCG.

[0022] In a preferred embodiment of the two-way liquid fermentation method for preparing tea extract described in this application, the fermentation temperature in step S2 is 31~33℃. And / or, the fermentation speed is 175~185 rpm; And / or, the aeration rate during fermentation is 1.2~1.4 vvm (the ratio of the volume of air introduced per minute per cubic meter of fermentation liquid to the volume of fermentation liquid).

[0023] This application utilizes the aforementioned fermentation temperature range. Under suitable temperature conditions, microorganisms grow vigorously, the fermentation cycle is reasonable, and the precursor substances in tea leaves can be fully converted into the target product, improving product quality and yield while ensuring the stability of the fermentation process. When the temperature is below the aforementioned range, the enzyme activity of microorganisms decreases, the metabolic rate slows down, and growth is inhibited. This leads to a prolonged fermentation cycle, and microorganisms may not be able to fully decompose the substrate, resulting in reduced product yield and decreased quality. Excessively high temperatures will cause protein denaturation and enzyme inactivation within the microorganisms, and may even lead to microbial death. This not only prevents the fermentation process from proceeding normally but may also produce harmful substances, affecting the quality and safety of the product.

[0024] Furthermore, for aerobic microbial fermentation, a suitable dissolved oxygen level (aeration rate of 1.2~1.4 vvm) ensures that microorganisms have sufficient oxygen for respiration, generating enough energy to support their growth and metabolic activities. Sufficient oxygen promotes the decomposition and transformation of organic matter in tea leaves by microorganisms, increasing the yield and quality of fermentation products. When dissolved oxygen is insufficient (aeration rate <1.0 vvm), the growth and metabolism of aerobic microorganisms are restricted, and they may switch to anaerobic respiration or fermentation pathways, producing byproducts such as lactic acid and ethanol. These byproducts affect the quality of the fermentation products, prolong the fermentation cycle, and reduce product yield.

[0025] In a preferred embodiment of the two-way liquid fermentation method for preparing tea extract described in this application, in step S2, the pH of the fermentation broth is adjusted to 4.0~4.5.

[0026] This application utilizes the aforementioned pH range, which allows microorganisms to normally absorb nutrients and carry out efficient metabolic activities, converting components in tea leaves into the target product and ensuring the high quality and stability of the fermentation product. When the pH value is below the aforementioned range, the acidity within the microbial cells increases, potentially inactivating some enzymes and affecting the microbial metabolic pathways. Simultaneously, cell membrane permeability may also change, leading to difficulties in nutrient absorption and hindered secretion of metabolic products. This will reduce the quality and yield of the fermentation product and may even lead to fermentation failure. Conversely, an excessively high pH value increases the alkalinity within the microbial cells, similarly affecting enzyme activity and cell membrane function. Microbial growth and metabolism are inhibited, resulting in reduced fermentation product production and decreased quality.

[0027] In this application, by precisely controlling the fermentation conditions, including temperature, pH, and dissolved oxygen levels (by setting the aeration rate to 1.2~1.4 vvm), the quality and stability of the fermentation product are improved, the risk of contamination by other microorganisms is reduced, and an optimal environment for microbial growth and metabolism is provided, ensuring the efficient and stable fermentation process. Compared with traditional fermentation methods, the fermentation cycle of this application is shortened, the quality and stability of the tea extract in the product are significantly improved, and the purity of EGCG is significantly increased.

[0028] In some specific embodiments, the solvents for adjusting the pH of the fermentation broth include a sodium hydroxide solution with a mass concentration of 1 mol / L and a hydrochloric acid solution with a mass concentration of 1 mol / L.

[0029] During the fermentation process, samples were taken every 6 to 12 hours. The content of EGCG in the fermentation broth was detected by high performance liquid chromatography, and the growth of microorganisms was detected by microscopic observation and dilution plating method.

[0030] Preferably, in step S3, the centrifugation speed is 8000~10000 r / min, and the centrifugation time is 15~20 min; And / or, the molecular weight cutoff of the ultrafiltration is 10,000 to 50,000; the pressure is 0.2 to 0.5 MPa.

[0031] The above-mentioned centrifugation conditions can remove large particulate impurities and bacterial cell debris. After collecting the supernatant, it is filtered through an ultrafiltration membrane with a molecular weight cutoff of 10,000 to 50,000 at a pressure of 0.2 to 0.5 MPa to remove small molecule impurities and some proteins to obtain a concentrated solution.

[0032] In a preferred embodiment of the two-way liquid fermentation method for tea extract described in this application, the extractant in step S3 includes ethyl acetate; And / or, the volume ratio of the concentrate to the extractant is 1:(1~2); And / or, in step S3, rotary evaporation is carried out at a temperature of 40~50℃ and a vacuum degree of 0.08~0.09 MPa.

[0033] This application also includes a step of purifying the tea extract (containing EGCG): The crude tea extract obtained above was further purified. First, silica gel column chromatography was used, with 200-300 mesh silica gel as the stationary phase and chloroform-methanol (volume ratio 9:1-7:3) as the eluent for gradient elution. The elution flow rate was controlled at 1-2 mL / min, and eluent was collected every 10-20 mL. Thin-layer chromatography (TLC) was used to monitor the components in the eluent, and the eluents containing EGCG were combined. The combined eluent was concentrated by rotary evaporation and further purified by high-performance liquid chromatography (HPLC). A C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was used, with acetonitrile-water (volume ratio 15:85-25:75, containing 0.1% formic acid) as the mobile phase, the flow rate controlled at 1.0 mL / min, and the detection wavelength at 280 nm. The eluent corresponding to the chromatographic peaks with high EGCG purity was collected, and the mobile phase was removed by concentration using a rotary evaporator to obtain a high-purity EGCG product. The EGCG purity was found to be over 99%.

[0034] This application uses the above-mentioned two-way liquid fermentation method for preparing tea extract to prepare tea extract.

[0035] The tea extract provided in this application contains fermented lactic acid (≥0.10 mg / mL), propyl gallate (≥3 μg / mL), octyl gallate (≥2.0 μg / mL), tryptophan derivative (≥1.0 μg / mL), quercetin-3-O-rutin glycoside (≥1.5 μg / mL), and quercetin-3-O-glucuronide (≥3 μg / mL).

[0036] Furthermore, the crude tea extract can contain up to 21.0% EGCG, and the purity of the EGCG sample can reach 99.8%.

[0037] This application uses a two-way liquid fermentation method to prepare tea extract. The two-way interaction between the plant and the microorganism can induce the tea extract to produce more novel active substances, exert better anti-inflammatory and oil-controlling effects, and significantly improve the extraction rate and stability of EGCG. It is suitable for anti-aging and soothing repair products for sensitive skin.

[0038] This application also provides the application of the above-mentioned two-way liquid fermentation method for preparing tea extract in improving the content and purity of epigallocatechin gallate.

[0039] The two-way liquid fermentation system of this application places tea extract and a bacterial solution containing a complex of lactic acid bacteria and yeast in a closed liquid environment for fermentation. This two-way liquid fermentation system combines the advantages of liquid fermentation, such as uniform nutrient distribution, high microbial metabolic efficiency, and strong controllability of process parameters, and can also promote the bidirectional transformation between tea and microorganisms. The tea extract not only provides nutrients for fungi, but also changes its own structural composition and produces new effects under the influence of fungal metabolites and cytokines, which can improve the utilization rate of components and reduce waste. In addition, compared with solid-state fermentation, this system avoids the problems of open systems being prone to contamination by other microorganisms and the difficulty in controlling the fermentation endpoint and quality, making it more suitable for large-scale industrial production.

[0040] Compared with the prior art, this application has the following beneficial effects: This application provides a two-way liquid fermentation method for preparing tea extract and its application. First, the tea leaves undergo plasma pretreatment to retain their vibrant green color and nutritional components, which helps increase the content of epigallocatechin gallate (EGCG) in the tea extract. Further, the dried tea leaves are subjected to gradient pulverization and grading, increasing the initial dissolution rate of core components such as EGCG in the tea extract. This application also employs a composite strain of lactic acid bacteria and yeast for synergistic fermentation of the mixture. This synergistic fermentation utilizes a multi-probiotic combination system, forming a metabolic cascade relationship between different strains. The metabolites of one strain become nutrient substrates for another, constructing a mutually dependent and highly efficient metabolic model. This synergistic effect can deeply transform the components of the tea extract, inducing the production of more novel active substances and exerting novel effects, while significantly improving the extraction rate of EGCG. Using the two-way liquid fermentation method of this application, the EGCG content in tea extract can be increased, significantly improving the extraction rate and purity of EGCG. Attached Figure Description

[0041] Figure 1 High-performance liquid chromatography chromatogram of EGCG in tea extract; Figure 2 The images show the facial redness reduction effect of Application Example 3 and Comparative Application Example 1. Detailed Implementation

[0042] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0043] In the following examples and comparative examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Furthermore, the raw materials used in each parallel experiment are the same.

[0044] Example 1: A two-way liquid fermentation method for preparing tea extract This embodiment provides a two-way liquid fermentation method for tea extract, including the following steps: S1. Select fresh green tea leaves free from pests, diseases, and impurities. After screening, weigh 100g and spread it evenly on a tray. Vacuum pack and seal. Start the low-temperature plasma reaction equipment for preheating for 10 minutes. Place the vacuum-packed tea leaves into the reactor. Use air as the working gas. Set the discharge voltage to 12kV, the processing time to 10 minutes, the gas flow rate to 2L / min, and the distance between the electrode and the tea leaves to 10mm. Simultaneously control the ambient temperature to 20℃ and the humidity to 40%. Monitor the temperature and humidity in real time during the process. After pretreatment, remove the tea leaves and place them in a 40℃ drying oven for 2 hours until the moisture content reaches 3%, obtaining the dried tea leaves. After drying, the tea leaves are crushed in a jaw crusher at 500 rpm for 5 minutes until they reach a mesh size of 20, thus breaking down the outer skin structure and obtaining the first-grade tea. The first-grade tea is then fed into an air jet mill for ultrafine grinding at an inlet air pressure of 0.6 MPa and a grinding pressure of 0.3 MPa until it reaches a mesh size of 70, achieving a cell wall breakage rate of over 85%, thus obtaining the second-grade tea. The second-grade tea is then passed through a cyclone classifier at an inlet air volume of 30 m³ / min. 3 The tea leaves were screened at a grading frequency of 20Hz to obtain uniform particles with a mesh size of 80 mesh, resulting in the third grade tea. Deionized water and the third grade tea were then thoroughly mixed at a mass ratio of 1:1.5 and stirred at 150 rpm for 15 minutes in a mixer to ensure the raw materials and water were fully infiltrated, forming a homogeneous mixture.

[0045] S2. Select a rigorously screened and cultivated compound microbial strain, composed of lactic acid bacteria and yeast in a 1:1 ratio. On a sterile operating table, inoculate the compound microbial strain into the treated mixture at an inoculation mass ratio of 2% (v / v). Quickly transfer the inoculated mixture to a fermenter equipped with agitation and aeration devices. Set the fermentation temperature to 31℃ and maintain this temperature precisely using a constant temperature control system. The agitator continuously stirs at 175 rpm. The dissolved oxygen level of the fermentation system is controlled by adjusting the aeration rate, which is set to 1.2 vvm (the ratio of air volume introduced per cubic meter of fermentation broth per minute to the volume of fermentation broth). Simultaneously, use acid-base adjusters (1 mol / L sodium hydroxide solution and 1 mol / L hydrochloric acid solution) to control the pH of the fermentation system, maintaining a pH of 4.3 to obtain the fermentation broth. During the fermentation process, samples were taken every 6 to 12 hours. The content of EGCG in the fermentation broth was detected by high performance liquid chromatography, and the growth of microorganisms was detected by microscopic observation and dilution plating method.

[0046] S3. After fermentation, the fermentation broth is centrifuged at 8000 r / min for 15 minutes to remove large particulate impurities and bacterial cell debris. The supernatant is collected and filtered through an ultrafiltration membrane with a molecular weight cutoff of 10000-50000 at a pressure of 0.2 MPa to remove small molecule impurities and some proteins, thus obtaining a concentrated solution. Ethyl acetate is added to the concentrated solution at a volume ratio of 1:1, shaken for 5 minutes, and allowed to stand for 30-60 minutes to separate the layers. The extraction is repeated twice, and the organic phases are combined. Finally, the solvent is removed by rotary evaporation at 40℃ and a vacuum of 0.08 MPa to obtain a crude tea extract containing EGCG.

[0047] S4. The crude tea extract underwent further purification. First, silica gel column chromatography was used, with 200-mesh silica gel as the stationary phase and a gradient elution of chloroform-methanol (9:1, v / v) as the eluent. The elution flow rate was controlled at 1 mL / min, and eluent was collected every 10 mL. Thin-layer chromatography (TLC) was used to monitor the components in the eluent, and the eluents containing EGCG were combined. The combined eluents were concentrated using a rotary evaporator and further purified using high-performance liquid chromatography (HPLC). A C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was used, with acetonitrile-water (15:85, v / v, containing 0.1% formic acid) as the mobile phase, the flow rate controlled at 1.0 mL / min, and the detection wavelength at 280 nm. The eluent corresponding to the chromatographic peak with high EGCG purity was collected, and the mobile phase was removed by rotary evaporation to obtain the EGCG product.

[0048] Example 2: A two-way liquid fermentation method for preparing tea extract This embodiment provides a two-way liquid fermentation method for tea extract, including the following steps: S1. Select fresh green tea leaves free from pests, diseases, and impurities. After screening, weigh 100g and spread it evenly on a tray. Vacuum pack and seal. Start the low-temperature plasma reaction equipment for preheating for 15 minutes. Place the vacuum-packed tea leaves into the reactor. Use air as the working gas. Set the discharge voltage to 13.6kV, the processing time to 20 minutes, the gas flow rate to 2L / min, and the distance between the electrode and the tea leaves to 20mm. Simultaneously control the ambient temperature to 30℃ and the humidity to 60%. Monitor the temperature and humidity in real time during the process. After pretreatment, remove the tea leaves and place them in a 50℃ drying oven to dry for 3 hours until the moisture content reaches 5%, obtaining the dried tea leaves. After drying, the tea leaves are crushed in a jaw crusher at 800 r / min for 8 minutes until they reach a mesh size of 40, thus breaking down the outer skin structure and obtaining the first-grade tea. The first-grade tea is then fed into an air jet mill for ultrafine grinding at an inlet air pressure of 0.8 MPa and a grinding pressure of 0.5 MPa until it reaches a mesh size of 120, achieving a cell wall breakage rate of over 85%, thus obtaining the second-grade tea. The second-grade tea is then passed through a cyclone classifier at an inlet air volume of 50 m³ / min. 3The tea leaves were screened at a grading frequency of 30Hz to obtain uniform particles with a mesh size of 140 mesh, resulting in the third-grade tea. Deionized water was then thoroughly mixed with the third-grade tea at a mass ratio of 1:4, and stirred in a mixer at 200 rpm for 20 minutes to ensure complete immersion of the raw materials in water, forming a homogeneous mixture.

[0049] S2. Select a rigorously screened and cultivated compound microbial strain, composed of lactic acid bacteria and yeast in a 1:2 ratio. On a sterile operating table, inoculate the compound microbial strain into the treated mixture at an inoculation mass ratio of 5% (v / v). Quickly transfer the inoculated mixture to a fermenter equipped with agitation and aeration devices. Set the fermentation temperature to 33℃ and maintain this temperature precisely using a constant temperature control system. The agitator continuously stirs at 185 rpm. The dissolved oxygen level of the fermentation system is controlled by adjusting the aeration rate, which is set to 1.4 vvm (the ratio of air volume introduced per cubic meter of fermentation broth per minute to the volume of fermentation broth). Simultaneously, use acid-base adjusters (1 mol / L sodium hydroxide solution and 1 mol / L hydrochloric acid solution) to control the pH of the fermentation system, maintaining the pH at 4.5 to obtain the fermentation broth. During the fermentation process, samples were taken every 6 to 12 hours. The content of EGCG in the fermentation broth was detected by high performance liquid chromatography, and the growth of microorganisms was detected by microscopic observation and dilution plating method.

[0050] S3. After fermentation, the fermentation broth was centrifuged at 10,000 r / min for 20 min to remove large particulate impurities and bacterial cell debris. The supernatant was collected and filtered through an ultrafiltration membrane with a molecular weight cutoff of 10,000-50,000 at a pressure of 0.5 MPa to remove small molecule impurities and some proteins, thus obtaining a concentrated solution. Ethyl acetate was added to the concentrated solution at a volume ratio of 1:2, shaken for 10 min, and allowed to stand for 30-60 minutes to separate the layers. The extraction was repeated 3 times and the organic phases were combined. Finally, the solvent was removed by rotary evaporation at 50℃ and a vacuum of 0.09 MPa to obtain a crude tea extract containing EGCG.

[0051] S4. The crude tea extract underwent further purification. First, silica gel column chromatography was used, with 300-mesh silica gel as the stationary phase and a gradient elution of chloroform-methanol (7:3, v / v) as the eluent. The elution flow rate was controlled at 2 mL / min, and eluent was collected every 20 mL. Thin-layer chromatography (TLC) was used to monitor the components in the eluent, and the eluents containing EGCG were combined. The combined eluents were concentrated using a rotary evaporator and then further purified using high-performance liquid chromatography (HPLC). A C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was used, with acetonitrile-water (25:75, v / v, containing 0.1% formic acid) as the mobile phase, the flow rate controlled at 1.0 mL / min, and the detection wavelength at 280 nm. The eluent corresponding to the chromatographic peak with higher EGCG purity was collected, and the mobile phase was removed by rotary evaporation to obtain the EGCG product.

[0052] Example 3: A two-way liquid fermentation method for preparing tea extract This embodiment provides a two-way liquid fermentation method for tea extract, including the following steps: S1. Select fresh green tea leaves free from pests, diseases, and impurities. After screening, weigh 100g and spread it evenly on a tray. Vacuum pack and seal. Start the low-temperature plasma reaction equipment for preheating for 15 minutes. Place the vacuum-packed tea leaves into the reactor. Use air as the working gas. Set the discharge voltage to 13kV, the processing time to 15 minutes, the gas flow rate to 2L / min, and the distance between the electrode and the tea leaves to 15mm. Simultaneously control the ambient temperature to 25℃ and the humidity to 50%. Monitor the temperature and humidity in real time during the process. After pretreatment, remove the tea leaves and place them in a 45℃ drying oven for 2.5 hours until the moisture content reaches 4%, obtaining the dried tea leaves. The dried tea leaves are crushed in a jaw crusher at 700 r / min for 6 minutes until they reach a mesh size of 30, thus breaking down the outer skin structure and obtaining the first-grade tea. The first-grade tea is then fed into an air jet mill for ultrafine grinding at an inlet air pressure of 0.7 MPa and a grinding pressure of 0.4 MPa until it reaches a mesh size of 100, achieving a cell wall breakage rate of over 85%, thus obtaining the second-grade tea. The second-grade tea is then passed through a cyclone classifier at an inlet air volume of 40 m³ / min. 3 The tea leaves were screened at a grading frequency of 25Hz to obtain uniform particles with a mesh size of 120, resulting in the third grade tea. Deionized water was then thoroughly mixed with the third grade tea at a mass ratio of 1:3, and stirred in a mixer at 180 rpm for 18 minutes to ensure complete immersion of the raw materials in water, forming a homogeneous mixture.

[0053] S2. Select a rigorously screened and cultivated compound microbial strain, composed of lactic acid bacteria and yeast in a 1:1.5 ratio. On a sterile operating table, inoculate the compound microbial strain into the treated mixture at an inoculation mass ratio of 3.5% (v / v). Quickly transfer the inoculated mixture to a fermenter equipped with agitation and aeration devices. Set the fermentation temperature to 32℃ and maintain this temperature precisely using a constant temperature control system. The agitator continuously stirs at 180 rpm. The dissolved oxygen level of the fermentation system is controlled by adjusting the aeration rate, which is set to 1.3 vvm (the ratio of air volume introduced per cubic meter of fermentation broth per minute to the volume of fermentation broth). Simultaneously, use acid-base adjusters (1 mol / L sodium hydroxide solution and 1 mol / L hydrochloric acid solution) to control the pH of the fermentation system, maintaining the pH at 4.3 to obtain the fermentation broth. During the fermentation process, samples were taken every 8 hours, and the EGCG content in the fermentation broth was detected by high performance liquid chromatography. At the same time, the growth of microorganisms was detected by microscopic observation and dilution plating method.

[0054] S3. After fermentation, the fermentation broth was centrifuged at 9000 r / min for 18 min to remove large particulate impurities and bacterial cell debris. The supernatant was collected and filtered through an ultrafiltration membrane with a molecular weight cutoff of 30000 at a pressure of 0.35 MPa to remove small molecule impurities and some proteins, thus obtaining a concentrated solution. Ethyl acetate was added to the concentrated solution at a volume ratio of 1:1.5, shaken for 8 min, and allowed to stand for 30-60 minutes to separate the layers. The extraction was repeated 2-3 times and the organic phases were combined. Finally, the solvent was removed by rotary evaporation at 45℃ and a vacuum of 0.085 MPa to obtain a crude tea extract containing EGCG.

[0055] S4. The crude tea extract underwent further purification. First, silica gel column chromatography was used, with 250-mesh silica gel as the stationary phase and a gradient elution of chloroform-methanol (8:2, v / v) as the eluent. The elution flow rate was controlled at 1.5 mL / min, and eluent was collected in 15 mL increments. Thin-layer chromatography (TLC) was used to monitor the components in the eluent, and the eluents containing EGCG were combined. The combined eluents were concentrated using a rotary evaporator and then further purified using high-performance liquid chromatography (HPLC). A C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was used, with acetonitrile-water (20:80, v / v, containing 0.1% formic acid) as the mobile phase, the flow rate controlled at 1.0 mL / min, and the detection wavelength at 280 nm. The eluent corresponding to the chromatographic peak with high EGCG purity was collected and freeze-dried to obtain the EGCG product.

[0056] Example 4: A two-way liquid fermentation method for preparing tea extract Compared with Example 3, the method of Example 4 differs in that the fermentation temperature in step S2 is different, and the fermentation temperature is 25°C. The remaining steps are the same as those in Example 3.

[0057] Example 5: A two-way liquid fermentation method for preparing tea extract Compared with Example 3, the method of Example 5 differs in that the fermentation temperature in step S2 is different, and the fermentation temperature is 40°C. The remaining steps are the same as those in Example 3.

[0058] Example 6: A two-way liquid fermentation method for preparing tea extract Compared with Example 3, the method of Example 6 differs in that the ventilation rate in step S2 is different, with a ventilation rate of 0.9 vvm. The remaining steps are the same as those in Example 3.

[0059] Example 7: A two-way liquid fermentation method for preparing tea extract Compared with Example 3, the method of Example 7 differs in that the ventilation rate in step S2 is different, and the ventilation rate is 1.8 vvm. The other steps are the same as those in Example 3.

[0060] Example 8: A two-way liquid fermentation method for preparing tea extract Compared with Example 3, the method of Example 8 differs in that the pH value in step S2 is different, with a pH value of 6.0, while the other steps are the same as in Example 3.

[0061] Example 9: A two-way liquid fermentation method for preparing tea extract Compared with Example 3, the method of Example 9 differs in that the pH value in step S2 is different, with a pH value of 7.5, while the other steps are the same as in Example 3.

[0062] Example 10: A two-way liquid fermentation method for preparing tea extract Compared with Example 3, the method of Example 10 differs in that step S1 is different, adopting a two-stage process, while the remaining steps are the same as in Example 3.

[0063] Step S1 is: Fresh green tea leaves free from pests, diseases, and impurities were selected. After screening, 100g of the leaves were weighed and spread evenly on a tray, then vacuum-sealed. The low-temperature plasma reactor was preheated for 15 minutes. The vacuum-sealed tea leaves were then placed into the reactor, using air as the working gas. The discharge voltage was set to 13kV, the processing time to 15 minutes, the gas flow rate to 2L / min, and the distance between the electrode and the tea leaves to 15mm. The ambient temperature was controlled at 25℃ and the humidity at 50%, with real-time monitoring of temperature and humidity during the process. After pretreatment, the tea leaves were removed and placed in a 45℃ drying oven for 2.5 hours until the moisture content reached 4%, yielding the dried tea leaves.

[0064] Take the dried tea leaves and crush them in a jaw crusher at 700 r / min for 6 minutes until they are pulverized to 30 mesh, thus destroying their skin structure and obtaining the first grade tea. Pass the first grade tea into a cyclone classifier with an air intake of 40 m³ / min. 3 The tea leaves were screened at a grading frequency of 25Hz to obtain uniform particles with a mesh size of 100 mesh, resulting in the second-grade tea. Deionized water and the second-grade tea were then thoroughly mixed at a mass ratio of 1:3. The mixture was stirred in a mixer at 180 rpm for 18 minutes to ensure complete immersion of the raw materials in water, forming a homogeneous mixture.

[0065] Example 11: A two-way liquid fermentation method for preparing tea extract Compared with Example 3, the method of Example 11 is different in that the compound strain in step S2 is composed of lactic acid bacteria and yeast in a ratio of 1:0.5, and the remaining steps are the same as in Example 3.

[0066] Example 12: A two-way liquid fermentation method for preparing tea extract Compared with Example 3, the method of Example 12 is different in that the compound strain in step S2 is composed of lactic acid bacteria and yeast in a ratio of 1:5, and the remaining steps are the same as in Example 3.

[0067] Example 13: A two-way liquid fermentation method for preparing tea extract Compared with Example 3, the method of Example 13 differs in that step S1 is different, the mesh counts of the first-grade tea, the second-grade tea, and the third-grade tea are different, and the remaining steps are the same as those of Example 3.

[0068] Step S1 is as follows: Fresh green tea leaves free from pests, diseases, and impurities were selected. After screening, 100g of the leaves were weighed and spread evenly on a tray, then vacuum-sealed. The low-temperature plasma reactor was preheated for 15 minutes. The vacuum-sealed tea leaves were then placed into the reactor, using air as the working gas. The discharge voltage was set to 13kV, the processing time to 15 minutes, the gas flow rate to 2L / min, and the distance between the electrode and the tea leaves to 15mm. The ambient temperature was controlled at 25℃ and the humidity at 50%, with real-time monitoring of temperature and humidity during the process. After pretreatment, the tea leaves were removed and placed in a 45℃ drying oven for 2.5 hours until the moisture content reached 4%, yielding the dried tea leaves.

[0069] The dried tea leaves are crushed in a jaw crusher at 700 r / min for 6 minutes until they reach 60 mesh, thus breaking down their outer skin structure and obtaining the first-grade tea. The first-grade tea is then fed into an air jet mill for ultrafine grinding at an inlet pressure of 0.7 MPa and a grinding pressure of 0.4 MPa, until it reaches 140 mesh, achieving a cell wall breakage rate of over 85%, thus obtaining the second-grade tea. The second-grade tea is then passed through a cyclone classifier at an inlet air volume of 40 m³ / min.3 The tea leaves were screened at a grading frequency of 25Hz to obtain uniform particles with a mesh size of 160 mesh, resulting in the third-grade tea. Deionized water was then thoroughly mixed with the third-grade tea at a mass ratio of 1:3, and stirred in a mixer at 180 rpm for 18 minutes to ensure complete immersion of the raw materials in water, forming a homogeneous mixture.

[0070] Comparative Example 1 Compared with Example 3, Comparative Example 1 differs in step S1, where the tea leaves are not pretreated. Specifically, the tea leaves are placed in a 45°C drying oven and dried for 2.5 hours until the moisture content reaches 4%, thus obtaining dried tea leaves. The remaining steps are the same as in Example 3.

[0071] Comparative Example 2 Compared with Example 3, Comparative Example 2 differs in that step S1 is different; instead of gradient pulverization and grading, the dried tea leaves are pulverized to a mesh size of 120. The processed tea leaves are then thoroughly mixed with deionized water and stirred in a mixer at 180 rpm for 18 minutes to ensure the raw materials are fully soaked in water and form a homogeneous mixture. The remaining steps are the same as in Example 3.

[0072] Comparative Example 3 Compared with Example 3, the difference in Comparative Example 3 is that step S2 is different, and the compound bacterial strain is lactic acid bacteria and Bacillus. The remaining steps are the same as in Example 3.

[0073] Comparative Example 4 Compared with Example 3, the difference in Comparative Example 4 is that step S2 is different, the compound strain is acetic acid bacteria and yeast, and the remaining steps are the same as in Example 3.

[0074] Comparative Example 5 Compared with Example 3, Comparative Example 5 differs in that step S2 is different; instead of using a compound bacterial strain, only lactic acid bacteria are used. The inoculation mass ratio remains unchanged, while the remaining steps are the same as in Example 3.

[0075] Comparative Example 6 Compared with Example 3, Comparative Example 6 differs in that step S2 is different; instead of using a compound strain, only yeast is used, while the inoculation mass ratio remains unchanged. The remaining steps are the same as in Example 3.

[0076] Example 1: Identification of EGCG-containing metabolites in crude tea extract by high-resolution mass spectrometry (UPLC-TOF-MS / MS) thing The crude EGCG-containing tea extracts prepared in Examples 1-13 were reconstituted and then separated using a UPLC system (ACQUITY HSS T3 column, acetonitrile-0.1% formic acid gradient elution). TOF-MS / MS was then used to acquire primary and secondary mass spectrometry data in positive and negative ion modes (scanning range m / z 50-1200, collision energy gradient 15-45 eV). Finally, the data were processed using software (peak extraction, molecular formula prediction, fragment matching), and new metabolites were screened by differential analysis with the unfermented control group. The structures were verified using standards.

[0077] Results: By comparing the mass spectrometry data of the unfermented control group and the fermented sample, 5-15 new metabolites may have been identified. These components were not detected in unfermented tea leaves or were present at levels below the mass spectrometry detection limit (e.g., <0.1 μg / mL), but increased significantly after fermentation (e.g., >1 μg / mL).

[0078] The unfermented control group (based on each example) was as follows: Replace step S2 with the following steps: The mixture prepared in step S1 (tea powder and deionized water mixed at a mass ratio of 1:3) is transferred to a water extraction tank equipped with stirring and temperature control. The water extraction temperature is set to 85°C and is precisely maintained by the constant temperature control system. The stirring paddle is continuously stirred at a speed of 120 rpm to fully dissolve the effective components of the tea. The water extraction time is 60 minutes.

[0079] The specific results are shown in Table 1.

[0080] Table 1 Experimental Example 2: EGCG Content in Tea Extract and Verification of EGCG Purity by High Performance Liquid Chromatography (HPLC) Determination of EGCG content in tea extract: Weigh and record the weight of the crude tea extract. Preliminary enrichment of the crude tea extract was performed by silica gel column chromatography and thin-layer chromatography, followed by fine separation by high performance liquid chromatography. The eluent was collected and freeze-dried to obtain high-purity EGCG product. Weigh and record the weight of EGCG.

[0081] EGCG content (%) = EGCG weight / crude tea extract weight × 100%.

[0082] EGCG purity was verified by high performance liquid chromatography (HPLC): Column: ODS column, 5 μm particle size, 4.6 × 250 mm; Mobile phase: acetonitrile: ethyl acetate: water (0.05% H3PO4) = 86:12:2 (V / V), pH = 3.0; Flow rate: 1.0 mL / min; Column temperature: 24℃; Detection wavelength: 276 nm; Injection volume: 10 μL.

[0083] Sample preparation: Weigh the EGCG products prepared in Examples 1-13 and Comparative Examples 1-6, dissolve them in the mobile phase, vortex mix, and then sonicate to degas for 10 minutes. Filter using a 0.22 μm filter membrane to avoid particle clogging of the chromatographic column.

[0084] Injection analysis: Simultaneously inject EGCG standard (known purity ≥98%) and sample solution, and compare retention time and peak shape. Perform 3-5 consecutive injections, calculate peak area RSD (relative standard deviation), and ensure RSD ≤2% to guarantee method reproducibility.

[0085] Purity calculation: If the impurities are similar to the EGCG response factor, the purity can be directly calculated by the percentage of peak area.

[0086] Formula: Purity (%) = (EGCG peak area / total peak area) × 100%.

[0087] The specific results are shown in Table 2. The high-performance liquid chromatogram of EGCG in the tea extract of Example 3 is shown below. Figure 1 As shown.

[0088] Table 2 Results: The crude tea extracts prepared in Examples 1-13 contained EGCG content ≥17.1%, and the purity of the prepared EGCG products was ≥93.4%. The purity of the EGCG products in Comparative Examples 1-6 was lower than that in Examples 1-13.

[0089] Among them, the EGCG content in the crude tea extract prepared in Examples 1-3 and the purity of the prepared EGCG products were better than those in Examples 4-12. Examples 4-5 used different fermentation temperatures, which were lower or higher than the specified fermentation temperature (31-33℃). Examples 6-7 used different aeration rates, which were lower or higher than the specified aeration rate (1.2-1.4 vvm). Examples 8-9 used different pH values, with the pH value being higher than 4.5. The purity of the prepared EGCG products was lower than that of Examples 1-3. This shows that by precisely controlling the fermentation conditions, and accurately controlling the temperature, pH, and dissolved oxygen during the fermentation process (by setting the aeration rate to 1.2-1.4 vvm), the EGCG content in the crude tea extract and the purity of the EGCG products can be significantly improved.

[0090] Example 10 used a two-stage processing method, and the EGCG content in the crude tea extract and the purity of the prepared EGCG product were lower than those in Examples 1-3. The two-way liquid fermentation method of this application abandons the single crushing mode and adopts a gradient process of "coarse crushing-ultrafine crushing and screening", which can improve the EGCG content in the crude tea extract and the purity of the prepared EGCG product. Example 13 used a crushing size that was not within the scope of this application, and the EGCG content in the crude tea extract and the purity of the prepared EGCG product were lower than those in Examples 1-3.

[0091] The ratio of lactic acid bacteria and yeast in the compound strains of Examples 11-12 is different, and the EGCG content in the crude tea extract and the purity of the prepared EGCG product are not as good as those in Examples 1-3. In this application, lactic acid bacteria and yeast are used in a suitable mass ratio range for synergistic fermentation, which can improve the purity of EGCG product.

[0092] Comparative Example 1 did not pretreat the tea leaves, or Comparative Example 2 underwent gradient crushing and grading. The EGCG content in the crude tea extract and the purity of the prepared EGCG product were lower than those in Examples 1-14. In Comparative Examples 3-6, the compound microbial strains used other combinations of microorganisms or a single microorganism, and the purity of the EGCG product was lower. This indicates that specific combinations of microorganisms can better improve the EGCG content in the crude tea extract and the purity of the EGCG product.

[0093] Experiment 3: In vitro anti-inflammatory test Cell culture: Seed the cell suspension into 12-well cell culture plates at a density of 5 x 10⁶ cells per well. 4 Add 1 mL of DMDM ​​medium to each well and incubate at 37°C in a CO2 incubator for 18-24 hours. Discard the original culture medium in the wells and add the following culture medium according to the experimental design, and continue incubation for 24 hours.

[0094] Negative control group (NC): 1% fetal bovine serum + DMDM.

[0095] Model control group (M): 1 μg / mL lipopolysaccharide + 1% fetal bovine serum + DMDM.

[0096] Test sample group: different test samples (EGCG products of Examples 1-13 and Comparative Examples 1-6) + 1 μg / mL lipopolysaccharide + 1% fetal bovine serum + DMDM.

[0097] ELISA detection of inflammatory factor TNF-α: Collect cell culture medium from each group, centrifuge at 4℃ for 5 min at a speed of 12000×g, take the supernatant and use the corresponding ELISA kit to detect the amount of TNF-α in each group. The specific operation should be performed according to the operation requirements of the corresponding kit.

[0098] Table 3 The results are shown in Table 3: The TNF-α content of the EGCG products prepared in Examples 1-13 was 72-87 pg / mL, while the TNF-α content of the EGCG products in Comparative Examples 1-6 was lower than that in Examples 1-14.

[0099] Among them, the TNF-α content of the EGCG products prepared in Examples 1-3 was better than that of the EGCG products prepared in Examples 4-12. Examples 4-5 used different fermentation temperatures, which were lower or higher than the fermentation temperature (31-33℃). Examples 6-7 used different aeration rates, which were lower or higher than the aeration rate (1.2-1.4 vvm). Examples 8-9 used different pH values, with the pH value being higher than 4.5. The TNF-α content of the EGCG products prepared in these examples was lower than that of Examples 1-3. This shows that by precisely controlling the fermentation conditions, and accurately controlling the temperature, pH, and dissolved oxygen during the fermentation process (by setting the aeration rate to 1.2-1.4 vvm), the TNF-α content of the EGCG products can be significantly improved.

[0100] Example 10 used a two-stage treatment, and the TNF-α content of the prepared EGCG product was lower than that of Examples 1-3. The two-way liquid fermentation method of this application abandons the single crushing mode and adopts a gradient process of "coarse crushing-ultrafine crushing and screening", which can improve the purity of the EGCG product. In Examples 11-12, the ratio of lactic acid bacteria and yeast in the compound strains was different, and the TNF-α content of the prepared EGCG product was lower than that of Examples 1-3. This application uses lactic acid bacteria and yeast in an appropriate mass ratio range for synergistic fermentation, which can improve the TNF-α content of the EGCG product. In Example 13, the size of each crushing stage was not within the range of this application, and the TNF-α content of the prepared EGCG product was lower than that of Examples 1-3.

[0101] In Comparative Example 1, the tea leaves were not pretreated, or in Comparative Example 2, the tea leaves were subjected to gradient crushing and grading. The TNF-α content of the prepared EGCG products was lower than that in Examples 1-14. In Comparative Examples 3-6, the compound microbial strains used other microbial combinations or single microbial strains. The TNF-α content of the EGCG products was lower, indicating that specific microbial combinations can better improve the TNF-α content of EGCG products.

[0102] Test Example 4: Patch Test Human skin patch testing verifies the safety and gentleness of EGCG products: Thirty volunteers were recruited, 15 men and 15 women, aged 20-50 years. A closed patch test method was used. Equal volumes (0.025 mL) of the test sample (EGCG products from Examples 1-13 and Comparative Examples 1-6) were placed in a specific patch applicator. The patch was then applied to the volunteer's arm with hypoallergenic adhesive tape, gently pressed to ensure even application to the skin, and left for 24 hours. A blank control group consisted of distilled water. After 24 hours, the patch applicator was removed, and skin reactions were observed and recorded at 0.5 h, 24 h, and 48 h. The severity of adverse skin reactions is shown in Table 4 below.

[0103] Table 4 After testing, the EGCG products of Examples 1-13 and Comparative Examples 1-6 of this application all showed negative reactions after human patch testing, indicating that they are safe and non-irritating to human skin, demonstrating that the samples are mild.

[0104] Experimental Example 5: Verification of Oil Control and Anti-inflammatory Effects on the Human Body Acne scar test: We will recruit 30 volunteers, 15 men and 15 women, aged 20-50, with excessive facial oil secretion (e.g., oil secretion ≥100μg / cm² in the forehead, nose, and cheek areas) and mild to moderate inflammation (e.g., acne, erythema).

[0105] Sample usage: Volunteers applied the sample (EGCG products of Examples 1-13 and Comparative Examples 1-6) to their face once in the morning and once in the evening, with each application amount being 0.5g.

[0106] Testing items: Data were collected from volunteers on days 0, 14, and 28 after the sample was collected. After the visit, the volunteers washed their faces with facial cleanser and sat quietly for 30 minutes in an air-conditioned room with a temperature of 21±1℃ and a humidity of 50±10%.

[0107] Using the Sebumeter SM815 skin oil meter, the probe was placed vertically in contact with the skin for 30 seconds. Three measurements were taken and the average value was recorded. The initial oil content (μg / cm²) was recorded.

[0108] Using a Mexameter MX18 skin colorimeter, the a* value (red pigment content) of the facial erythema area was measured before and after use, reflecting the severity of inflammatory erythema.

[0109] Oil improvement rate (%) = (initial oil content - oil content after 28 days) / initial oil content × 100%.

[0110] Improvement rate of α value (%) = (initial α value - α value after 28 days) / initial α value × 100%.

[0111] The results are shown in Table 5.

[0112] Table 5 The results are shown in Table 5: The EGCG products prepared in Examples 1-3 showed an oil improvement rate of 37.3%-41.5% and an a* value improvement rate of 31.5%-36.8% after 28 days of use, demonstrating better oil control and anti-inflammatory effects in the human body. Examples 4-5 used different fermentation temperatures (below or above the set fermentation temperature, 31-33℃), Examples 6-7 used different aeration rates (below or above the set aeration rate, 1.2-1.4 vvm), and Examples 8-9 used different pH values ​​(pH above 4.5). The resulting EGCG products showed lower oil improvement rates and a* value improvement rates after 28 days of use compared to Examples 1-3. This indicates that by precisely controlling the fermentation conditions, including temperature, pH, and dissolved oxygen (by setting the aeration rate to 1.2-1.4 vvm), the quality and stability of the fermentation products can be improved, reducing the risk of contamination by other microorganisms. Furthermore, these conditions provide an optimal environment for microbial growth and metabolism, ensuring efficient and stable fermentation. Compared with traditional fermentation methods, the fermentation cycle of this application can be shortened, the quality and stability of tea extract in the product can be significantly improved, and the purity of EGCG can be significantly improved, thereby enhancing the oil control and anti-inflammatory effects of the product.

[0113] Example 10 employs a two-stage processing method, which is inferior to Examples 1-3. The bidirectional liquid fermentation method of this application abandons the single-stage pulverization mode and adopts a gradient process of "coarse crushing-ultrafine pulverization and grading." First, the pulverized tea leaves are pulverized to 20-40 mesh using a jaw crusher to disrupt the surface structure of the tea leaves. Then, an air jet mill is used for ultrafine pulverization, controlling the particle size distribution of the first-stage tea leaves to be within 1000 μm, increasing the cell wall breakage rate to over 85%. Finally, a cyclone classifier is used to screen uniform particles, avoiding uneven mixing caused by fine powder agglomeration. This bidirectional liquid fermentation method can improve the initial dissolution rate of core components such as EGCG. Example 13 uses pulverization sizes outside the scope of this application, and the resulting EGCG product has inferior oil-controlling and anti-inflammatory effects compared to Examples 1-3.

[0114] The ratio of lactic acid bacteria to yeast in the compound strains of Examples 11-12 differs, resulting in lower oil content and a* value compared to Examples 1-3. This application employs a suitable mass ratio of lactic acid bacteria and yeast for synergistic fermentation, forming a metabolic cascade between different strains. Metabolites from one strain become nutrient substrates for another, constructing a mutually dependent and highly efficient metabolic model. This synergistic effect can deeply transform tea extract components, inducing the production of more novel active substances and exerting novel effects. Simultaneously, it significantly improves the extraction rate of EGCG, thereby enhancing the oil-controlling and anti-inflammatory effects of EGCG products.

[0115] Comparative Example 1, which did not pretreat the tea leaves, or Comparative Example 2, which underwent gradient pulverization and grading, resulted in EGCG products with lower oil improvement rates and a* value improvement rates after 28 days of use compared to Examples 1-14. Comparative Examples 3-6, which used other strain combinations or single strains, also resulted in EGCG products with lower oil improvement rates and a* value improvement rates after 28 days of use compared to Examples 1-14. This indicates that specific strain combinations can help improve the oil control and anti-inflammatory effects in the human body. The facial redness reduction effect images of Application Example 3 and Comparative Application Example 1 are shown below. Figure 2 As shown.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A two-way liquid fermentation method for preparing tea extract, characterized in that, Includes the following steps: S1. The tea leaves are pretreated with plasma, dried, and then subjected to gradient pulverization and grading to obtain tea powder; then the tea powder is mixed with deionized water to obtain a mixture. S2. Inoculate the bacterial culture of the compound strain of lactic acid bacteria and yeast into the mixture in step S1 for fermentation to obtain fermentation broth; S3. Centrifuge the fermentation broth from step S2, collect the supernatant and ultrafilter it to obtain a concentrated liquid; add an extractant to the concentrated liquid and rotary evaporate it to obtain crude tea extract, then purify it to obtain tea extract.

2. The two-way liquid fermentation method for preparing tea extract as described in claim 1, characterized in that, In step S1, the plasma pretreatment includes: The tea leaves were pretreated under conditions of 12~13.6 kV voltage, 20~30℃ temperature, and 40~60% humidity for 10~20 minutes.

3. The two-way liquid fermentation method for preparing tea extract as described in claim 1, characterized in that, In step S1, the gradient pulverization and grading process includes: The dried tea leaves are crushed in a jaw crusher at a speed of 500-800 r / min for 5-8 minutes until they are crushed to 20-40 mesh, thus obtaining the first grade of tea. The first-grade tea leaves are ultra-finely pulverized using an airflow mill with an inlet pressure of 0.6~0.8 MPa and a pulverizing pressure of 0.3~0.5 MPa, until the tea leaves are pulverized to 70~120 mesh, thus obtaining the second-grade tea leaves. The second-grade tea leaves are screened using a cyclone grading machine with an air intake of 30-50 m³ / h and a grading frequency of 20-30 Hz to obtain uniform particles with a mesh size of 80-140 mesh, thus obtaining the third-grade tea leaves.

4. The two-way liquid fermentation method for preparing tea extract as described in claim 1, characterized in that, In step S1, the mass ratio of deionized water to tea powder is 1:(1.5~5).

5. The two-way liquid fermentation method for tea extract as described in claim 1, characterized in that, In step S2, the mass ratio of lactic acid bacteria to yeast is 1:(1~2).

6. The two-way liquid fermentation method for preparing tea extract as described in claim 1, characterized in that, In step S2, the inoculation mass ratio of the bacterial solution is 2-5%.

7. The two-way liquid fermentation method for preparing tea extract as described in claim 1, characterized in that, In step S2, the fermentation temperature is 31~33℃; And / or, the fermentation speed is 175~185 rpm; And / or, the aeration rate during fermentation is 1.2~1.4 vvm; And / or, adjust the pH of the fermentation broth to 4.0~4.

5.

8. The two-way liquid fermentation method for preparing tea extract as described in claim 1, characterized in that, In step S3, the extractant includes ethyl acetate; And / or, the volume ratio of the concentrate to the extractant is 1:(1~2); And / or, in step S3, rotary evaporation is carried out at a temperature of 40~50℃ and a vacuum degree of 0.08~0.09 MPa.

9. The tea extract is prepared by the two-way liquid fermentation method for preparing tea extract as described in any one of claims 1 to 8.

10. The application of the two-way liquid fermentation method for preparing tea extract as described in any one of claims 1 to 8 in improving the content and purity of epigallocatechin gallate ester.

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