Method for resource utilization of tea residue

Abstract

The application relates to the technical field of tea residue resource utilization, and discloses a method for resource utilization of tea residue, which comprises the following steps: 1) mixing pretreated tea residue powder with a eutectic solvent, with a material-liquid ratio of 1g: 30-70mL; 2) adding a composite enzyme into the mixture according to 1-5% of the mass of the tea residue powder in the mixture to perform enzymolysis; 3) ultrasonically extracting the enzymolysis product in step 2), centrifuging, and collecting supernatant; and 4) separating and purifying the supernatant in step 3). The method is based on the synergistic extraction of the eutectic solvent, combines the composite enzyme enzymolysis and the ultrasonic extraction process, improves the extraction efficiency of functional components in the tea residue, and enables subsequent separation and purification through efficient extraction, so that the tea residue is further developed and utilized, a new idea is provided for industrialization, and the method has positive significance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of resource utilization of tea residue, and particularly relates to a method for resource utilization of tea residue. BACKGROUND

[0002] Tea is rich in various bioactive substances, and the most important active substance is tea polyphenol. Tea polyphenol is a general term for polyphenols including catechins, flavonoids, anthocyanidins and phenolic acids, and has beneficial effects such as anticancer, anti-inflammatory, antioxidant and antiviral effects. Flavonoids are one of the core components of tea polyphenols and belong to a subclass of polyphenols. For example, flavonoids include quercetin, kaempferol and other monomers and glycoside forms thereof; flavonols such as rutin are also important components of tea polyphenols and have various pharmacological activities such as anti-inflammatory, antibacterial, hypoglycemic, hypolipidemic, antihypertensive, antioxidant, anti-atherosclerotic, hepatoprotective and antitumor activities.

[0003] Tea residue is a product after tea leaves are extracted, and is often discarded in large quantities, resulting in waste and environmental pollution. However, tea residue is still rich in a large amount of tea polyphenol, especially flavonoids, which have high development value. This is because tea polyphenol (including flavonoids) in tea leaves exists in two forms, i.e. “free state” (which can be directly dissolved in water or a conventional solvent) and “bound state” (which is combined with proteins, polysaccharides and cellulose through hydrogen bonds and hydrophobic interactions). In the brewing process, the free-state components are almost completely dissolved, while the bound-state components cannot be directly separated because they are tightly combined with macromolecules and can only remain in the tea residue. Most of the existing methods for extracting tea polyphenol from tea leaves are not suitable for extracting tea polyphenol from tea residue. Therefore, a new method is needed for extracting tea polyphenol, especially flavonoids, from tea residue.

[0004] How to turn a large amount of tea residue into treasure and improve the added value of tea residue is the purpose of the present application, and how to effectively extract and separate these functional components by using a simple, low-cost and effective method is the main direction of the present application. SUMMARY

[0005] In order to overcome the problems of low extraction efficiency, low resource utilization rate, environmental pollution and high cost in the prior art, the present application provides a method for resource utilization of tea residue. The method is based on the synergistic extraction of eutectic solvent, combined with the use of complex enzyme enzymolysis and ultrasonic extraction process, which improves the extraction efficiency of functional components in tea residue. Efficient extraction enables subsequent separation and purification, thereby further developing and utilizing tea residue, providing new ideas for industrialization and having positive significance.

[0006] In order to achieve the above purpose, the present application provides a method for resource utilization of tea residue, comprising the following steps:

[0007] 1) mixing the pretreated tea residue powder with a deep eutectic solvent, the ratio of material to liquid being 1g:30-70mL; wherein the deep eutectic solvent has a water content of 20-50% by volume;

[0008] 2) adding a compound enzyme to the mixture of step 1) for enzymolysis, the amount of the compound enzyme being 1-5% of the mass of the tea residue powder in the mixture; wherein the compound enzyme comprises 5-8 parts by weight of an extraction enzyme and 2-5 parts by weight of a protective enzyme, the extraction enzyme comprises 5-7 parts by weight of a cellulase and 3-5 parts by weight of a pectinase, and the protective enzyme comprises 4-7 parts by weight of an acid protease and 1-2 parts by weight of SOD;

[0009] 3) ultrasonic extraction of the enzymolysis product of step 2), centrifugation, and collection of the supernatant;

[0010] 4) separation and purification of the supernatant of step 3).

[0011] Compared with the prior art, the present application has at least the following beneficial effects:

[0012] (1) The present application first applies a deep eutectic solvent to the extraction of functional components in tea residue, and uses the strong hydrogen bonding ability to form new hydrogen bonds between the hydrogen bond donors / acceptors in the molecular structure of the deep eutectic solvent and the hydroxyl groups and carbonyl groups of the functional components in the tea residue in a combined state (combined with proteins, polysaccharides and cellulose through hydrogen bonds and hydrophobic interactions), thereby breaking the bonds between them and proteins and polysaccharides and releasing the originally "insoluble" combined state components.

[0013] (2) The present application simultaneously adds a protective enzyme and an extraction enzyme into the extraction system of tea residue, thereby inhibiting the activities of polyphenol oxidase (PPO) and peroxidase (POD), achieving the synergistic effect of "protection-enzymolysis-extraction", solving the "enzyme activity compatibility" (avoiding mutual interference) and "action timing matching" (ensuring the simultaneous effect of breaking the wall and protection), and simplifying the operation process and achieving excellent economy.

[0014] (3) Based on the synergistic extraction of the deep eutectic solvent, combined with the enzymolysis of the compound enzyme and the ultrasonic extraction process, the present application improves the extraction efficiency of the functional components in the tea residue, and the efficient extraction enables subsequent separation and purification, thereby further developing and utilizing the tea residue and providing a new idea for industrialization, which has a positive significance. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is the tea residue collected in an embodiment of the present application;

[0016] Figure 2 is the pretreated tea residue powder in an embodiment of the present application;

[0017] Figure 3 is the prepared deep eutectic solvent in an embodiment of the present application;

[0018] Figure 4 is a mixture of tea residue powder and deep eutectic solvent in an embodiment of the present application;

[0019] Figure 5 is the supernatant after centrifugation in an embodiment of the present application. DETAILED DESCRIPTION

[0020] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges and the values are approximate values and should be understood to include values approximately near these ranges and values within these ranges. For ranges with endpoints, the endpoints are included in the range. For ranges without endpoints, the range extends to the endpoints of the underlying values. The endpoints of the ranges and the values are not limited to the precise values stated. The endpoints of the ranges and the values should be understood to include values approximately near these ranges and values within these ranges. The endpoints of the ranges and the values can be combined with each other to form one or more new ranges, which should be considered within the scope of the present disclosure.

[0021] According to one aspect of the present application, a method for resource utilization of tea residue is provided, comprising the following steps:

[0022] 1) mixing the pretreated tea residue powder with a deep eutectic solvent, the ratio of material to liquid being 1g:30-70mL; wherein the deep eutectic solvent has a water content of 20-50% by volume;

[0023] 2) adding a composite enzyme to the mixture of step 1) for enzymatic hydrolysis, the amount of the composite enzyme being 1-5% of the mass of the tea residue powder in the mixture; wherein the composite enzyme comprises 5-8 parts by weight of an extraction enzyme and 2-5 parts by weight of a protective enzyme, the extraction enzyme comprises 5-7 parts by weight of a cellulase and 3-5 parts by weight of a pectinase, and the protective enzyme comprises 4-7 parts by weight of an acid protease and 1-2 parts by weight of SOD;

[0024] 3) ultrasonic extraction of the enzymatic hydrolysis product of step 2), centrifugation, and collection of the supernatant;

[0025] 4) separation and purification of the supernatant of step 3).

[0026] In the present application, the raw material is selected to be tea residue. In addition to the purpose of "waste to treasure", the inventors have further considered that in addition to tea polyphenols, the finished tea also contains a large amount of impurity components such as protein, polysaccharide, caffeine, pigment, etc. These components also interact with the deep eutectic solvent, resulting in the "effective action site" of the deep eutectic solvent being occupied by impurities, and part of the deep eutectic solvent preferentially dissolving the impurities, thereby reducing the extraction efficiency of the target components. After brewing, most of the soluble impurities (such as caffeine, free protein, and small molecular polysaccharide) have been lost with the tea soup, and the deep eutectic solvent can more "accurately" act on the combined functional components, reducing the competition reaction with impurities, thereby improving the extraction rate of functional components in tea residue.

[0027] In the present application, the inventors found that tea dregs contain polyphenol oxidase (PPO) and peroxidase (POD) by nature: PPO can directly catalyze the oxidation of tea polyphenols into quinone substances, and POD uses H2O2 as a substrate to accelerate the oxidation of tea polyphenols. Therefore, anti-browning enzymes (essentially "enzyme inhibitors" enzymes) are needed to inhibit the activity of PPO / POD to protect tea polyphenols from "blocking endogenous oxidase". In an embodiment of the present application, the "enzyme inhibitor" enzyme, i.e. the protective enzyme, uses acid protease and SOD (superoxide dismutase) to act together.

[0028] In the present application, the inventors screened acid protease and SOD (superoxide dismutase) enzymes that maintain high activity in the pH range of 4.0-6.0 and the temperature range of 40-60℃. These two enzymes are combined only to inhibit the activity of polyphenol oxidase (PPO) and peroxidase (POD), and will not hydrolyze the extraction enzyme, nor will it affect the extraction effect of the extraction enzyme, i.e. cellulase and pectinase, which can efficiently break the wall and solve the adhesion. On this basis, the inventors experimentally obtained the appropriate weight ratio. In an embodiment of the present application, the composite enzyme includes 5-7 parts by weight of extraction enzyme and 3-5 parts by weight of protective enzyme, the extraction enzyme includes 5-6 parts by weight of cellulase and 4-5 parts by weight of pectinase, and the protective enzyme includes 5-6 parts by weight of acid protease and 1-2 parts by weight of SOD. The present application simultaneously adds protective enzymes and extraction enzymes into the extraction system of tea dregs, realizes the synergistic effect of "protection-enzymolysis-extraction", solves the "enzyme activity compatibility" (avoiding mutual interference) and "action timing matching" (ensuring the synchronization of breaking the wall and protection), realizes, and simplifies the operation process, and has excellent economy.

[0029] In the present application, the method of resource utilization of tea dregs is based on the synergistic extraction of eutectic solvent, combined with the use of composite enzyme enzymolysis and ultrasonic extraction process, which improves the extraction efficiency of functional components in tea dregs. Efficient extraction enables subsequent separation and purification, thereby further developing and utilizing tea dregs, providing new ideas for industrialization, and having positive significance.

[0030] According to an embodiment of the present application, the separation and purification of step 4) comprises the following scheme A: the supernatant of step 3) is added with a precipitant at a ratio of 1-5% w / v, the system is made to produce flocculent precipitate by mechanical stirring, the stirring is continued until the volume of the precipitate accounts for 70-90% of the total volume of the system, a buffer is added to make the pH of the reaction system 5.5-6.5, the stirring is continued for 10-30 min, and then centrifugation is performed; the precipitate is dissolved, filtered, centrifuged, and extracted, the organic layer extract is collected, vacuum dried, and the product tea polyphenol solid powder is obtained.

[0031] In some embodiments, the precipitant includes at least one of ZnCl2, AlCl3, FeCl3, and CaCl2.

[0032] In some embodiments, the buffer solution comprises an aqueous solution of NaHCO3 with a mass fraction of 10-20%.

[0033] In some embodiments, the dissolution is performed using 1-3 mol / L sulfuric acid.

[0034] In some embodiments, the extraction is performed using an equal volume of ethyl acetate.

[0035] In this invention, there are no special requirements for the centrifugation conditions. Generally, the precipitate can be removed after centrifugation at 10000 r / min for 10 min.

[0036] In this invention, the conditions for operations such as dissolving the precipitate, filtration, centrifugation, and extraction are all applicable to the conventional conditions for such operations in this field, and will not be elaborated further here.

[0037] In this invention, there are no special requirements for the vacuum drying conditions. Generally, vacuum drying is carried out at 45-75℃ for 3-6 hours.

[0038] According to one embodiment of the present invention, the separation and purification in step 4) includes the following scheme B: Take the supernatant from step 3) and add it to the macroporous resin at a ratio of 5-10% w / v, shake it in a water bath, and then elute the adsorbed macroporous resin sequentially with water and Na2CO3 aqueous solution with pH 8.5-9.5 as eluents. Collect the eluent of Na2CO3 aqueous solution, adjust the pH to 3-5, and then elute sequentially with water and 60-80% v / v ethanol aqueous solution. Collect the eluent of ethanol aqueous solution, concentrate and dry it to obtain the product flavonoid solid powder.

[0039] In some embodiments, the macroporous resin includes at least one of AB-8, D101, X-5, and NKA-9.

[0040] In this invention, the macroporous resin used generally undergoes the following pretreatment: the macroporous resin is soaked in 95% ethanol for 24 hours, then soaked in 5% HCl solution and 4% NaOH solution for 3 hours respectively, and washed with deionized water until it is odorless and neutral during each soaking. This pretreatment is then used for future reference. This is merely one pretreatment method and does not limit the scope of this invention.

[0041] In some embodiments, the conditions for the water bath oscillation include a temperature of 20-30°C.

[0042] In some embodiments, the conditions for water bath oscillation include a rotation speed of 50-200 r / min.

[0043] In some embodiments, the conditions for water bath oscillation include a time of 12-36 hours.

[0044] In this invention, the conditions for operations such as elution, concentration and drying are all applicable to the conventional conditions for such operations in this field, and will not be elaborated further here.

[0045] According to one embodiment of the present invention, in step 1), the pretreatment includes: drying the collected tea dregs at 40-50℃, grinding them, and storing them at -15 to -25℃ for later use.

[0046] According to one embodiment of the present invention, in step 1), the eutectic solvent includes a hydrogen bond acceptor and a hydrogen bond donor.

[0047] According to one embodiment of the present invention, in step 1), the hydrogen bond acceptor includes choline chloride and / or betaine.

[0048] According to one embodiment of the present invention, in step 1), the hydrogen bond donor includes at least one of ethylene glycol, 1,4-butanediol, glycerol, 1,3-butanediol, levulinic acid, malic acid, urea, glycerol, and glucose.

[0049] According to one embodiment of the present invention, in step 1), the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:4-6.

[0050] According to one embodiment of the present invention, in step 2), the conditions for enzymatic hydrolysis include adjusting the pH of the system to 4-6.

[0051] According to one embodiment of the present invention, in step 2), the conditions for enzymatic hydrolysis include: the enzymatic hydrolysis temperature is 40-60℃.

[0052] According to one embodiment of the present invention, in step 2), the conditions for enzymatic hydrolysis include: the enzymatic hydrolysis time is 2-3 hours.

[0053] According to one embodiment of the present invention, in step 2), the conditions for enzymatic hydrolysis include: boiling for 3-4 minutes after enzymatic hydrolysis to inactivate the enzyme and obtain the enzymatic hydrolysis product.

[0054] According to one embodiment of the present invention, in step 2), the cellulase is 10,000-30,000 U / g.

[0055] According to one embodiment of the present invention, in step 2), the pectinase is 0.5-1.5 million U / g.

[0056] According to one embodiment of the present invention, in step 2), the acidic protease is 20,000-40,000 U / g.

[0057] According to one embodiment of the present invention, in step 2), the SOD is 0.1-0.5 million U / g.

[0058] According to one embodiment of the present invention, in step 3), the conditions for ultrasonic extraction include a temperature of 40-60°C.

[0059] According to one embodiment of the present invention, in step 3), the conditions for ultrasonic extraction include a power of 200-400w.

[0060] According to one embodiment of the present invention, in step 3), the conditions for ultrasonic extraction include a time of 40-60 min.

[0061] The following will explore the optimal implementation conditions of the present invention through exploratory examples, and compare the experimental effects of the present invention with those of the examples and comparative examples. 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.

[0062] In the following exploratory examples and comparative examples:

[0063] Tea dregs are solid residues produced during the brewing, processing, or use of tea leaves;

[0064] Rutin and gallic acid standards (purity ≥98%) were provided by Beijing Bailingwei Technology Co., Ltd.; choline chloride, betaine, cellulase, pectinase, acidic protease, superoxide dismutase (SOD), zinc chloride (ZnCl2), aluminum chloride (AlCl3), ferric chloride (FeCl3), calcium chloride (CaCl2), Folin-Ciocalteu, 1,4-butanediol, levulinic acid, urea, malic acid, glycerol, glucose, and 1,3-butanediol were all analytical grade and sourced from Shanghai Aladdin Biochemical Technology Co., Ltd.; ethylene glycol, glycerol, sodium nitrite, aluminum nitrate, sodium hydroxide, sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), sulfuric acid, and ethyl acetate were all analytical grade and sourced from Sichuan Xilong Scientific Co., Ltd.; methanol and ethanol were analytical grade and purchased from Beijing Bailingwei Technology Co., Ltd.; AB-8, D101, X-5, and NKA-9 macroporous resins were purchased from Beijing Solarbio Technology Co., Ltd.

[0065] The instruments used in the following exploratory examples and comparative examples are from:

[0066] SpectraMax Paradigm microplate reader (Shanghai Meigu Molecular Instruments Co., Ltd.);

[0067] KQ-800KDE High-Power CNC Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0068] BSA224S electronic analytical balance (Sartorius Trading Co., Ltd., Germany);

[0069] TGL-16aR High-Speed ​​Tabletop Refrigerated Centrifuge (Shanghai Anting Scientific Instrument Factory);

[0070] JXFSTPRP-CL Fully Automatic Sample Cryogenic Grinding Instrument (Shanghai Jingxin Industrial Development Co., Ltd.);

[0071] B15-3 Intelligent Thermostatic Timed Magnetic Stirrer (Shanghai Jingxin Industrial Development Co., Ltd.);

[0072] Vacuum freeze-drying concentrator (Thermo Fisher Scientific, Inc., USA);

[0073] All water was prepared using the Milli-Q pure water preparation system.

[0074] In the following exploratory examples and comparative examples:

[0075] Extraction rate calculation: Take the supernatant obtained in step 3), dilute it according to the concentration by a dilution factor of N, then measure the absorbance value according to the method in the standard curve, and calculate the extraction amount of polyphenols and flavonoids respectively according to the standard curve:

[0076] ;

[0077] In the formula: W is the extraction amount of polyphenols and flavonoids, mg / g; C is the concentration of polyphenols and flavonoids after being substituted into the standard curve, µg / mL; V is the volume of the supernatant, mL; N is the dilution factor; M is the mass of tea residue powder, g.

[0078] Tea residue pretreatment: such as Figures 1 to 2 As shown, the collected tea dregs are dried in a 45℃ oven, ground into tea powder, and stored at -20℃ for later use.

[0079] Preparation Example 1

[0080] Preparation of polyphenol standard curve: Weigh 100 mg of gallic acid standard into a 100 mL volumetric flask, dilute to the mark with water, and shake well to obtain a 1 mg / mL gallic acid standard solution. Take 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, and 5.0 mL of each solution into 100 mL volumetric flasks, dilute to the mark with water, and shake well to prepare stock solutions with concentrations of 10 µg / mL, 20 µg / mL, 30 µg / mL, 40 µg / mL, and 50 µg / mL, respectively. Take 1 mL of each stock solution into a 10 mL test tube, add 5 mL of 10% Folin-Ciocalteu reagent, react for 5 min, then add 4 mL of 7.5% sodium carbonate solution, dilute to the mark with water, shake well, and incubate at room temperature in the dark for 30 min. Using water as a reference solution, the absorbance was measured by spectrophotometer at a wavelength of 765 nm. A standard curve was plotted with gallic acid concentration (μg / mL) on the x-axis and absorbance (A) on the y-axis. Linear fitting was performed using the least squares method to obtain the regression equation; the gallic acid standard curve equation can be expressed as A = 0.0034C + 0.1112(R² + π / 2)². 2 =0.9900), where C is the mass concentration (μg / mL) and A is the absorbance value.

[0081] Preparation Example 2

[0082] Preparation of flavonoid standard curve: Weigh 100 mg of rutin standard into a 100 mL volumetric flask, dilute to the mark with water, and shake well to obtain a 1 mg / mL rutin standard solution. Dilute 4 mL of the standard solution with water to 25 mL to prepare a 0.16 mg / mL rutin standard solution. Pipette 0, 0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 mL of the 0.16 mg / mL rutin standard solution into separate 10 mL volumetric flasks. Add 0.6 mL of sodium nitrite solution, shake well, and let stand for 6 min. Then add 0.5 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 min. Add 3 mL of 1 mol / L NaOH solution, dilute to the mark with distilled water to 10 mL, shake well, and let stand for 15 min. Using water as a reference solution, measure the absorbance at 510 nm. Plot a standard curve with rutin mass concentration (μg / mL) on the x-axis and absorbance (A) on the y-axis. Linear fitting was performed using the least squares method to obtain the regression equation; the rutin standard curve equation can be expressed as A = 0.0078C + 5 × 10⁻⁶. -4 (R) 2 =0.9997), where C is the mass concentration (μg / mL) and A is the absorbance value.

[0083] Exploration Example 1

[0084] 1) Take 0.1g of pretreated tea residue powder and place it in 18 test tubes. Add 18 kinds of eutectic solvents listed in Table 1 below and mix them. The material-to-liquid ratio is 1g:40mL.

[0085] 2) Add 0.002g of a complex enzyme to the mixture in step 1) for enzymatic hydrolysis, wherein the complex enzyme comprises 6 parts by weight of an extractive enzyme and 4 parts by weight of a protective enzyme; the extractive enzyme comprises 5 parts by weight of cellulase and 5 parts by weight of pectinase, and the protective enzyme comprises 4 parts by weight of acidic protease and 1 part by weight of SOD.

[0086] 3) Extract the enzymatic hydrolysis product from step 2) at 40°C with ultrasonic power of 300W for 1 hour, then centrifuge at 10000r / min for 10 minutes and collect the supernatant.

[0087] Based on a hydrogen bond acceptor to hydrogen bond donor molar ratio of 1:2 and a water content of 20%, 18 different types of eutectic solvents were designed (Table 1). Stirring was performed using a magnetic stirrer, and the water bath temperature was controlled at 80℃. After heating for 1 hour, as follows... Figure 3 As shown, a transparent and homogeneous liquid is formed and no crystals precipitate at room temperature.

[0088] Table 1 Different types of eutectic solvents

[0089]

[0090] The extraction yields of polyphenols and flavonoids were calculated based on the standard curves, and the effects of different types of eutectic solvents on the extraction content of polyphenols and flavonoids were obtained (Table 2). The optimal eutectic solvent was then selected.

[0091] Table 2. Effects of different eutectic solvents on the extraction yield (mg / g) of polyphenols and flavonoids from tea residue.

[0092]

[0093] As shown in Table 2, DES-16 (betaine / urea) had the best extraction effect, with an extraction yield of 152.12 mg / g of polyphenols and 19.57 mg / g of flavonoids, both significantly higher than the extraction effects of other eutectic solvents. This may be because the diffusion power of this combination is close to the polarity of the tea residue polyphenol and flavonoid extracts, which is conducive to the dissolution and diffusion of polyphenols and flavonoids.

[0094] Therefore, in subsequent exploratory examples, betaine / urea was used as the optimal eutectic extraction solvent for extracting polyphenols and flavonoids from tea residue.

[0095] Exploration Example 2

[0096] 1) Take 0.1g of pretreated tea residue powder and place it in 7 test tubes. Add an equal amount of eutectic solvent (the molar ratio of betaine to urea is 1:2, and the water content is 20%) and mix. The material-to-liquid ratio is 1g:40mL.

[0097] 2) Add 0g, 0.001g, 0.002g, 0.003g, 0.004g, 0.005g, and 0.006g of a complex enzyme to the seven test tubes in step 1) for enzymatic hydrolysis. The complex enzyme comprises 6 parts by weight of an extracting enzyme and 4 parts by weight of a protective enzyme. The extracting enzyme comprises 5 parts by weight of cellulase and 5 parts by weight of pectinase. The protective enzyme comprises 4 parts by weight of acidic protease and 1 part by weight of SOD.

[0098] 3) Extract the enzymatic hydrolysis product from step 2) at 40°C with ultrasonic power of 300W for 1 hour, then centrifuge at 10000r / min for 10 minutes and collect the supernatant.

[0099] Effects of different amounts of compound enzymes on the extraction yield of polyphenols and flavonoids:

[0100] Table 3 shows that the extraction yields of polyphenols and flavonoids initially increased and then decreased with increasing enzyme concentration. This may be because increasing enzyme concentration disrupts cell walls, leading to the continuous dissolution of polyphenols and flavonoids. However, excessive enzyme concentration can damage the structure of polyphenols and flavonoids, resulting in a decrease in their extraction yield. The extraction efficiency of polyphenols and flavonoids was highest when the enzyme concentration was 3%. Therefore, adding 3% of the compound enzyme is the optimal enzyme concentration for extracting polyphenols and flavonoids from tea residue.

[0101] Table 3. Effects of different amounts of compound enzymes on the extraction yield (mg / g) of polyphenols and flavonoids from tea residue.

[0102]

[0103] Exploration Example 3

[0104] 1) Take 0.1g of pretreated tea residue powder and place it in 5 test tubes. Add equal amounts of eutectic solvent (betaine:urea molar ratio of 1:2, water content of 20%) to each test tube and mix. The material-to-liquid ratio is 1g:40mL.

[0105] 2) Add 0.003g of the complex enzyme to each of the five test tubes from step 1) for enzymatic hydrolysis; wherein the complex enzyme comprises different weight ratios: 4 parts by weight of extracting enzyme and 6 parts by weight of protective enzyme, 5 parts by weight of extracting enzyme and 5 parts by weight of protective enzyme, 6 parts by weight of extracting enzyme and 4 parts by weight of protective enzyme, 7 parts by weight of extracting enzyme and 3 parts by weight of protective enzyme, and 8 parts by weight of extracting enzyme and 2 parts by weight of protective enzyme; the extracting enzyme comprises 5 parts by weight of cellulase and 5 parts by weight of pectinase, and the protective enzyme comprises 5 parts by weight of acidic protease and 1 part by weight of SOD;

[0106] 3) Extract the enzymatic hydrolysis product from step 2) at 40°C with ultrasonic power of 300W for 1 hour, then centrifuge at 10000r / min for 10 minutes and collect the supernatant.

[0107] Effects of different compound enzyme ratios on the extraction yield of polyphenols and flavonoids:

[0108] Table 4. Effect of compound enzyme ratio on the extraction yield (mg / g) of polyphenols and flavonoids from tea residue.

[0109]

[0110] Table 4 shows that the extraction yield of polyphenols and flavonoids initially increases and then decreases with increasing enzyme ratios. The extraction yield is low at ratios of 4:6 and 5:5, possibly due to excessive protective enzymes, resulting in light oxidation but insufficient cell wall disruption. A ratio of 6:4 yields moderate extraction, with moderate protective enzyme activity and moderate cell wall disruption. The highest extraction yield of polyphenols and flavonoids is achieved at a ratio of 7:3, likely a universally optimal solution based on the dynamic balance between enzymatic hydrolysis efficiency and oxidative protection—this ratio best matches the raw material characteristics of tea residue (fiber structure, oxidase activity), enzyme functional division, and extraction process requirements, ultimately achieving the comprehensive goal of high polyphenol and flavonoid extraction yield and low oxidative loss. An 8:2 ratio also results in low extraction, possibly due to excessive extracting enzymes, leading to good cell wall disruption but heavy oxidation. Therefore, 7:3 is the optimal enzyme ratio for extracting polyphenols and flavonoids from tea residue.

[0111] Exploration Example 4

[0112] 1) Take 0.1g of pretreated tea residue powder and place it in 11 test tubes. Add an equal amount of eutectic solvent (the molar ratio of betaine to urea is 1:2, and the water content is 20%) and mix. The material-to-liquid ratio is 1g:40mL.

[0113] 2) Add 0.003g of the complex enzyme to each of the 11 test tubes from step 1) for enzymatic hydrolysis; wherein the complex enzyme comprises 7 parts by weight of extracting enzyme and 3 parts by weight of protective enzyme; the extracting enzyme comprises different weight ratios as follows: 4 parts by weight of cellulase and 5 parts by weight of pectinase, 5 parts by weight of cellulase and 5 parts by weight of pectinase, 6 parts by weight of cellulase and 5 parts by weight of pectinase, 5 parts by weight of cellulase and 4 parts by weight of pectinase, 7 parts by weight of cellulase and 5 parts by weight of pectinase, 6 parts by weight of cellulase and 4 parts by weight of pectinase, 5 parts by weight of cellulase and 3 parts by weight of pectinase, 7 parts by weight of cellulase and 4 parts by weight of pectinase, 6 parts by weight of cellulase and 3 parts by weight of pectinase, 7 parts by weight of cellulase and 3 parts by weight of pectinase, and 8 parts by weight of cellulase and 3 parts by weight of pectinase; the protective enzyme comprises 5 parts by weight of acidic protease and 1 part by weight of SOD.

[0114] 3) Extract the enzymatic hydrolysis product from step 2) at 40°C with ultrasonic power of 300W for 1 hour, then centrifuge at 10000r / min for 10 minutes and collect the supernatant.

[0115] Effects of different extraction enzyme ratios on the extraction yield of polyphenols and flavonoids:

[0116] Under the optimal compound enzyme ratio, the proportion of extraction enzymes (cellulase and pectinase) was optimized, and the results are shown in Table 5. When the ratio of cellulase to pectinase was 4:5, there was too much pectinase, and the cellulose skeleton was not sufficiently destroyed. When the ratio was 6:4, cellulase fully destroyed the skeleton, and pectinase simultaneously decomposed the interstitial matrix, resulting in the highest extraction yield. When the ratios were 7:3 and 8:3, there was too much cellulase, and the pectin interstitial matrix encapsulated the cellulose, preventing the enzyme from contacting the skeleton, resulting in low extraction yields. Therefore, a cellulase to pectinase ratio of 6:4 is the optimal extraction enzyme ratio for extracting polyphenols and flavonoids from tea residue.

[0117] Table 5. Effect of extraction enzyme ratio on the extraction yield (mg / g) of polyphenols and flavonoids from tea residue.

[0118]

[0119] Exploration Example 5

[0120] 1) Take 0.1g of pretreated tea residue powder and place it in 11 test tubes. Add an equal amount of eutectic solvent (the molar ratio of betaine to urea is 1:2, and the water content is 20%) and mix. The material-to-liquid ratio is 1g:40mL.

[0121] 2) Add 0.003g of the complex enzyme to each of the 11 test tubes in step 1) for enzymatic hydrolysis; wherein the complex enzyme comprises 7 parts by weight of extracting enzyme and 3 parts by weight of protective enzyme; the extracting enzyme comprises 6 parts by weight of cellulase and 4 parts by weight of pectinase; the protective enzyme comprises different weight ratios, namely: 4 parts by weight of acidic protease and 2 parts by weight of SOD, 5 parts by weight of acidic protease and 2 parts by weight of SOD, 6 parts by weight of acidic protease and 2 parts by weight of SOD, 7 parts by weight of acidic protease and 2 parts by weight of SOD, 4 parts by weight of acidic protease and 1 part by weight of SOD, 5 parts by weight of acidic protease and 1 part by weight of SOD, 6 parts by weight of acidic protease and 1 part by weight of SOD, and 7 parts by weight of acidic protease and 1 part by weight of SOD;

[0122] 3) Extract the enzymatic hydrolysis product from step 2) at 40°C with ultrasonic power of 300W for 1 hour, then centrifuge at 10000r / min for 10 minutes and collect the supernatant.

[0123] Effects of different protective enzyme ratios on the extraction yield of polyphenols and flavonoids:

[0124] Under the optimal ratio of compound enzymes and extraction enzymes, the proportion of protective enzymes (acidic protease and SOD) was optimized. The results are shown in Table 6. When the ratios were 4:2, 5:2, 6:2, 7:2, and 4:1, the acidic protease was insufficient, resulting in low extraction yields. The highest extraction yields of polyphenols and flavonoids were achieved at a ratio of 5:1. However, at ratios of 6:1 and 7:1, there was an excess of acidic protease and an insufficient amount of SOD, which prevented the removal of reactive oxygen species, leading to low extraction yields. Therefore, a 5:1 ratio of acidic protease to SOD is the optimal protective enzyme ratio for extracting polyphenols and flavonoids from tea residue.

[0125] Table 6. Effect of protective enzyme ratio on the extraction yield (mg / g) of polyphenols and flavonoids from tea residue.

[0126]

[0127] Exploration Example 6

[0128] 1) Take 0.1g of pretreated tea residue powder and place it in 7 test tubes. Add eutectic solvents with a betaine:urea molar ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, and 1:7 respectively and mix them. The material-to-liquid ratio is 1g:40mL. The water content of the eutectic solvent is 20%.

[0129] 2) Add 0.003g of a complex enzyme (4.2 parts by weight of cellulase, 2.8 parts by weight of pectinase, 1.5 parts by weight of acidic protease and 0.3 parts by weight of SOD) to the mixture in step 1) for enzymatic hydrolysis;

[0130] 3) Extract the enzymatic hydrolysis product from step 2) at 40°C with ultrasonic power of 300W for 1 hour, then centrifuge at 10000r / min for 10 minutes and collect the supernatant.

[0131] Effects of different molar ratios of eutectic solvents on the extraction yield of polyphenols and flavonoids:

[0132] Table 7 shows that as the molar ratio of urea gradually increases, the extraction efficiency of both polyphenols and flavonoids exhibits a trend of first increasing and then decreasing. The optimal extraction efficiency is achieved when the betaine:urea ratio is 1 mol:5 mol, with an extraction yield of 178.76 mg / g for polyphenols and 30.32 mg / g for flavonoids. This is likely because a betaine:urea ratio of 1 mol:5 mol more effectively extracts polyphenols and flavonoids from tea residue to the upper phase, improving separation purity. Therefore, a eutectic solvent ratio of 1 mol:5 mol for betaine is the optimal molar ratio for extracting polyphenols and flavonoids from tea residue.

[0133] Table 7. Effect of low eutectic solvent molar ratio on the extraction yield (mg / g) of polyphenols and flavonoids from tea residue.

[0134]

[0135] Exploration Example 7

[0136] 1) Take 0.1g of pretreated tea residue powder and place it in 7 test tubes. Add eutectic solvents with water contents of 0%, 10%, 20%, 30%, 40%, 50%, and 60% respectively and mix them. The material-to-liquid ratio is 1g:40mL. The molar ratio of betaine to urea is 1:5.

[0137] 2) Add 0.003g of a complex enzyme (4.2 parts by weight of cellulase, 2.8 parts by weight of pectinase, 1.5 parts by weight of acidic protease and 0.3 parts by weight of SOD) to the mixture in step 1) for enzymatic hydrolysis;

[0138] 3) Extract the enzymatic hydrolysis product from step 2) at 40°C with ultrasonic power of 300W for 1 hour, then centrifuge at 10000r / min for 10 minutes and collect the supernatant.

[0139] Effects of different water content eutectic solvents on the extraction yield of polyphenols and flavonoids:

[0140] Table 8 shows that the concentrations of polyphenols and flavonoids in the eutectic solvent system initially increase and then decrease with increasing water content. When the water content is below 30%, the system viscosity is high, which limits the dispersion of tea residue powder and the mass transfer rate of polyphenols and flavonoids, leading to a decrease in extraction efficiency. When the water content exceeds 50%, the hydrogen bond network of the eutectic solvent may be disrupted, resulting in a decrease in the solvent's solubility for polyphenols and flavonoids, and consequently, a reduction in extraction efficiency. Therefore, a water content of 30% is the optimal water content for extracting polyphenols and flavonoids from tea residue.

[0141] Table 8. Effect of water content in eutectic solvent on the extraction yield (mg / g) of polyphenols and flavonoids from tea residue.

[0142]

[0143] Exploration Example 8

[0144] 1) Take 0.1g of pretreated tea residue powder and place it in 8 test tubes. Add eutectic solvent to each tube and mix. The material-to-liquid ratios are 1g:10mL, 1g:20mL, 1g:30mL, 1g:40mL, 1g:45mL, 1g:50mL, 1g:60mL, and 1g:70mL, respectively. The molar ratio of betaine to urea is 1:5, and the water content of the eutectic solvent is 30%.

[0145] 2) Add 0.003g of a complex enzyme (4.2 parts by weight of cellulase, 2.8 parts by weight of pectinase, 1.5 parts by weight of acidic protease and 0.3 parts by weight of SOD) to the mixture in step 1) for enzymatic hydrolysis;

[0146] 3) Extract the enzymatic hydrolysis product from step 2) at 40°C with ultrasonic power of 300W for 1 hour, then centrifuge at 10000r / min for 10 minutes and collect the supernatant.

[0147] Effect of different material-to-liquid ratios on the extraction yield of polyphenols and flavonoids:

[0148] As shown in Table 9, the yields of flavonoids and polyphenols gradually increased as the solid-liquid ratio (g / mL) increased from 1:10 to 1:45. When the solid-liquid ratio exceeded 1:50 g / mL, the extraction efficiency of polyphenols and flavonoids decreased significantly. This was because the solvent excessively dissolved impurities such as proteins, polysaccharides, and pigments. These impurities may form complexes with polyphenols and flavonoids or occupy the active sites of the solvent through competitive adsorption, thereby reducing the extraction efficiency of polyphenols and flavonoids. Therefore, a solid-liquid ratio of 1:45 g / mL is the optimal ratio for extracting polyphenols and flavonoids from tea residue.

[0149] Table 9. Effects of different solid-liquid ratios (g / mL) on the extraction yield (mg / g) of polyphenols and flavonoids from tea residue.

[0150]

[0151] Analysis of variance

[0152] Based on the single-factor experiments, the factors that significantly affect the extraction yield of polyphenols and flavonoids from tea residue were screened using analysis of variance. The results are shown in Tables 10 and 11. For polyphenols, the results showed that the ratio of compound enzymes had the greatest impact on the extraction yield, followed by the amount of compound enzymes added. The ratio of protective enzymes, the ratio of extractive enzymes, the molar ratio, the material-liquid ratio, and the water content had relatively small effects on tea polyphenols. For flavonoids, the amount of compound enzymes added had the greatest impact on the extraction yield, followed by the ratio of compound enzymes, the ratio of protective enzymes, the ratio of extractive enzymes, and finally the molar ratio, the material-liquid ratio, and the water content.

[0153] Table 10 Results of Polyphenol Analysis of Variance

[0154]

[0155] Table 11 Results of ANOVA for Flavonoids

[0156]

[0157] Example 1

[0158] 1) such as Figure 4 As shown, 0.1g of pretreated tea residue powder was placed in a test tube and mixed with a eutectic solvent (the ratio of material to liquid was 1g:45mL, the molar ratio of betaine to urea was 1:5, and the water content was 30%).

[0159] 2) Add 0.003g of a complex enzyme (4.2 parts by weight of cellulase, 2.8 parts by weight of pectinase, 1.5 parts by weight of acidic protease and 0.3 parts by weight of SOD) to the mixture in step 1) for enzymatic hydrolysis;

[0160] 3) Extract the enzymatic hydrolysis product from step 2) using ultrasound at 300W at 40℃ for 1 hour, then centrifuge at 10000r / min for 10 minutes and collect the product as shown in the image. Figure 5 The supernatant shown;

[0161] 4) Separate and purify the supernatant from step 3) using scheme A;

[0162] Scheme A: Add ZnCl2 as a precipitant to the supernatant of tea leaves. A flocculent precipitate will appear and gradually increase until it fills the entire extract. Add 15% NaHCO3 buffer to adjust the pH of the reaction system to 6.0, and continue precipitation for 20 minutes. Then, centrifuge at 10000 rpm for 10 minutes and collect the precipitate. Dissolve the precipitate in 2 mol / L sulfuric acid, filter, and centrifuge to remove impurities such as tea proteins and tea polysaccharides, yielding a transparent orange solution. Extract this solution with ethyl acetate (1:1 volume ratio of ethyl acetate to tea polyphenol solution) to obtain an organic layer containing dissolved tea polyphenols. Dry this layer under vacuum at 60℃ for 4 hours to obtain a solid powder of tea polyphenols. The purity of this solid powder was determined to be 89.62% by the Folin-Ciocalteu colorimetric method.

[0163] Example 2

[0164] 1) Take 0.1g of pretreated tea residue powder and place it in a test tube. Add a eutectic solvent (the ratio of material to liquid is 1g:45mL, the molar ratio of betaine to urea is 1:5, and the water content is 30%) and mix.

[0165] 2) Add 0.003g of a complex enzyme (4.2 parts by weight of cellulase, 2.8 parts by weight of pectinase, 1.5 parts by weight of acidic protease and 0.3 parts by weight of SOD) to the mixture in step 1) for enzymatic hydrolysis;

[0166] 3) Extract the enzymatic hydrolysis product from step 2) at 40°C with ultrasonic power of 300W for 1 hour, then centrifuge at 10000r / min for 10 minutes and collect the supernatant.

[0167] 4) Separate and purify the supernatant from step 3) using scheme B;

[0168] Scheme B: Macroporous resin AB-8 was soaked in 95% ethanol for 24 hours, then soaked in 5% HCl solution and 4% NaOH solution for 3 hours each time, and washed with deionized water until odorless and neutral. This pretreatment was then used for later use. 2.0 g of resin and 25 mL of crude tea residue extract were placed in a 150 mL sealed Erlenmeyer flask and shaken in a water bath at 26℃ and 100 r / min for 24 hours for adsorption. The adsorbed macroporous resin was eluted sequentially with 400 mL of water and 200 mL of Na2CO3 aqueous solution (pH 9). The eluent from the Na2CO3 aqueous solution was collected, and the pH was adjusted to 4. The loading flow rate was 10 mL / min. Subsequently, it was eluted sequentially with 200 mL of water and 400 mL of 70% ethanol aqueous solution. The eluent from the 70% ethanol aqueous solution was collected, concentrated, and dried to obtain tea flavonoid powder. The purity of this solid powder was tested using the aluminum trichloride-sodium nitrite colorimetric method and found to be 89.53%.

[0169] The purity of polyphenols and flavonoids was determined according to the following formula in the examples:

[0170]

[0171] Comparative Example 1

[0172] Effects of different extraction methods on the extraction yield of polyphenols and flavonoids

[0173] The extraction methods used were those listed in the table below, with other conditions remaining unchanged. The results are shown in the table below:

[0174] Table 12 Effects of different extraction methods on the extraction yield (mg / g) of polyphenols and flavonoids from tea residue

[0175]

[0176] Table 12 shows that the ultrasound-enzyme-assisted eutectic solvent extraction method significantly improves the extraction efficiency of polyphenols and flavonoids compared with other methods. In terms of extraction enzymes, cellulase and pectinase are the "core tools for cell wall deconstruction" in tea residue extraction. The former is responsible for destroying the skeleton, and the latter is responsible for unblocking the channels. The combination of the two can release the encapsulated polyphenols / flavonoids. In terms of protective enzymes, acidic protease protects the dissolution potential of active ingredients (breaking the encapsulation and reducing impurity interference), while SOD protects the structure and function of active ingredients (inhibiting oxidation and retaining biological activity). The two work together to ensure the extraction effect and significantly improve the extraction rate and efficiency of polyphenols and flavonoids. The microjets and shock waves generated by ultrasound can directly break down plant cell walls, releasing intracellular polyphenols and flavonoids. The mechanical vibration of ultrasound promotes the penetration of eutectic solvents into the plant tissue, increasing the solute-solvent contact area. The hydrogen bonds inside the eutectic solvent can form strong interactions with the hydroxyl and carbonyl groups of polyphenols / flavonoids, significantly improving solubility. Therefore, the ultrasound-enzyme-assisted eutectic solvent method is a fast, efficient, and environmentally friendly method for extracting polyphenols and flavonoids from tea residue.

[0177] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for resource-based utilization of tea dregs, characterized in that, Includes the following steps: 1) Mix the pretreated tea residue powder with a eutectic solvent at a ratio of 1g:30-70mL; wherein the volumetric water content of the eutectic solvent is 20-50%. 2) Add a compound enzyme to the mixture at 1-5% of the mass of tea residue powder in step 1) for enzymatic hydrolysis; wherein the compound enzyme comprises 5-8 parts by weight of extracting enzyme and 2-5 parts by weight of protective enzyme, the extracting enzyme comprises 5-7 parts by weight of cellulase and 3-5 parts by weight of pectinase, and the protective enzyme comprises 4-7 parts by weight of acidic protease and 1-2 parts by weight of SOD. 3) Extract the enzymatic hydrolysis product from step 2) by sonication, centrifuge, and collect the supernatant; 4) Separate and purify the supernatant from step 3); The separation and purification described in step 4) includes the following scheme A: Option A: Take the supernatant from step 3) and add a precipitant at a ratio of 1-5% w / v. Stir mechanically to produce a flocculent precipitate. Continue stirring until the precipitate accounts for 70-90% of the total volume of the system. Add buffer solution to adjust the pH of the reaction system to 5.5-6.

5. Continue stirring for 10-30 minutes and centrifuge. Dissolve the precipitate, filter, centrifuge, extract, collect the organic layer extract, and vacuum dry to obtain the product, tea polyphenol solid powder. Alternatively, the separation and purification described in step 4) may include the following scheme B: Option B: Take the supernatant from step 3) and add it to the macroporous resin at a ratio of 5-10% w / v. Shake in a water bath. After adsorption, elute the macroporous resin sequentially with water and Na2CO3 aqueous solution with pH 8.5-9.

5. Collect the Na2CO3 aqueous solution eluent, adjust the pH to 3-5, and then elute sequentially with water and 60-80% v / v ethanol aqueous solution. Collect the ethanol aqueous solution eluent, concentrate and dry to obtain the product flavonoid solid powder.

2. The method according to claim 1, characterized in that, The precipitant described in Scheme A includes at least one of ZnCl2, AlCl3, FeCl3, and CaCl2; and / or The buffer solution described in Scheme A comprises a 10-20% (w / w) NaHCO3 aqueous solution; and / or The dissolution described in Scheme A uses 1-3 mol / L sulfuric acid; and / or The extraction described in Scheme A uses an equal volume of ethyl acetate.

3. The method according to claim 1, characterized in that, The macroporous resin described in Scheme B includes at least one of AB-8, D101, X-5, and NKA-9; and / or The conditions for water bath oscillation described in Scheme B include: The temperature is 20-30℃; and / or Rotational speed is 50-200 r / min; and / or The time is 12-36 hours.

4. The method according to claim 1, characterized in that, In step 1), The pretreatment includes: drying the collected tea dregs at 40-50℃, grinding them, and storing them at -15 to -25℃ for later use.

5. The method according to claim 1, characterized in that, In step 1), The eutectic solvent includes hydrogen bond acceptors and hydrogen bond donors; The hydrogen bond acceptors include choline chloride and / or betaine; and / or The hydrogen bond donor includes at least one selected from ethylene glycol, 1,4-butanediol, glycerol, 1,3-butanediol, levulinic acid, malic acid, urea, glycerol, and glucose; and / or The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:4-6.

6. The method according to claim 1, characterized in that, In step 2), The conditions for the enzymatic hydrolysis include: Adjust the pH of the system to 4-6; and / or The enzymatic hydrolysis temperature is 40-60℃; and / or The enzymatic hydrolysis time is 2-3 hours; and / or After enzymatic hydrolysis, boil for 3-4 minutes to deactivate the enzyme and obtain the enzymatic hydrolysis product.

7. The method according to claim 1, characterized in that, In step 2), The cellulase concentration is 10,000-30,000 U / g; and / or The pectinase concentration is 0.5-1.5 million U / g; and / or The acidic protease has a concentration of 20,000-40,000 U / g; and / or The SOD concentration is 0.1-0.5 million U / g.

8. The method according to claim 1, characterized in that, In step 3), The conditions for ultrasonic extraction include: Temperature is 40-60℃; and / or Power of 200-400W; and / or The time is 40-60 minutes.

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