Process method for extracting trilobatin from sweet tea tender leaves

Abstract

The invention discloses a process method for extracting trilobatin from sweet tea tender leaves, and belongs to the technical field of trilobatin separation. The method comprises the following steps: countercurrent extraction, chromatographic separation and crystallization purification, and specifically comprises the following steps: by taking tender leaves of strigose hydrangea juvenile leaves as a raw material, treating the tender leaves of strigose hydrangea juvenile leaves by adopting an aqueous solution of mixed enzyme and a surfactant, and countercurrent extraction, so that the solubility of trilobatin in water is improved, the trilobatin is fully dissolved out, and the use of a large amount of organic extraction solvents is avoided; and based on the molecular sieve action of macroporous resin and the principle that the solubility of trilobatin in a low-temperature aqueous solution is reduced, the removal of components such as polysaccharide, protein and phloretin in the sweet tea tender leaves is realized, and high-purity trilobatin crystals are obtained. The method has the advantages of simple and efficient process, no need of specific equipment and suitability for industrial production, and has a wide application prospect in the field of trilobatin extraction.

Description

Technical Field

[0001] This invention relates to the field of trifolin separation technology, and in particular to a process for extracting trifolin from tender leaves of sweet tea. Background Technology

[0002] Tilobatin is a natural dihydrochalcone compound and a newly emerging functional sweetener. It is listed as a Recognized As Safe Substance (#2171) by the Joint FAO / WHO Expert Committee on Food Additives and is recognized as such by the American Association of Flavor and Extract Manufacturers (#4674). It is non-hygroscopic, non-deliquescent, 300 times sweeter than sucrose, and highly stable. It can replace sucrose and produces products with a unique flavor. Tilobatin was first discovered as a sweetener in 1961 from North American begonias (*Trilobatin is a dihydrochalcone compound). Malus spp. cv. It was first discovered in Adams, and then again in 1980 in plants of the Symplococephala family (…). Symplocos microcalyx Hay.), in Litsea cubeba in 1982 [ Lithocarpus litseifolius It was discovered in [Hance] Chun. Currently, Litsea cubeba (commonly known as sweet tea) has been cultivated on a large scale, providing ample resources for the development and utilization of trifolin.

[0003] Chinese invention patent CN108948104A discloses a process for extracting trifolin from Litsea cubeba leaves. The steps include S1: raw material crushing, S2: heating separation, S3: vacuum concentration, S4: extraction precipitation, and S5: crystallization. First, selected Litsea cubeba leaves are crushed. The crushed coarse powder is extracted with water, heated under reflux for separation, and the separated liquid is filtered to separate the residue and filtrate. After multiple extractions, the filtrates are combined and concentrated under vacuum. The concentrated solution is then extracted, followed by hot saturated crystallization. After multiple crystallizations, trifolin crystals are finally obtained. This invention uses water as the extraction solvent and a unique heated reflux separator combined with n-butanol for extraction to achieve high-purity trifolin. However, the process using non-standard equipment has poor applicability in large-scale production and places high demands on operator training, equipment maintenance, and compatibility with other subsystems. The above process uses n-butanol, which has good selectivity for trifolin, but n-butanol has a certain degree of toxicity, posing a safety hazard for the production of food or pharmaceutical additives (such as trifolin). Its extraction products cannot be used directly in food or pharmaceuticals and must undergo solvent removal and solvent conversion processes, which increases the complexity of the process. Furthermore, the treatment of waste n-butanol requires specific procedures, increasing environmental protection costs.

[0004] Chinese invention patent CN116063361A discloses an extraction method for trifolin from *Cephalotaxus fortunei*, as well as the application of trifolin. The method includes: extracting trifolin from *Cephalotaxus fortunei* leaves using a sonic-assisted reflux method at 55-75°C with an ethanol solution containing 40%-80% by volume, to obtain a first extract with a first purity of trifolin; purifying the first extract using an adsorption resin and an ethanol solution containing 50% by volume, to obtain a second extract with a second purity of trifolin; determining the trifolin content in the second extract using liquid chromatography; calculating the second purity value of trifolin based on the calculated trifolin content; and identifying the target trifolin as the second extract when the second purity value meets a preset purity standard for trifolin. This application uses ethanol as the extraction solvent and employs an ultrasonic cleaner combined with silica gel chromatography to separate and purify the trifolin. However, the extraction requires a large amount of ethanol, resulting in high solvent costs. Furthermore, the silica gel chromatography column used for purification needs to be replaced each time, leading to high economic costs and cumbersome, time-consuming operations. The chromatography process itself is also time-consuming, reducing extraction efficiency. This process is suitable for small-scale preparations in laboratories or for fine purification scenarios where cost is not a primary concern. Its application is limited in cost-sensitive, high-efficiency industrial preparation environments.

[0005] In summary, this paper provides a process for extracting trifolin from tender leaves of sweet tea, which achieves high-purity trifolin extraction in an efficient and convenient manner, overcomes the dependence on specific equipment, and avoids the use of large amounts of organic extraction solvents in the process. This is of great significance for expanding the application of trifolin. Summary of the Invention

[0006] In view of the above-mentioned deficiencies of the prior art, the present invention provides a process for extracting trifolin from tender leaves of sweet tea, comprising the following steps: (1) Countercurrent extraction: Take tender leaves of sweet tea, add an aqueous solution containing mixed enzymes and surfactants, perform countercurrent extraction, concentrate under reduced pressure, and obtain crude extract of sweet tea; The mixed enzymes include two or more of cellulase, pectinase, and trypsin; the surfactants are one or more of Tween, alkyl glycosides, and sodium stearate. (2) Chromatographic separation: The crude extract of sweet tea was loaded onto a macroporous adsorption resin, and then eluted with pure water and ethanol in a gradient. The eluted portion of trifolin was collected, concentrated under reduced pressure, and the ethanol was removed to obtain the refined extract. (3) Crystallization and purification: The extract is cooled by a program to obtain trifolin crystals, thus completing the extraction of trifolin.

[0007] Preferably, in step (1), the solid-liquid mass ratio of the sweet tea leaves to the aqueous solution containing mixed enzymes and surfactants is 1:10 to 1:40.

[0008] Preferably, in step (1), the concentration of the mixed enzyme is 50~120 g / L.

[0009] More preferably, the mixed enzyme is cellulase and pectinase, and the mass ratio of cellulase to pectinase is 1:4 to 3:1.

[0010] Preferably, in step (1), the concentration of the surfactant is 1 wt.% to 2 wt.%.

[0011] More preferably, the surfactant is an alkyl glycoside.

[0012] Alkyl glycosides are mild and safe surfactants with wide availability. They can be used in the food, cosmetics, and pharmaceutical industries, and also optimize extraction efficiency. They are a type of surfactant that is particularly suitable for the process of this invention.

[0013] Preferably, in step (1), the countercurrent extraction time is 60~90 min and the extraction temperature is 40~70 ℃.

[0014] Preferably, in step (2), the macroporous adsorption resin used is AB-8 macroporous adsorption resin.

[0015] AB-8 macroporous adsorption resin can be repeatedly regenerated and reused, meeting the needs of large-scale industrial production. Furthermore, it boasts high safety, uses water / ethanol as the eluent, and is relatively simple to operate, easily automatable, significantly reducing operating costs and process complexity.

[0016] Preferably, in step (2), elution is performed using aqueous ethanol solutions with concentrations of 20 vol.%, 40 vol.%, 60 vol.%, and 80 vol.%, and the trifolin elution fraction obtained from the 60 vol.% aqueous ethanol solution is collected, concentrated under reduced pressure, and the ethanol is removed to obtain a refined extract.

[0017] Preferably, in step (3), the programmed cooling steps are as follows: from 60 ℃ to 40 ℃, the temperature is reduced by 10~12 ℃ per hour; from 40 ℃ to 10 ℃, the temperature is reduced by 6~8 ℃ per hour; from 10 ℃ to 4 ℃, the temperature is reduced by 2~4 ℃ per hour to obtain trifolin glycoside crystals.

[0018] Based on the above technical solutions, the design concept and principle of this invention are as follows: This invention uses a mixed enzyme to treat the tender leaves of sweet tea, which can destroy the cell walls of plant cells and the layer of pectin on the surface of sweet tea leaves under relatively mild conditions, so as to fully dissolve trifolin; and combine surfactants with trifolin components to form micelles in aqueous solution, thereby improving the solubility of trifolin in water and avoiding the use of a large amount of organic extraction solvents; then, a crude extract of sweet tea is obtained by countercurrent extraction.

[0019] Existing technologies typically employ heating and decocting to process sweet tea leaves after enzyme solution treatment, and surfactants are used in the processing of essential oils containing non-polar volatile components. It is important to note that, unlike essential oil extraction which usually requires the destruction of volatile structures, trifolin extraction requires addressing the binding of glycosides to lipids / cell walls. This invention combines enzymatic hydrolysis with surfactants to achieve a synergistic effect. Surfactants reduce surface tension, enhance enzyme permeability to the cell wall, and promote the dissolution of trifolin. Countercurrent extraction is the core process in this step. This invention utilizes the countercurrent flow of solution and material to achieve efficient mass transfer, enabling continuous and efficient material contact, thereby reducing extraction time and energy consumption. In countercurrent extraction, the solvent and material are in countercurrent contact, maintaining a consistently high concentration gradient, thus accelerating the diffusion of trifolin from plant cells to the solvent. Compared to traditional soaking or decocting, countercurrent extraction avoids the limitation of trifolin extraction caused by solvent saturation, and also amplifies the effect of surfactants enhancing the solubility of trifolin, resulting in more thorough dissolution. Furthermore, compared to existing methods that use heating and boiling to treat enzyme solutions, this invention utilizes countercurrent extraction to overcome the technical obstacle of localized overheating in traditional boiling methods, and avoids prolonged high-temperature treatment by using continuous flow. After the cell walls are broken down during the enzymatic hydrolysis stage, countercurrent extraction rapidly dissolves the target substance, reducing the risk of oxidation or degradation and maintaining component stability, making it particularly suitable for the extraction of heat-sensitive components such as trifolin.

[0020] After obtaining the crude extract of sweet tea, this invention utilizes the molecular sieving effect of macroporous resin such as AB-8, based on the principle that the solubility of trifolin decreases in low-temperature aqueous solutions, to remove polysaccharides, proteins, phlorizin, and other components from the tender leaves of sweet tea through chromatographic separation and crystallization purification, obtaining high-purity trifolin crystals. For the processing of the refined extract, existing processes include drying (spray drying, freeze drying, etc.) or conventional gradient cooling crystallization to obtain trifolin. However, conventional drying methods easily form highly hygroscopic amorphous powders rather than crystals, leading to the decomposition of some components when high-temperature processing is involved. Conventional gradient cooling easily produces polycrystalline turbidity. Under the specific programmed cooling parameters of this invention, trifolin has high solubility at 60 ℃. This stage is designed with a relatively fast cooling rate of 10~12 ℃ / h, which can quickly cross its high solubility range, inducing crystal nucleus formation while avoiding prolonged high-temperature exposure. Compared with conventional cooling crystallization, this design significantly shortens the heat-sensitive period, which is beneficial for ensuring product activity. For the intermediate temperature range of 40 ℃ to 10 ℃, this is the critical stage for the growth of trifolin crystals. The solubility of trifolin decreases non-linearly with decreasing temperature; excessively rapid cooling can lead to a sudden increase in supersaturation, resulting in the formation of small, irregular crystals. This invention designs the cooling rate to 6~8 ℃ / h, matching the slope of the trifolin solubility curve, to ensure orderly crystal growth and improve crystal uniformity. Finally, in the low temperature range of 10 ℃ to 4 ℃, near the crystallization endpoint, the change in trifolin solubility slows down, but the reduced molecular motion at low temperatures can easily lead to crystal aggregation. A cooling rate of 2~4 ℃ / h allows molecules to fully arrange themselves within the crystal lattice, reducing internal stress and preventing crystal cracking or the inclusion of solvent impurities, ultimately yielding high-purity trifolin crystals.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects: This invention provides a process for extracting trifolin from tender leaves of sweet tea. The process involves treating the tender leaves with an aqueous solution of a mixed enzyme and surfactant, followed by countercurrent extraction. This improves the solubility of trifolin in water, ensuring complete dissolution and avoiding the use of large amounts of organic extraction solvents. Subsequently, utilizing the molecular sieving effect of macroporous resin and the principle that the solubility of trifolin decreases in low-temperature aqueous solutions, polysaccharides, proteins, phlorizin, and other components in the tender leaves of sweet tea are removed, yielding high-purity trifolin crystals. This method is simple, efficient, requires no specialized equipment, and is suitable for industrial production. Attached Figure Description

[0022] Figure 1 This is the high-performance liquid chromatography (HPLC) chromatogram of the crude extract of tender leaves of sweet tea 1; Figure 2 This is the HPLC chromatogram of trifolin crystals; Figure 3This is the mass spectrum ion diagram of trifolin crystals. Detailed Implementation

[0023] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0024] Example 1 This embodiment provides a process for extracting trifolin from tender leaves of sweet tea, the steps of which are as follows: (1) Countercurrent extraction: The cleaned young leaves of Litsea cubeba were added to an aqueous solution of alkyl glycoside, cellulase and pectinase at a solid-liquid mass ratio of 1:20, and the mixture was extracted countercurrently for 60 min at an extraction temperature of 40℃. The mass concentrations of alkyl glycoside, cellulase and pectinase in the aqueous solution were 1%, 40 g / L and 60 g / L, respectively. The extract was concentrated under reduced pressure to obtain a crude extract of sweet tea 1. (2) Chromatographic separation: The crude extract of sweet tea obtained in step (1) was loaded onto AB-8 macroporous adsorption resin, and then eluted with 6 column volumes of pure water, 20% ethanol, 40% ethanol, 60% ethanol and 80% ethanol in sequence. The 60% ethanol eluent was collected, concentrated under reduced pressure, and the ethanol was removed to obtain the refined extract. (3) The refined extract obtained in step (2) was placed in a cooling crystallizer and cooled by program: from 60 ℃ to 40 ℃, the temperature was reduced by 10 ℃ per hour; from 40 ℃ to 10 ℃, the temperature was reduced by 8 ℃ per hour; from 10 ℃ to 4 ℃, the temperature was reduced by 2 ℃ per hour, and trifolin crystals were obtained with a yield of 80.2%.

[0025] This embodiment follows the steps in the literature (Zhang Yazhou, Wang Tao, Zhu Jingjing, et al. Study on the determination of phlorizin and trifolin content in Litsea cubeba. 2018, 27(18):5-8), and uses high performance liquid chromatography for characterization. The test results of trifolin crystals in the crude extract of sweet tea tender leaves are as follows: Figure 1 , 2 As shown. The crystallization of trifolin was tested using mass spectrometry ion-pairing assay, and the results are as follows. Figure 3 As shown above, this method successfully produces high-purity trifolin crystals. The mass spectrometry ion spectrum of the trifolin crystals shows that 435.1321 represents the [MH] ratio of trifolin. -1 Peak, 273.0784, mass-to-charge ratio of [MH] for the nucleus structure of phlorizin, the parent nucleus of trifolin. -1 The peak confirmed that the crystal was composed of trifolin.

[0026] Example 2 This embodiment provides a process for extracting trifolin from tender leaves of sweet tea, the steps of which are as follows: (1) Countercurrent extraction: The cleaned young leaves of Litsea cubeba were added to an aqueous solution of alkyl glycoside, cellulase and pectinase at a solid-liquid mass ratio of 1:20, and the mixture was extracted countercurrently for 70 min at a temperature of 50 ℃. The mass concentrations of alkyl glycoside, cellulase and pectinase in the aqueous solution were 1.2%, 40 g / L and 60 g / L, respectively. The extract was concentrated under reduced pressure to obtain a crude extract of sweet tea. (2) Chromatographic separation: The crude extract of sweet tea obtained in step (1) was loaded onto AB-8 macroporous adsorption resin, and then eluted with 7 column volumes of pure water, 20% ethanol, 40% ethanol, 60% ethanol and 80% ethanol in sequence. The 60% ethanol eluent was collected, concentrated under reduced pressure, and the ethanol was removed to obtain the refined extract. (3) The purified extract obtained in step (2) was placed in a cooling crystallizer and cooled by a programmed temperature reduction: from 60 ℃ to 40 ℃ at a rate of 10 ℃ per hour; from 40 ℃ to 10 ℃ at a rate of 8 ℃ per hour; and from 10 ℃ to 4 ℃ at a rate of 2 ℃ per hour, to obtain trifolin crystals with a yield of 82.3%. The trifolin crystals were tested using the characterization method as described in Example 1, and the target trifolin component was obtained in this example.

[0027] Example 3 This embodiment provides a process for extracting trifolin from tender leaves of sweet tea, the steps of which are as follows: (1) Countercurrent extraction: The cleaned young leaves of Litsea cubeba were added to an aqueous solution of alkyl glycoside, cellulase and pectinase at a solid-liquid mass ratio of 1:20, and the mixture was extracted countercurrently for 70 min at a temperature of 55 ℃. The mass concentrations of alkyl glycoside, cellulase and pectinase in the aqueous solution were 1.5%, 40 g / L and 60 g / L, respectively. The extract was concentrated under reduced pressure to obtain a crude extract of sweet tea. (2) Chromatographic separation: The crude extract of sweet tea obtained in step (1) is loaded onto AB-8 macroporous adsorption resin, and then eluted with 8 column volumes of pure water, 20% ethanol, 40% ethanol, 60% ethanol and 80% ethanol in sequence. The 60% ethanol eluent is collected, concentrated under reduced pressure, and the ethanol is removed to obtain the refined extract. (3) The purified extract obtained in step (2) was placed in a cooling crystallizer and cooled by a programmed cooling process: from 60 ℃ to 40 ℃ at a rate of 10 ℃ per hour; from 40 ℃ to 10 ℃ at a rate of 8 ℃ per hour; and from 10 ℃ to 4 ℃ at a rate of 2 ℃ per hour, to obtain trifolin crystals with a yield of 80%. The trifolin crystals were tested using the characterization method as described in Example 1, and the target trifolin component was obtained in this example.

[0028] Example 4 This embodiment provides a process for extracting trifolin from tender leaves of sweet tea, the steps of which are as follows: (1) Countercurrent extraction: The cleaned young leaves of Litsea cubeba were added to an aqueous solution of alkyl glycoside, cellulase and pectinase at a solid-liquid mass ratio of 1:20, and the mixture was extracted countercurrently for 80 min at a temperature of 55 ℃. The mass concentrations of alkyl glycoside, cellulase and pectinase in the aqueous solution were 1.6%, 40 g / L and 60 g / L, respectively. The extract was concentrated under reduced pressure to obtain a crude extract of sweet tea. (2) Chromatographic separation: The crude extract of sweet tea obtained in step (1) was loaded onto AB-8 macroporous adsorption resin, and then eluted with 9 column volumes of pure water, 20% ethanol, 40% ethanol, 60% ethanol and 80% ethanol in sequence. The 60% ethanol eluent was collected, concentrated under reduced pressure, and the ethanol was removed to obtain the refined extract. (3) The purified extract obtained in step (2) was placed in a cooling crystallizer and cooled by a programmed temperature reduction: from 60 ℃ to 40 ℃ at a rate of 10 ℃ per hour; from 40 ℃ to 10 ℃ at a rate of 8 ℃ per hour; and from 10 ℃ to 4 ℃ at a rate of 2 ℃ per hour, to obtain trifolin crystals with a yield of 85.6%. The trifolin crystals were tested using the characterization method as described in Example 1, and the target trifolin component was obtained in this example.

[0029] Example 5 This embodiment provides a process for extracting trifolin from tender leaves of sweet tea, the steps of which are as follows: (1) Countercurrent extraction: The cleaned young leaves of Litsea cubeba were added to an aqueous solution of alkyl glycoside, cellulase and pectinase at a solid-liquid mass ratio of 1:20, and the mixture was extracted countercurrently for 90 min at a temperature of 60 ℃. The mass concentrations of alkyl glycoside, cellulase and pectinase in the aqueous solution were 1.8%, 40 g / L and 60 g / L, respectively. The extract was concentrated under reduced pressure to obtain a crude extract of sweet tea. (2) Chromatographic separation: The crude extract of sweet tea obtained in step (1) is loaded onto AB-8 macroporous adsorption resin, and then eluted with 8 column volumes of pure water, 20% ethanol, 40% ethanol, 60% ethanol and 80% ethanol in sequence. The 60% ethanol eluent is collected, concentrated under reduced pressure, and the ethanol is removed to obtain the refined extract. (3) The purified extract obtained in step (2) was placed in a cooling crystallizer and cooled by a programmed cooling process: from 60 ℃ to 40 ℃ at a rate of 10 ℃ per hour; from 40 ℃ to 10 ℃ at a rate of 8 ℃ per hour; and from 10 ℃ to 4 ℃ at a rate of 2 ℃ per hour, to obtain trifolin crystals with a yield of 91.2%. The trifolin crystals were tested using the characterization method described in Example 1, and the target trifolin component was obtained in this example.

[0030] Example 6 This embodiment provides a process for extracting trifolin from tender leaves of sweet tea, the steps of which are as follows: (1) Countercurrent extraction: The cleaned young leaves of Litsea cubeba were added to an aqueous solution of alkyl glycoside, cellulase and pectinase at a solid-liquid mass ratio of 1:20, and the mixture was extracted countercurrently for 90 min at a temperature of 55 ℃. The mass concentrations of alkyl glycoside, cellulase and pectinase in the aqueous solution were 2%, 40 g / L and 60 g / L, respectively. The extract was concentrated under reduced pressure to obtain a crude extract of sweet tea. (2) Chromatographic separation: The crude extract of sweet tea obtained in step (1) was loaded onto AB-8 macroporous adsorption resin, and then eluted with 10 column volumes of pure water, 20% ethanol, 40% ethanol, 60% ethanol and 80% ethanol in sequence. The 60% ethanol eluent was collected, concentrated under reduced pressure, and the ethanol was removed to obtain the refined extract. (3) The purified extract obtained in step (2) was placed in a cooling crystallizer and cooled by a programmed temperature reduction: from 60 ℃ to 40 ℃ at a rate of 10 ℃ per hour; from 40 ℃ to 10 ℃ at a rate of 8 ℃ per hour; and from 10 ℃ to 4 ℃ at a rate of 2 ℃ per hour, to obtain trifolin crystals with a yield of 90.5%. The trifolin crystals were tested using the characterization method as described in Example 1, and the target trifolin component was obtained in this example.

[0031] Since losses are inevitable during the extraction process, and crystal quality and activity are easily limited by existing processes, the extraction yield of conventional processes in this field is typically around 80%. This invention achieves the extraction of high-purity trifolin crystals with a yield >80%, and some embodiments reach a high yield level >90%, with the highest being 91.2%. The product quality and yield of this method are significantly superior to existing extraction processes.

[0032] In summary, this process utilizes an aqueous solution of mixed enzymes and surfactants to treat tender tea leaves and employs countercurrent extraction, thereby improving the solubility of trifolin in water and ensuring its complete dissolution, thus avoiding the use of large amounts of organic extraction solvents. Furthermore, by leveraging the molecular sieving effect of macroporous resin and the principle that the solubility of trifolin decreases in low-temperature aqueous solutions, the process removes polysaccharides, proteins, phlorizin, and other components from tender tea leaves, yielding high-purity trifolin crystals. This invention offers advantages such as a simple and efficient process, requiring no specialized equipment, and suitability for industrial production, demonstrating broad application prospects in the field of trifolin extraction.

[0033] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A process for extracting trifolin from tender leaves of sweet tea, characterized in that, Includes the following steps: (1) Countercurrent extraction: Take tender leaves of sweet tea, add an aqueous solution containing mixed enzymes and surfactants, perform countercurrent extraction, concentrate under reduced pressure, and obtain crude extract of sweet tea; The mixed enzymes include two or more of cellulase, pectinase, and trypsin; the surfactants are one or more of Tween, alkyl glycosides, and sodium stearate. (2) Chromatographic separation: The crude extract of sweet tea was loaded onto a macroporous adsorption resin, and then eluted with pure water and ethanol in a gradient. The eluted portion of trifolin was collected, concentrated under reduced pressure, and the ethanol was removed to obtain the refined extract. (3) Crystallization and purification: The extract is cooled by a program to obtain trifolin crystals, thus completing the extraction of trifolin.

2. The process for extracting trifolin from tender leaves of sweet tea according to claim 1, characterized in that: In step (1), the solid-liquid mass ratio of the sweet tea tender leaves to the aqueous solution containing mixed enzymes and surfactants is 1:10 to 1:

40.

3. The process for extracting trifolin from tender leaves of sweet tea according to claim 1, characterized in that: In step (1), the concentration of the mixed enzyme is 50~120 g / L.

4. The process for extracting trifolin from tender leaves of sweet tea according to claim 3, characterized in that: The mixed enzyme is cellulase and pectinase, with a mass ratio of cellulase to pectinase of 1:4 to 3:

1.

5. The process for extracting trifolin from tender leaves of sweet tea according to claim 1, characterized in that: In step (1), the concentration of the surfactant is 1 wt.% to 2 wt.%.

6. The process for extracting trifolin from tender leaves of sweet tea according to claim 5, characterized in that: The surfactant is an alkyl glycoside.

7. The process for extracting trifolin from tender leaves of sweet tea according to claim 1, characterized in that: In step (1), the countercurrent extraction time is 60~90 min and the extraction temperature is 40~70 ℃.

8. The process for extracting trifolin from tender leaves of sweet tea according to claim 1, characterized in that: In step (2), the macroporous adsorption resin used is AB-8 macroporous adsorption resin.

9. The process for extracting trifolin from tender leaves of sweet tea according to claim 1, characterized in that: In step (2), a gradient elution is performed using pure water and aqueous solutions of ethanol with concentrations of 20 vol.%, 40 vol.%, 60 vol.%, and 80 vol.%. The trifolin elution fraction obtained from the 60 vol.% aqueous solution of ethanol is collected, concentrated under reduced pressure, and the ethanol is removed to obtain the refined extract.

10. The process for extracting trifolin from tender leaves of sweet tea according to claim 1, characterized in that, In step (3), the temperature reduction process is as follows: from 60 ℃ to 40 ℃, the temperature is reduced by 10~12 ℃ per hour; from 40 ℃ to 10 ℃, the temperature is reduced by 6~8 ℃ per hour; from 10 ℃ to 4 ℃, the temperature is reduced by 2~4 ℃ per hour to obtain trifolin glycoside crystals.

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