Production method for ultrasonic-assisted low-temperature extraction of plant extract

Abstract

The invention relates to the technical field of plant active ingredient extraction, and particularly discloses an ultrasonic-assisted low-temperature extraction plant extract production method, which comprises the following steps: raw material pretreatment, composite solvent system preparation, ultrasonic-assisted low-temperature extraction, and low-temperature enrichment and purification. An enzyme-mediator-oligosaccharide system is used for preparing a composite pre-modifier to pretreat a plant raw material, then a hydrophilic deep eutectic solvent-low carbon alcohol-natural cosolvent composite system is combined with wetting-crushing double-stage ultrasonic extraction, and the plant extract is obtained through natural clarification, low-temperature vacuum concentration and macroporous resin purification. According to the method, heat-sensitive components are protected through full-chain low-temperature control, the plant extraction efficiency is improved through composite pre-modification and the hydrophilic deep eutectic solvent, the product purity is improved through secondary purification, the solvent is environmentally friendly and degradable, the process is easy to industrialize, the defects that heat-sensitive components are easy to degrade, the extraction efficiency is low and the like in the prior art are effectively overcome, and the method is suitable for industrial production. The method is suitable for plant active ingredient extraction.

Description

Technical Field

[0001] This invention relates to the field of plant active ingredient extraction technology, specifically to a production method for ultrasonic-assisted low-temperature extraction of plant extracts. Background Technology

[0002] Plant extracts are rich in active ingredients such as polyphenols, flavonoids, alkaloids, saponins and volatile oils, and have a variety of physiological functions such as anti-oxidation, anti-inflammation and immune regulation. They are widely used in food, pharmaceuticals, cosmetics and other fields.

[0003] Currently, mainstream plant extraction processes mainly include solvent reflux extraction, Soxhlet extraction, and hot reflux extraction. These methods typically require high temperatures of 60-100℃, which can easily lead to oxidation, isomerization, polymerization, or degradation of heat-sensitive active ingredients. Furthermore, traditional processes often use single-polarity solvents, making it difficult to meet the dissolution requirements of different polarity active ingredients in plants, resulting in low yields of target components. Some processes use organic solvents such as methanol, chloroform, and ethyl acetate to improve extraction efficiency, posing a risk of solvent toxicity residues and incurring high costs and energy consumption for subsequent solvent recovery.

[0004] While existing ultrasound-assisted extraction methods can enhance mass transfer, they generally employ a single, continuous, fixed-power mode. Ultrasonic heat generation can easily cause local temperatures to exceed 40°C, exacerbating the loss of active ingredients. Furthermore, the lack of matching raw material pretreatment and integrated post-extraction purification methods results in extracts containing large amounts of impurities such as starch, pectin, and protein, requiring multiple post-processing steps. This not only complicates the process and increases costs but also causes irreversible loss of active ingredients. Existing low-temperature extraction helps protect active ingredients, but at low temperatures, solvent viscosity increases, diffusion is limited, mass transfer efficiency is low, and extraction times often reach 2-8 hours, making it difficult to meet industrial-scale efficiency requirements. Some technologies improve flowability by adding co-solvents or surfactants, potentially introducing safety or environmental hazards. In addition, low solvent recovery rates and reliance on strong acids or bases to adjust pH are inconsistent with green production trends. Based on the above, this application provides a method for producing plant extracts using ultrasound-assisted low-temperature extraction. Summary of the Invention

[0005] In order to overcome the shortcomings of existing plant extract extraction technologies, such as easy degradation of heat-sensitive components, low extraction efficiency, poor environmental friendliness of solvents, insufficient product purity, and complex processes, this application provides a production method for ultrasonic-assisted low-temperature extraction of plant extracts.

[0006] In a first aspect, this application provides a method for producing plant extracts by ultrasonic-assisted low-temperature extraction, employing the following technical solution: A method for producing plant extracts by ultrasound-assisted low-temperature extraction includes the following steps: S1. Raw material pretreatment: Vacuum freeze-dry the plant raw materials, ultrafine pulverize them, and sieve them to obtain plant micro powder; spray the plant micro powder with a composite premodifier, shake and incubate to obtain pretreated plant micro powder; S2. Preparation of composite solvent system: The hydrophilic deep eutectic solvent, low carbon alcohol and natural co-solvent are mixed evenly to obtain the composite solvent system; S3. Ultrasonic-assisted low-temperature extraction: After the pretreated plant micro powder is mixed evenly with the composite solvent system, the first stage of continuous ultrasonic treatment and the second stage of pulsed ultrasonic treatment are carried out in sequence to obtain a turbid extract. A natural clarifying agent is added and mixed evenly, and then allowed to stand to obtain a clear extract. S4. Low-temperature enrichment and purification: The clarified extract is concentrated under low-temperature reduced pressure to obtain a concentrate. The concentrate is then purified by adsorption with macroporous resin and freeze-dried under vacuum to obtain the plant extract.

[0007] Preferably, in step S1, the temperature for vacuum freeze drying is -50℃ to -40℃, the vacuum degree is 10-30Pa, and the drying is carried out until the moisture content is ≤5%; the temperature for ultrafine pulverization is -15℃ to -5℃, and the time is 10-12min; and the material is passed through a 180-220 mesh sieve.

[0008] Preferably, in step S1, the composite pre-modifier is sprayed evenly onto the plant micropowder by atomization, and the spraying amount is 10-15% of the mass of the plant micropowder; after spraying, it is shaken and incubated at 4-10℃ for 15-30 minutes.

[0009] Preferably, the preparation method of the composite premodifier in step S1 is as follows: Laccase and enzyme stabilizer were dissolved in citrate-disodium hydrogen phosphate buffer to obtain prepreg A; oligosaccharide and phenolic acid mediator were then dissolved in citrate-disodium hydrogen phosphate buffer to obtain prepreg B; before use, prepreg A and prepreg B were mixed evenly and incubated by shaking to obtain composite premodifier.

[0010] Preferably, the enzyme stabilizer is selected from one or more of trehalose, mannitol, and xylitol; the oligosaccharide is selected from one or more of chitosan oligosaccharide, xylooligosaccharide, and fructooligosaccharide; and the phenolic acid mediator is selected from one or more of gallic acid, ferulic acid, p-coumaric acid, and sinapic acid.

[0011] Preferably, the mass ratio of laccase, enzyme stabilizer and citrate-disodium hydrogen phosphate buffer is 1:2-5:160-180.

[0012] Preferably, the mass ratio of the oligosaccharide, phenolic acid mediator, and citrate-disodium hydrogen phosphate buffer is 1:0.5-2:80-150.

[0013] Preferably, the volume ratio of pre-prepared liquid A to pre-prepared liquid B is 1:0.8-1.5.

[0014] The principle of the composite pre-modifier: Through the catalytic action of laccase, the cell wall components on the surface of plant microparticles are mildly degraded and modified under the mediation of phenolic acid mediators, which destroys the dense structure of the cell wall and improves the penetration efficiency of subsequent solvents; oligosaccharides can bind to the active groups on the surface of plant cells, reducing the adsorption loss of target components in the cells, while enzyme stabilizers can maintain the activity stability of laccase during the pretreatment process and avoid the inhibition of enzyme activity by low temperature or buffer environment, ultimately achieving efficient pretreatment of plant raw materials and providing favorable conditions for subsequent extraction.

[0015] Preferably, in step S2, the volume ratio of the hydrophilic deep eutectic solvent, the low-carbon alcohol, and the natural co-solvent is 65-75:20-30:2-4.

[0016] Preferably, in step S2, the lower alcohol is isopropanol or 1,2-propanediol; the natural co-solvent is a 3-5% (w / w) aqueous solution of dipotassium glycyrrhizate.

[0017] Preferably, the method for preparing the hydrophilic deep eutectic solvent in step S2 is as follows: The glycerol-lactic acid-malic acid ternary system was mixed with betaine to obtain a mixed system. L-proline was added to the mixed system and stirred until a homogeneous and transparent liquid was formed. The mixture was then aged at a constant temperature to obtain a hydrophilic eutectic solvent.

[0018] Preferably, the glycerol-lactic acid-malic acid ternary system is composed of glycerol, lactic acid and malic acid in a molar ratio of 2:1-2:0.5-1.

[0019] Preferably, the mass ratio of the glycerol-lactic acid-malic acid ternary system to betaine is 2.2-2.8:1; and the mass ratio of the mixed system to L-proline is 20-40:1.

[0020] Preferably, the constant temperature curing temperature is 50-60℃ and the time is 1-2 hours.

[0021] The principle of hydrophilic deep eutectic solvents: Hydrophilic deep eutectic solvents are prepared using a strategy of "first constructing a highly efficient hydrogen bond donor system, then introducing a single hydrogen bond acceptor." The core is to precisely control the matching degree between hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs). The glycerol-lactic acid-malic acid ternary system is a highly efficient hydrogen bond donor (HBD) system. Mixing the three components in a specific molar ratio forms a stable HBD aggregate containing multiple hydroxyl and carboxyl groups, providing ample hydrogen bond donor sites. Betaine, as a mild and stable hydrogen bond acceptor (HBA), has a zwitterionic structure containing both quaternary ammonium cations and carboxyl groups, effectively accepting hydrogen bonds from HBDs and regulating the system's polarity and charge distribution. After mixing the HBD system with betaine, a uniform, low-melting-point deep eutectic network is formed through intermolecular hydrogen bonding. The subsequently added L-proline, with its amino and carboxyl groups, can embed into this hydrogen bond network, further optimizing the network structure, reducing viscosity, and enhancing the selective solubility of plant active ingredients.

[0022] Preferably, in step S3, the mass ratio of pretreated plant micro powder to composite solvent system is 1:10-15; and the mass ratio of turbid extract to natural clarifying agent is 100:1.5-2.5.

[0023] Preferably, the natural clarifying agent in step S3 is composed of chitosan, sodium carboxymethyl cellulose, and water in a mass ratio of 1:2 to 4:50.

[0024] Preferably, in step S3, the power of the first stage continuous ultrasound treatment is 180-220W, the frequency is 25-30kHz, and the time is 20-30min; the power of the second stage pulsed ultrasound treatment is 280-320W, the frequency is 35-40kHz, the ultrasound lasts for 30-40s, the intermittent mode lasts for 15-25s, and the time is 30-50min.

[0025] The principle of the first-stage continuous ultrasonic treatment and the second-stage pulsed ultrasonic treatment: A two-stage ultrasonic design of "wetting-disruption" is adopted, achieving stepwise enhanced mass transfer through gradient control of power and frequency. The first stage uses low-power, low-frequency continuous ultrasonic treatment, utilizing the microjets generated by low-frequency vibrations to drive the composite solvent system to penetrate the pretreated plant micropowder, achieving uniform wetting of the intercellular spaces. The second stage switches to a high-power, high-frequency pulsed ultrasonic mode. The cavitation effect generated by high-intensity ultrasound breaks down residual cell wall structures, promoting the release of target components from the cells. The intervals between treatments balance the system temperature and provide time for the dissolved target components to diffuse into the solvent, thus balancing extraction efficiency and the stability of heat-sensitive components.

[0026] Preferably, in step S4, the vacuum degree of the low-temperature vacuum concentration is -0.095MPa to -0.1MPa, the temperature is -10℃ to -5℃, and the concentration is to 12-15% of the initial volume.

[0027] Preferably, the specific steps of macroporous resin adsorption and purification in step S4 are as follows: A chromatography column was packed with AB-8 macroporous resin containing 20-30% of the concentrate volume. The concentrate was diluted with deionized water to a solid content of 5-8%, and the pH was adjusted to 5.5-5.7. After filtration through a 0.45 μm filter membrane, the concentrate was loaded onto the chromatography column at a flow rate of 2-3 BV / h. After loading, the column was washed with 2-3 BV of deionized water at a flow rate of 3-4 BV / h to remove impurities. The column was then eluted with a 40-60% (v / v) low-carbon alcohol solution at a flow rate of 1.5-2.5 BV / h, and the eluent was collected. The collected eluent was concentrated under reduced pressure at -0.095 MPa to -0.1 MPa and -20°C to -10°C until it reached 12-15% of the initial volume of the eluent, thus completing the purification process.

[0028] Secondly, this application provides a plant extract prepared by the above-described ultrasonic-assisted low-temperature extraction method for plant extracts.

[0029] In summary, this application has the following beneficial effects: (1) A pioneering "enzyme-mediator-oligosaccharide" composite pre-modification strategy was developed to achieve targeted and gentle deconstruction of the cell wall. By combining laccase, phenolic acid mediator, oligosaccharides, and enzyme stabilizers in a specific buffer system, a composite pre-modifier with biocatalytic activity was formed. This system can precisely modify the lignin-cellulose network on the surface of plant microparticles at low temperatures, selectively weakening the dense structure of the cell wall. At the same time, oligosaccharides shield intracellular active sites through hydrogen bonds or electrostatic interactions, significantly reducing the non-specific adsorption loss of target components. This pretreatment method effectively avoids the damage to raw materials caused by various factors, laying a structural foundation for subsequent efficient extraction.

[0030] (2) Innovative construction of a "segmented system synergistic" hydrophilic deep eutectic solvent with both high solubility and environmental friendliness: Breaking through the traditional single HBD-HBA pairing mode, glycerol, lactic acid and malic acid are first premixed to form a multi-hydrogen bond donor (HBD) system, and then mixed with a single hydrogen bond acceptor (HBA) betaine, and L-proline is introduced as a hydrogen bond network regulator. This design forms a more stable, lower melting point and higher polarity solvent network through cross-system hydrogen bond recombination, effectively overcoming the limitations of traditional DES in terms of polarity coverage, mass transfer efficiency or component selectivity, significantly improving the broad-spectrum solubility of various plant active ingredients such as polyphenols, flavonoids, and organic acids, while maintaining excellent biocompatibility and biodegradability.

[0031] (3) A two-stage ultrasonic process of "wetting-breaking" is proposed to achieve efficient mass transfer and cell wall disruption at low temperature: The ultrasonic process is divided into two functionally distinct stages: The first stage uses low-power, low-frequency continuous ultrasonic treatment to promote the uniform penetration of the composite solvent into the intercellular space by utilizing the micro-jet effect; The second stage switches to high-power, high-frequency pulse mode to precisely break the residual cell structure and release intracellular components through the controllable cavitation effect; The interval effectively suppresses local temperature rise and ensures that the temperature is maintained in the stable range of heat-sensitive components throughout the process, taking into account both extraction efficiency and component integrity.

[0032] (4) Achieve low-temperature control throughout the entire chain from pulverization to concentration, systematically protecting heat-sensitive active substances: the ultra-fine pulverization of raw materials is carried out at -15℃ to -5℃ to prevent the loss of volatile or oxidatively sensitive components; extraction and clarification are completed below room temperature; concentration is carried out at low temperature and reduced pressure at -10℃ to -5℃. The entire process has no heating steps, completely avoiding the degradation, isomerization or polymerization of components caused by traditional high-temperature operations such as reflux and distillation, thereby improving the retention rate of heat-sensitive components (such as ginsenosides, anthocyanins, etc.).

[0033] (5) Integrating natural clarification and targeted resin purification to obtain high-purity, high-recovery final products: Chitosan and sodium carboxymethyl cellulose aqueous solution are used as natural clarifiers. Through charge neutralization and bridging, impurities such as proteins and tannins are efficiently removed, avoiding secondary pollution introduced by chemical flocculants. The subsequent macroporous resin purification process is highly compatible with the composite solvent system. By precisely controlling the sample loading pH, flow rate and elution alcohol concentration, the selective enrichment of target components is achieved. The purity of the product is improved compared with conventional methods, and the elution solvent can be recycled, reducing production costs. The high-purity final product is obtained by subsequent vacuum freeze drying. Detailed Implementation

[0034] The present application will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0035] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0036] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0037] The laccase was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., model: FDY-2204.

[0038] Example 1 A method for producing plant extracts by ultrasound-assisted low-temperature extraction includes the following steps: S1. Raw material pretreatment: Green tea raw materials are freeze-dried at -50℃ and 10Pa vacuum until the moisture content is 5%, then ultra-finely pulverized at -15℃ for 10 minutes and passed through a 180-mesh sieve to obtain plant micro powder; a composite pre-modifying agent is evenly sprayed onto the plant micro powder by atomization, with a spray amount of 10% of the mass of the plant micro powder, and then shaken and incubated at 4℃ for 15 minutes to obtain pretreated plant micro powder; S2. Preparation of composite solvent system: A hydrophilic deep eutectic solvent, isopropanol and a 3% (w / w) dipotassium glycyrrhizate aqueous solution are mixed evenly to obtain a composite solvent system. S3. Ultrasonic-assisted low-temperature extraction: After mixing the pretreated plant micro powder with the composite solvent system at a mass ratio of 1:10, the first stage of continuous ultrasonic treatment was carried out at 180W and 25kHz for 20min, followed by a second stage of pulsed ultrasonic treatment at 280W and 35kHz for 30s and 15s intermittent mode for 30min, resulting in a turbid extract. Natural clarifying agent (composed of chitosan, sodium carboxymethyl cellulose and water at a mass ratio of 1:2:50) was added to the turbid extract at a mass ratio of 100:1.5, mixed evenly, and allowed to stand at 4℃ for 16h to obtain a clear extract. S4. Low-Temperature Enrichment and Purification: The clarified extract was concentrated under reduced pressure at -0.095 MPa and -10°C to 12% of its initial volume, yielding a concentrated solution. An AB-8 macroporous resin column was packed with 20% of the concentrated solution volume. The concentrated solution was diluted with deionized water to a solid content of 5%, and the pH was adjusted to 5.5 with 0.1 mol / L citric acid solution. After filtration through a 0.45 μm filter membrane, the solution was loaded onto the chromatography column at a flow rate of 2 BV / h. After loading, the solution was... 2 BV of deionized water was used to rinse and remove impurities at a flow rate of 3 BV / h; then, 40% isopropanol solution was used to elute at a flow rate of 1.5 BV / h, and the eluent was collected; the collected eluent was concentrated under low-temperature reduced pressure at -0.095 MPa and -20 °C to 12% of the initial volume of the eluent, thus completing the macroporous resin adsorption purification. Subsequently, the obtained concentrate was freeze-dried under vacuum at a cold trap temperature of -50 °C and a vacuum degree of 8 Pa for 36 h to obtain green tea extract.

[0039] The preparation method of the composite premodifier is as follows: Laccase and trehalose are dissolved in 20mM, pH 4.8 citrate-disodium hydrogen phosphate buffer (the mass ratio of laccase, trehalose and citrate-disodium hydrogen phosphate buffer is 1:2:160) to obtain prepreg A; chitosan oligosaccharide and gallic acid are then dissolved in 20mM, pH 5 citrate-disodium hydrogen phosphate buffer (the mass ratio of chitosan oligosaccharide, gallic acid and citrate-disodium hydrogen phosphate buffer is 1:0.5:80) to obtain prepreg B; before use, prepreg A and prepreg B are mixed evenly at a volume ratio of 1:0.8, and then incubated at 4℃ with shaking for 15 min to obtain the composite premodifier.

[0040] The preparation method of the hydrophilic deep eutectic solvent is as follows: a glycerol-lactic acid-malic acid ternary system with a mass ratio of 2.2:1 (prepared by stirring and mixing glycerol, lactic acid and malic acid in a molar ratio of 2:1:0.5 at 50℃ for 30 min) is mixed with betaine to obtain a mixed system. L-proline is added to the mixed system at a mass ratio of 20:1, and the mixture is stirred at 80 rpm until a homogeneous transparent liquid is formed. The mixture is then aged at 50℃ for 1 h to obtain the hydrophilic deep eutectic solvent.

[0041] Example 2 A method for producing plant extracts by ultrasound-assisted low-temperature extraction includes the following steps: S1. Raw material pretreatment: Green tea raw materials are freeze-dried at -45℃ and 20Pa vacuum until the moisture content is 5%, then ultra-finely pulverized at -10℃ for 11 minutes and passed through a 200-mesh sieve to obtain plant micro powder; a composite pre-modifying agent is evenly sprayed onto the plant micro powder by atomization, with a spray amount of 12% of the mass of the plant micro powder, and then shaken and incubated at 6℃ for 20 minutes to obtain pretreated plant micro powder; S2. Preparation of composite solvent system: A hydrophilic deep eutectic solvent, isopropanol and a 4% (w / w) dipotassium glycyrrhizate aqueous solution are mixed evenly to obtain a composite solvent system. S3. Ultrasonic-assisted low-temperature extraction: After the pretreated plant micro powder and the composite solvent system are mixed evenly at a mass ratio of 1:12, the first stage of continuous ultrasonic treatment is carried out at 200W and 28kHz for 25min, and then the second stage of pulsed ultrasonic treatment is carried out at 300W, 38kHz, 35s ultrasonic and 20s intermittent mode for 40min to obtain a turbid extract. Natural clarifying agent (composed of chitosan, sodium carboxymethyl cellulose and water at a mass ratio of 1:3:50) is added to the turbid extract at a mass ratio of 100:2, mixed evenly, and allowed to stand at 6℃ for 20h to obtain a clear extract. S4. Low-Temperature Enrichment and Purification: The clarified extract was concentrated under reduced pressure at -0.098 MPa and -8°C to 13.5% of its initial volume. A 25% volume fraction of the concentrate was packed into an AB-8 macroporous resin chromatography column. The concentrate was diluted with deionized water to a solid content of 6%, and the pH was adjusted to 5.6 with 0.15 mol / L citric acid solution. After filtration through a 0.45 μm filter membrane, the concentrate was loaded onto the chromatography column at a flow rate of 2.5 BV / h. After loading, 2... The solution was rinsed with 0.5 BV of deionized water at a flow rate of 3.5 BV / h to remove impurities; then eluted with 50% isopropanol solution at a flow rate of 2 BV / h, and the eluent was collected; the collected eluent was concentrated again under low temperature reduced pressure at -0.098 MPa and -15 °C to 13.5% of the initial volume of the eluent, thus completing the macroporous resin adsorption purification. The resulting concentrate was then freeze-dried under vacuum at a cold trap temperature of -50 °C and a vacuum degree of 8 Pa for 36 h to obtain the green tea extract.

[0042] The preparation method of the composite premodifier is as follows: Laccase and trehalose are dissolved in 35mM, pH 5 citrate-disodium hydrogen phosphate buffer (the mass ratio of laccase, trehalose and citrate-disodium hydrogen phosphate buffer is 1:4:170) to obtain prepreg A; then chitosan oligosaccharide and gallic acid are dissolved in 30mM, pH 5.5 citrate-disodium hydrogen phosphate buffer (the mass ratio of chitosan oligosaccharide, gallic acid and citrate-disodium hydrogen phosphate buffer is 1:1.5:100) to obtain prepreg B; before use, prepreg A and prepreg B are mixed evenly at a volume ratio of 1:1.2, and then incubated at 6℃ with shaking for 20 min to obtain the composite premodifier.

[0043] The preparation method of the hydrophilic deep eutectic solvent is as follows: a glycerol-lactic acid-malic acid ternary system with a mass ratio of 2.5:1 (prepared by stirring and mixing glycerol, lactic acid and malic acid in a molar ratio of 2:1.5:0.75 at 50℃ for 35 min) is mixed with betaine to obtain a mixed system. L-proline is added to the mixed system at a mass ratio of 30:1, and the mixture is stirred at 100 rpm until a homogeneous transparent liquid is formed. The mixture is then aged at 55℃ for 1.5 h to obtain the hydrophilic deep eutectic solvent.

[0044] Example 3 A method for producing plant extracts by ultrasound-assisted low-temperature extraction includes the following steps: S1. Raw material pretreatment: Green tea raw materials are freeze-dried at -40℃ and 30Pa vacuum until the moisture content is 5%, then ultra-finely pulverized at -5℃ for 12 minutes and passed through a 220-mesh sieve to obtain plant micro powder; a composite pre-modifying agent is evenly sprayed onto the plant micro powder by atomization, with the spray amount being 15% of the mass of the plant micro powder, and then shaken and incubated at 10℃ for 30 minutes to obtain pretreated plant micro powder; S2. Preparation of composite solvent system: A hydrophilic deep eutectic solvent, isopropanol and a 5% dipotassium glycyrrhizate aqueous solution with a volume ratio of 75:30:4 were mixed evenly to obtain a composite solvent system. S3. Ultrasonic-assisted low-temperature extraction: After the pretreated plant micro powder and the composite solvent system are mixed evenly at a mass ratio of 1:15, the first stage of continuous ultrasonic treatment is carried out at 220W and 30kHz for 30min, and then the second stage of pulsed ultrasonic treatment is carried out at 320W and 40kHz for 40s and 25s intermittent mode for 50min to obtain a turbid extract. Natural clarifying agent (composed of chitosan, sodium carboxymethyl cellulose and water at a mass ratio of 1:4:50) is added to the turbid extract at a mass ratio of 100:2.5, mixed evenly, and allowed to stand at 8℃ for 24h to obtain a clear extract. S4. Low-Temperature Enrichment and Purification: The clarified extract was concentrated under reduced pressure at -0.1 MPa and -5°C to 15% of its initial volume, yielding a concentrated solution. An AB-8 macroporous resin column was packed with 30% of the concentrated solution volume. The concentrated solution was diluted with deionized water to a solid content of 8%, and the pH was adjusted to 5.7 with 0.2 mol / L citric acid solution. After filtration through a 0.45 μm filter membrane, the solution was loaded onto the chromatography column at a flow rate of 3 BV / h. After loading, the solution was purified using 3... BV was rinsed with deionized water at a flow rate of 4 BV / h to remove impurities; then eluted with 60% isopropanol solution at a flow rate of 2.5 BV / h, and the eluent was collected; the collected eluent was concentrated again under low temperature reduced pressure at -0.1 MPa and -10℃ to 15% of the initial volume of the eluent, thus completing the macroporous resin adsorption purification. Subsequently, the obtained concentrate was freeze-dried under vacuum at a cold trap temperature of -50℃ and a vacuum degree of 8 Pa for 36 h to obtain green tea extract.

[0045] The preparation method of the composite premodifier is as follows: Laccase and trehalose are dissolved in 50mM, pH 5.2 citrate-disodium hydrogen phosphate buffer (the mass ratio of laccase, trehalose and citrate-disodium hydrogen phosphate buffer is 1:5:180) to obtain prepreg A; chitosan oligosaccharide and gallic acid are then dissolved in 50mM, pH 6 citrate-disodium hydrogen phosphate buffer (the mass ratio of chitosan oligosaccharide, gallic acid and citrate-disodium hydrogen phosphate buffer is 1:2:150) to obtain prepreg B; before use, prepreg A and prepreg B are mixed evenly at a volume ratio of 1:1.5, and then incubated at 10℃ with shaking for 30 min to obtain the composite premodifier.

[0046] The preparation method of the hydrophilic deep eutectic solvent is as follows: a glycerol-lactic acid-malic acid ternary system with a mass ratio of 2.8:1 (prepared by stirring and mixing glycerol, lactic acid and malic acid in a molar ratio of 2:2:1 at 50℃ for 40 min) is mixed with betaine to obtain a mixed system. L-proline is added to the mixed system at a mass ratio of 40:1, and the mixture is stirred at 120 rpm until a homogeneous and transparent liquid is formed. The mixture is then aged at 60℃ for 2 h to obtain the hydrophilic deep eutectic solvent.

[0047] Comparative Example 1 A method for producing plant extracts by ultrasound-assisted low-temperature extraction includes the following steps: S1. Raw material pretreatment: Green tea raw materials are freeze-dried at -45℃ and 20Pa vacuum until the moisture content is 5%, then ultra-finely pulverized at -10℃ for 11 minutes and passed through a 200-mesh sieve to obtain plant micro powder; S2. Preparation of composite solvent system: A hydrophilic deep eutectic solvent, isopropanol and a 4% (w / w) dipotassium glycyrrhizate aqueous solution are mixed evenly to obtain a composite solvent system. S3. Ultrasonic-assisted low-temperature extraction: After the plant micro powder and the composite solvent system are mixed evenly at a mass ratio of 1:12, the first stage of continuous ultrasonic treatment is carried out at 200W and 28kHz for 25min, and then the second stage of pulsed ultrasonic treatment is carried out at 300W, 38kHz, 35s ultrasonic and 20s intermittent mode for 40min to obtain a turbid extract. Natural clarifying agent (composed of chitosan, sodium carboxymethyl cellulose and water at a mass ratio of 1:3:50) is added to the turbid extract at a mass ratio of 100:2, mixed evenly, and allowed to stand at 6℃ for 20h to obtain a clear extract. S4. Low-Temperature Enrichment and Purification: The clarified extract was concentrated under reduced pressure at -0.098 MPa and -8°C to 13.5% of its initial volume. A 25% volume fraction of the concentrate was packed into an AB-8 macroporous resin chromatography column. The concentrate was diluted with deionized water to a solid content of 6%, and the pH was adjusted to 5.6 with 0.15 mol / L citric acid solution. After filtration through a 0.45 μm filter membrane, the concentrate was loaded onto the chromatography column at a flow rate of 2.5 BV / h. After loading, 2... The solution was rinsed with 0.5 BV of deionized water at a flow rate of 3.5 BV / h to remove impurities; then eluted with 50% isopropanol solution at a flow rate of 2 BV / h, and the eluent was collected; the collected eluent was concentrated again under low temperature reduced pressure at -0.098 MPa and -15 °C to 13.5% of the initial volume of the eluent, thus completing the macroporous resin adsorption purification. The resulting concentrate was then freeze-dried under vacuum at a cold trap temperature of -50 °C and a vacuum degree of 8 Pa for 36 h to obtain the green tea extract.

[0048] The preparation method of the hydrophilic deep eutectic solvent is as follows: a glycerol-lactic acid-malic acid ternary system with a mass ratio of 2.5:1 (prepared by stirring and mixing glycerol, lactic acid and malic acid in a molar ratio of 2:1.5:0.75 at 50℃ for 35 min) is mixed evenly with betaine to obtain a mixed system. L-proline is added to the mixed system at a mass ratio of 30:1, and the mixture is stirred at 100 rpm until a homogeneous transparent liquid is formed. The mixture is then aged at 55℃ for 1.5 h to obtain the hydrophilic deep eutectic solvent.

[0049] Comparative Example 2 A method for producing plant extracts by ultrasound-assisted low-temperature extraction includes the following steps: S1. Raw material pretreatment: Green tea raw materials are freeze-dried at -45℃ and 20Pa vacuum until the moisture content is 5%, then ultra-finely pulverized at -10℃ for 11 minutes and passed through a 200-mesh sieve to obtain plant micro powder; a composite pre-modifying agent is evenly sprayed onto the plant micro powder by atomization, with a spray amount of 12% of the mass of the plant micro powder, and then shaken and incubated at 6℃ for 20 minutes to obtain pretreated plant micro powder; S2. Preparation of composite solvent system: A hydrophilic deep eutectic solvent, isopropanol and a 4% (w / w) dipotassium glycyrrhizate aqueous solution are mixed evenly to obtain a composite solvent system. S3. Ultrasonic-assisted low-temperature extraction: After the pretreated plant micro powder and the composite solvent system are mixed evenly at a mass ratio of 1:12, the first stage of continuous ultrasonic treatment is carried out at 200W and 28kHz for 25min, and then the second stage of pulsed ultrasonic treatment is carried out at 300W, 38kHz, 35s ultrasonic and 20s intermittent mode for 40min to obtain a turbid extract. Natural clarifying agent (composed of chitosan, sodium carboxymethyl cellulose and water at a mass ratio of 1:3:50) is added to the turbid extract at a mass ratio of 100:2, mixed evenly, and allowed to stand at 6℃ for 20h to obtain a clear extract. S4. Low-Temperature Enrichment and Purification: The clarified extract was concentrated under reduced pressure at -0.098 MPa and -8°C to 13.5% of its initial volume. A 25% volume fraction of the concentrate was packed into an AB-8 macroporous resin chromatography column. The concentrate was diluted with deionized water to a solid content of 6%, and the pH was adjusted to 5.6 with 0.15 mol / L citric acid solution. After filtration through a 0.45 μm filter membrane, the concentrate was loaded onto the chromatography column at a flow rate of 2.5 BV / h. After loading, 2... The solution was rinsed with 0.5 BV of deionized water at a flow rate of 3.5 BV / h to remove impurities; then eluted with 50% isopropanol solution at a flow rate of 2 BV / h, and the eluent was collected; the collected eluent was concentrated again under low temperature reduced pressure at -0.098 MPa and -15 °C to 13.5% of the initial volume of the eluent, thus completing the macroporous resin adsorption purification. The resulting concentrate was then freeze-dried under vacuum at a cold trap temperature of -50 °C and a vacuum degree of 8 Pa for 36 h to obtain the green tea extract.

[0050] The preparation method of the composite premodifier is as follows: laccase and trehalose are dissolved in 35mM, pH 5 citrate-disodium hydrogen phosphate buffer (the mass ratio of laccase, trehalose and citrate-disodium hydrogen phosphate buffer is 1:4:170) to obtain the composite premodifier.

[0051] The preparation method of the hydrophilic deep eutectic solvent is as follows: a glycerol-lactic acid-malic acid ternary system with a mass ratio of 2.5:1 (prepared by stirring and mixing glycerol, lactic acid and malic acid in a molar ratio of 2:1.5:0.75 at 50℃ for 35 min) is mixed with betaine to obtain a mixed system. L-proline is added to the mixed system at a mass ratio of 30:1, and the mixture is stirred at 100 rpm until a homogeneous transparent liquid is formed. The mixture is then aged at 55℃ for 1.5 h to obtain the hydrophilic deep eutectic solvent.

[0052] Comparative Example 3 A method for producing plant extracts by ultrasound-assisted low-temperature extraction includes the following steps: S1. Raw material pretreatment: Green tea raw materials are freeze-dried at -45℃ and 20Pa vacuum until the moisture content is 5%, then ultra-finely pulverized at -10℃ for 11 minutes and passed through a 200-mesh sieve to obtain plant micro powder; a composite pre-modifying agent is evenly sprayed onto the plant micro powder by atomization, with a spray amount of 12% of the mass of the plant micro powder, and then shaken and incubated at 6℃ for 20 minutes to obtain pretreated plant micro powder; S2. Preparation of composite solvent system: A hydrophilic deep eutectic solvent, isopropanol and a 4% (w / w) dipotassium glycyrrhizate aqueous solution are mixed evenly to obtain a composite solvent system. S3. Ultrasonic-assisted low-temperature extraction: After the pretreated plant micro powder and the composite solvent system are mixed evenly at a mass ratio of 1:12, the first stage of continuous ultrasonic treatment is carried out at 200W and 28kHz for 25min, and then the second stage of pulsed ultrasonic treatment is carried out at 300W, 38kHz, 35s ultrasonic and 20s intermittent mode for 40min to obtain a turbid extract. Natural clarifying agent (composed of chitosan, sodium carboxymethyl cellulose and water at a mass ratio of 1:3:50) is added to the turbid extract at a mass ratio of 100:2, mixed evenly, and allowed to stand at 6℃ for 20h to obtain a clear extract. S4. Low-Temperature Enrichment and Purification: The clarified extract was concentrated under reduced pressure at -0.098 MPa and -8°C to 13.5% of its initial volume. A 25% volume fraction of the concentrate was packed into an AB-8 macroporous resin chromatography column. The concentrate was diluted with deionized water to a solid content of 6%, and the pH was adjusted to 5.6 with 0.15 mol / L citric acid solution. After filtration through a 0.45 μm filter membrane, the concentrate was loaded onto the chromatography column at a flow rate of 2.5 BV / h. After loading, 2... The solution was rinsed with 0.5 BV of deionized water at a flow rate of 3.5 BV / h to remove impurities; then eluted with 50% isopropanol solution at a flow rate of 2 BV / h, and the eluent was collected; the collected eluent was concentrated again under low temperature reduced pressure at -0.098 MPa and -15 °C to 13.5% of the initial volume of the eluent, thus completing the macroporous resin adsorption purification. The resulting concentrate was then freeze-dried under vacuum at a cold trap temperature of -50 °C and a vacuum degree of 8 Pa for 36 h to obtain the green tea extract.

[0053] The preparation method of the composite premodifier is as follows: chitosan oligosaccharide and gallic acid are dissolved in 30mM, pH 5.5 citrate-disodium hydrogen phosphate buffer (the mass ratio of chitosan oligosaccharide, gallic acid and citrate-disodium hydrogen phosphate buffer is 1:1.5:100) to obtain the composite premodifier.

[0054] The preparation method of the hydrophilic deep eutectic solvent is as follows: a glycerol-lactic acid-malic acid ternary system with a mass ratio of 2.5:1 (prepared by stirring and mixing glycerol, lactic acid and malic acid in a molar ratio of 2:1.5:0.75 at 50℃ for 35 min) is mixed with betaine to obtain a mixed system. L-proline is added to the mixed system at a mass ratio of 30:1, and the mixture is stirred at 100 rpm until a homogeneous transparent liquid is formed. The mixture is then aged at 55℃ for 1.5 h to obtain the hydrophilic deep eutectic solvent.

[0055] Comparative Example 4 A method for producing plant extracts by ultrasound-assisted low-temperature extraction includes the following steps: S1. Raw material pretreatment: Green tea raw materials are freeze-dried at -45℃ and 20Pa vacuum until the moisture content is 5%, then ultra-finely pulverized at -10℃ for 11 minutes and passed through a 200-mesh sieve to obtain plant micro powder; a composite pre-modifying agent is evenly sprayed onto the plant micro powder by atomization, with a spray amount of 12% of the mass of the plant micro powder, and then shaken and incubated at 6℃ for 20 minutes to obtain pretreated plant micro powder; S2. Preparation of composite solvent system: A ternary system of glycerol-lactic acid-malic acid with a volume ratio of 70:25:3 (prepared by stirring and mixing glycerol, lactic acid and malic acid with a molar ratio of 2:1.5:0.75 at 50℃ for 35 min), isopropanol and a 4% dipotassium glycyrrhizate aqueous solution were mixed evenly to obtain a composite solvent system. S3. Ultrasonic-assisted low-temperature extraction: After the pretreated plant micro powder and the composite solvent system are mixed evenly at a mass ratio of 1:12, the first stage of continuous ultrasonic treatment is carried out at 200W and 28kHz for 25min, and then the second stage of pulsed ultrasonic treatment is carried out at 300W, 38kHz, 35s ultrasonic and 20s intermittent mode for 40min to obtain a turbid extract. Natural clarifying agent (composed of chitosan, sodium carboxymethyl cellulose and water at a mass ratio of 1:3:50) is added to the turbid extract at a mass ratio of 100:2, mixed evenly, and allowed to stand at 6℃ for 20h to obtain a clear extract. S4. Low-Temperature Enrichment and Purification: The clarified extract was concentrated under reduced pressure at -0.098 MPa and -8°C to 13.5% of its initial volume. A 25% volume fraction of the concentrate was packed into an AB-8 macroporous resin chromatography column. The concentrate was diluted with deionized water to a solid content of 6%, and the pH was adjusted to 5.6 with 0.15 mol / L citric acid solution. After filtration through a 0.45 μm filter membrane, the concentrate was loaded onto the chromatography column at a flow rate of 2.5 BV / h. After loading, 2... The solution was rinsed with 0.5 BV of deionized water at a flow rate of 3.5 BV / h to remove impurities; then eluted with 50% isopropanol solution at a flow rate of 2 BV / h, and the eluent was collected; the collected eluent was concentrated again under low temperature reduced pressure at -0.098 MPa and -15 °C to 13.5% of the initial volume of the eluent, thus completing the macroporous resin adsorption purification. The resulting concentrate was then freeze-dried under vacuum at a cold trap temperature of -50 °C and a vacuum degree of 8 Pa for 36 h to obtain the green tea extract.

[0056] The preparation method of the composite premodifier is as follows: Laccase and trehalose are dissolved in 35mM, pH 5 citrate-disodium hydrogen phosphate buffer (the mass ratio of laccase, trehalose and citrate-disodium hydrogen phosphate buffer is 1:4:170) to obtain prepreg A; then chitosan oligosaccharide and gallic acid are dissolved in 30mM, pH 5.5 citrate-disodium hydrogen phosphate buffer (the mass ratio of chitosan oligosaccharide, gallic acid and citrate-disodium hydrogen phosphate buffer is 1:1.5:100) to obtain prepreg B; before use, prepreg A and prepreg B are mixed evenly at a volume ratio of 1:1.2, and then incubated at 6℃ with shaking for 20 min to obtain the composite premodifier.

[0057] Comparative Example 5 A method for producing plant extracts by ultrasound-assisted low-temperature extraction includes the following steps: S1. Raw material pretreatment: Green tea raw materials are freeze-dried at -45℃ and 20Pa vacuum until the moisture content is 5%, then ultra-finely pulverized at -10℃ for 11 minutes and passed through a 200-mesh sieve to obtain plant micro powder; a composite pre-modifying agent is evenly sprayed onto the plant micro powder by atomization, with a spray amount of 12% of the mass of the plant micro powder, and then shaken and incubated at 6℃ for 20 minutes to obtain pretreated plant micro powder; S2. Preparation of composite solvent system: Betaine, isopropanol and dipotassium glycyrrhizate aqueous solution with a volume ratio of 70:25:3 were mixed evenly to obtain composite solvent system. S3. Ultrasonic-assisted low-temperature extraction: After the pretreated plant micro powder and the composite solvent system are mixed evenly at a mass ratio of 1:12, the first stage of continuous ultrasonic treatment is carried out at 200W and 28kHz for 25min, and then the second stage of pulsed ultrasonic treatment is carried out at 300W, 38kHz, 35s ultrasonic and 20s intermittent mode for 40min to obtain a turbid extract. Natural clarifying agent (composed of chitosan, sodium carboxymethyl cellulose and water at a mass ratio of 1:3:50) is added to the turbid extract at a mass ratio of 100:2, mixed evenly, and allowed to stand at 6℃ for 20h to obtain a clear extract. S4. Low-Temperature Enrichment and Purification: The clarified extract was concentrated under reduced pressure at -0.098 MPa and -8°C to 13.5% of its initial volume. A 25% volume fraction of the concentrate was packed into an AB-8 macroporous resin chromatography column. The concentrate was diluted with deionized water to a solid content of 6%, and the pH was adjusted to 5.6 with 0.15 mol / L citric acid solution. After filtration through a 0.45 μm filter membrane, the concentrate was loaded onto the chromatography column at a flow rate of 2.5 BV / h. After loading, 2... The solution was rinsed with 0.5 BV of deionized water at a flow rate of 3.5 BV / h to remove impurities; then eluted with 50% isopropanol solution at a flow rate of 2 BV / h, and the eluent was collected; the collected eluent was concentrated again under low temperature reduced pressure at -0.098 MPa and -15 °C to 13.5% of the initial volume of the eluent, thus completing the macroporous resin adsorption purification. The resulting concentrate was then freeze-dried under vacuum at a cold trap temperature of -50 °C and a vacuum degree of 8 Pa for 36 h to obtain the green tea extract.

[0058] The preparation method of the composite premodifier is as follows: Laccase and trehalose are dissolved in 35mM, pH 5 citrate-disodium hydrogen phosphate buffer (the mass ratio of laccase, trehalose and citrate-disodium hydrogen phosphate buffer is 1:4:170) to obtain prepreg A; then chitosan oligosaccharide and gallic acid are dissolved in 30mM, pH 5.5 citrate-disodium hydrogen phosphate buffer (the mass ratio of chitosan oligosaccharide, gallic acid and citrate-disodium hydrogen phosphate buffer is 1:1.5:100) to obtain prepreg B; before use, prepreg A and prepreg B are mixed evenly at a volume ratio of 1:1.2, and then incubated at 6℃ with shaking for 20 min to obtain the composite premodifier.

[0059] Comparative Example 6 A method for producing plant extracts by ultrasound-assisted low-temperature extraction includes the following steps: S1. Raw material pretreatment: Green tea raw materials are freeze-dried at -45℃ and 20Pa vacuum until the moisture content is 5%, then ultra-finely pulverized at -10℃ for 11 minutes and passed through a 200-mesh sieve to obtain plant micro powder; a composite pre-modifying agent is evenly sprayed onto the plant micro powder by atomization, with a spray amount of 12% of the mass of the plant micro powder, and then shaken and incubated at 6℃ for 20 minutes to obtain pretreated plant micro powder; S2. Preparation of composite solvent system: A hydrophilic deep eutectic solvent, isopropanol and a 4% (w / w) dipotassium glycyrrhizate aqueous solution are mixed evenly to obtain a composite solvent system. S3. Ultrasonic-assisted low-temperature extraction: After the pretreated plant micro powder and the composite solvent system are mixed evenly at a mass ratio of 1:12, the mixture is subjected to pulsed ultrasonic treatment at 300W, 38kHz, 35s ultrasonic and 20s intermittent mode for 65min to obtain a turbid extract. Natural clarifying agent (composed of chitosan, sodium carboxymethyl cellulose and water at a mass ratio of 1:3:50) is added to the turbid extract at a mass ratio of 100:2, mixed evenly, and allowed to stand at 6℃ for 20h to obtain a clear extract. S4. Low-Temperature Enrichment and Purification: The clarified extract was concentrated under reduced pressure at -0.098 MPa and -8°C to 13.5% of its initial volume. A 25% volume fraction of the concentrate was packed into an AB-8 macroporous resin chromatography column. The concentrate was diluted with deionized water to a solid content of 6%, and the pH was adjusted to 5.6 with 0.15 mol / L citric acid solution. After filtration through a 0.45 μm filter membrane, the concentrate was loaded onto the chromatography column at a flow rate of 2.5 BV / h. After loading, 2... The solution was rinsed with 0.5 BV of deionized water at a flow rate of 3.5 BV / h to remove impurities; then eluted with 50% isopropanol solution at a flow rate of 2 BV / h, and the eluent was collected; the collected eluent was concentrated again under low temperature reduced pressure at -0.098 MPa and -15 °C to 13.5% of the initial volume of the eluent, thus completing the macroporous resin adsorption purification. The resulting concentrate was then freeze-dried under vacuum at a cold trap temperature of -50 °C and a vacuum degree of 8 Pa for 36 h to obtain the green tea extract.

[0060] The preparation method of the composite premodifier is as follows: Laccase and trehalose are dissolved in 35mM, pH 5 citrate-disodium hydrogen phosphate buffer (the mass ratio of laccase, trehalose and citrate-disodium hydrogen phosphate buffer is 1:4:170) to obtain prepreg A; then chitosan oligosaccharide and gallic acid are dissolved in 30mM, pH 5.5 citrate-disodium hydrogen phosphate buffer (the mass ratio of chitosan oligosaccharide, gallic acid and citrate-disodium hydrogen phosphate buffer is 1:1.5:100) to obtain prepreg B; before use, prepreg A and prepreg B are mixed evenly at a volume ratio of 1:1.2, and then incubated at 6℃ with shaking for 20 min to obtain the composite premodifier.

[0061] The preparation method of the hydrophilic deep eutectic solvent is as follows: a glycerol-lactic acid-malic acid ternary system with a mass ratio of 2.5:1 (prepared by stirring and mixing glycerol, lactic acid and malic acid in a molar ratio of 2:1.5:0.75 at 50℃ for 35 min) is mixed with betaine to obtain a mixed system. L-proline is added to the mixed system at a mass ratio of 30:1, and the mixture is stirred at 100 rpm until a homogeneous transparent liquid is formed. The mixture is then aged at 55℃ for 1.5 h to obtain the hydrophilic deep eutectic solvent.

[0062] Performance testing The performance of the plant extracts (green tea extracts) prepared in Examples 1-3 and Comparative Examples 1-6 was tested.

[0063] Tea polyphenol extraction rate: The total content of tea polyphenols in the extract was determined by the Folin-Ciocalteu method. The extraction rate was calculated by combining the initial content of tea polyphenols in the raw material. The formula is: Extraction rate (%) = (Mass of tea polyphenols in the extract / Total mass of tea polyphenols in the raw material) × 100%; Tea polyphenol purity: The proportion of the target peak area to the total peak area is calculated by separating and purifying the tea polyphenol monomers (catechins, epicatechins, etc.) in the extract by high performance liquid chromatography (HPLC). Tea polyphenol retention rate: Based on the initial activity of tea polyphenols in the raw material, the antioxidant activity of tea polyphenols in the extract was determined by the DPPH free radical scavenging method, and the activity retention rate was calculated. Viscosity of the extraction system: The viscosity of the composite solvent system was measured using a rotational viscometer at the extraction temperature (4℃). Impurity residue: Protein content was determined by Coomassie Brilliant Blue G-250 staining method, and starch content was determined by anthrone colorimetric method.

[0064] The test results are shown in Table 1.

[0065] Table 1 Performance testing of green tea extracts prepared in Examples 1-3 and Comparative Examples 1-6 As can be seen from the data in Table 1, the green tea extracts prepared in Examples 1-3 all showed excellent performance in terms of core performance indicators: the extraction rate of tea polyphenols reached 82.3-86.7%, the purity was 91.5-93.8%, the retention rate was 95.2-96.5%, and the amount of impurities was low and the viscosity of the extraction system was suitable.

[0066] Comparative Example 1 omitted the pretreatment step of the composite pre-modifier, and the tea polyphenol extraction rate was only 68.5%, a decrease of 18.2% compared with Example 2. Other indicators showed no significant difference. The reason is that without the targeting effect of the "enzyme-mediator-oligosaccharide" composite pre-modifier, the dense structure of the plant cell wall was not gently deconstructed, and the composite solvent could not quickly penetrate into the cell, hindering the dissolution of the target component and resulting in a significant decrease in the extraction rate. However, since the subsequent low-temperature extraction and purification process was not changed, the purity, retention rate, and residual impurities did not fluctuate significantly.

[0067] The composite pre-modifier in Comparative Example 2 contained only pre-prepared solution A and lacked the mediators and oligosaccharides found in pre-prepared solution B. Its tea polyphenol extraction rate was 72.1%, a decrease of 14.6% compared to Example 2. This is because pre-prepared solution A only contained laccase and an enzyme stabilizer, lacking the mediating effect of phenolic acid mediators, which significantly reduced the degradation efficiency of laccase on the lignin-cellulose network of the cell wall. Simultaneously, the absence of oligosaccharides shielding intracellular active sites resulted in non-specific adsorption and loss of the target components. These two factors combined led to the decreased extraction rate.

[0068] The composite pre-modifier in Comparative Example 3 contained only pre-prepared solution B and lacked laccase and other enzymes found in pre-prepared solution A. Its tea polyphenol extraction rate was 70.3%, a decrease of 16.4% compared to Example 2. This is because pre-prepared solution B lacks the biocatalytic activity of laccase, and the phenolic acid mediator cannot effectively disassemble the cell wall structure on its own. It can only weakly improve the adsorption of the target component through oligosaccharides, failing to achieve the synergistic pretreatment effect of "enzyme-mediator-oligosaccharide," resulting in insufficient extraction efficiency.

[0069] Comparative Example 4's hydrophilic eutectic solvent was prepared using only a glycerol-lactic acid-malic acid ternary system and an aqueous solution of isopropanol and dipotassium glycyrrhizate. It contained no betaine, resulting in a tea polyphenol extraction rate of 65.8% and a purity of 87.2%. However, the extraction system exhibited a high viscosity of 52.8 mPa·s, indicating a significant overall decline in performance. This is because only a hydrogen bond donor system was used, without mixing with betaine hydrogen bond acceptors. This prevented the formation of a stable eutectic solvent hydrogen bond network, leading to insufficient solvent polarity and solubility, a significantly increased viscosity, and reduced mass transfer efficiency. Consequently, the dissolution of the target components decreased, and the separation of impurities was also affected.

[0070] Comparative Example 5, using a hydrophilic deep eutectic solvent prepared solely with an aqueous solution of betaine, isopropanol, and dipotassium glycyrrhizate, without a glycerol-lactic acid-malic acid ternary system, exhibited the lowest tea polyphenol extraction rate (63.2%) and retention rate (92.8%) among all groups. This is because the single hydrogen bond acceptor system lacks sufficient hydrogen bond donor sites, failing to form effective hydrogen bonds with polar target components such as tea polyphenols, resulting in extremely poor solubility. Simultaneously, the solvent system's insufficient stability weakens its protective effect on heat-sensitive components during low-temperature extraction, leading to a double decrease in both extraction and retention rates.

[0071] Comparative Example 6 only retained the second stage of pulsed ultrasonic treatment, resulting in a tea polyphenol retention rate of only 88.4%, a decrease of 8.1% compared to Example 2, and an extraction rate that also dropped to 75.6%. The reason is that the "wetting-disruption" two-stage design was not adopted. Although the single-stage high-power ultrasound can disrupt the cell wall, it lacks a low-power pre-permeation step, and the solvent does not uniformly wet the intercellular spaces, so some target components cannot be fully dissolved. In addition, continuous high-power ultrasound makes it difficult to control the local temperature rise, and heat-sensitive tea polyphenols undergo oxidative degradation, ultimately resulting in a simultaneous decrease in extraction and retention rates.

[0072] The above specific embodiments are merely explanations of this application and are not intended to limit this application. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for producing plant extracts by ultrasonic-assisted low-temperature extraction, characterized in that, Includes the following steps: S1. Raw material pretreatment: Vacuum freeze-dry the plant raw materials, ultrafine pulverize them, and sieve them to obtain plant micro powder; spray the plant micro powder with a composite premodifier, shake and incubate to obtain pretreated plant micro powder; S2. Preparation of composite solvent system: The hydrophilic deep eutectic solvent, low carbon alcohol and natural co-solvent are mixed evenly to obtain the composite solvent system; S3. Ultrasonic-assisted low-temperature extraction: After the pretreated plant micro powder is mixed evenly with the composite solvent system, the first stage of continuous ultrasonic treatment and the second stage of pulsed ultrasonic treatment are carried out in sequence to obtain a turbid extract. A natural clarifying agent is added and mixed evenly, and then allowed to stand to obtain a clear extract. S4. Low-temperature enrichment and purification: The clarified extract is concentrated under low-temperature reduced pressure to obtain a concentrate. The concentrate is then purified by adsorption with macroporous resin and freeze-dried under vacuum to obtain the plant extract.

2. The method for producing plant extracts by ultrasonic-assisted low-temperature extraction according to claim 1, characterized in that, In step S1, the temperature for vacuum freeze drying is -50℃ to -40℃, the vacuum degree is 10-30Pa, and the drying is carried out until the moisture content is ≤5%; the temperature for ultrafine pulverization is -15℃ to -5℃, and the time is 10-12min; and the material is passed through a 180-220 mesh sieve.

3. The method for producing plant extracts by ultrasonic-assisted low-temperature extraction according to claim 1, characterized in that, The preparation method of the composite premodifier in step S1 is as follows: Laccase and enzyme stabilizer were dissolved in citrate-disodium hydrogen phosphate buffer to obtain prepreg A; oligosaccharide and phenolic acid mediator were dissolved in citrate-disodium hydrogen phosphate buffer to obtain prepreg B; before use, prepreg A and prepreg B were mixed evenly and incubated by shaking to obtain composite premodifier.

4. The method for producing plant extracts by ultrasonic-assisted low-temperature extraction according to claim 3, characterized in that, The enzyme stabilizer is selected from one or more of trehalose, mannitol, and xylitol; the oligosaccharide is selected from one or more of chitosan oligosaccharide, xylooligosaccharide, and fructooligosaccharide; and the phenolic acid mediator is selected from one or more of gallic acid, ferulic acid, p-coumaric acid, and sinapic acid.

5. The method for producing plant extracts by ultrasonic-assisted low-temperature extraction according to claim 1, characterized in that, In step S2, the lower alcohol is isopropanol or 1,2-propanediol; the natural co-solvent is a 3-5% (w / w) dipotassium glycyrrhizate aqueous solution.

6. The method for producing plant extracts by ultrasonic-assisted low-temperature extraction according to claim 1, characterized in that, The method for preparing the hydrophilic deep eutectic solvent in step S2 is as follows: The glycerol-lactic acid-malic acid ternary system was mixed with betaine to obtain a mixed system. L-proline was added to the mixed system and stirred until a homogeneous and transparent liquid was formed. The mixture was then aged at a constant temperature to obtain a hydrophilic eutectic solvent.

7. The method for producing plant extracts by ultrasonic-assisted low-temperature extraction according to claim 1, characterized in that, In step S3, the natural clarifying agent consists of chitosan, sodium carboxymethyl cellulose, and water in a mass ratio of 1:2 to 4:

50.

8. The method for producing plant extracts by ultrasonic-assisted low-temperature extraction according to claim 1, characterized in that, In step S3, the power of the first stage of continuous ultrasound treatment is 180-220W, the frequency is 25-30kHz, and the time is 20-30min; the power of the second stage of pulsed ultrasound treatment is 280-320W, the frequency is 35-40kHz, the ultrasound lasts for 30-40s, the intermittent mode lasts for 15-25s, and the time is 30-50min.

9. The method for producing plant extracts by ultrasonic-assisted low-temperature extraction according to claim 1, characterized in that, In step S4, the vacuum degree of low-temperature vacuum concentration is -0.095MPa to -0.1MPa, the temperature is -10℃ to -5℃, and the concentration is reduced to 12-15% of the initial volume.

10. A plant extract prepared by the ultrasonic-assisted low-temperature extraction method for producing plant extracts according to any one of claims 1-9.

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