Method for extracting tea saponin from oil tea cake by coupling enzymatic hydrolysis with ultrasonic-assisted deep eutectic solvent and optimization method thereof

Abstract

The present invention discloses a method for extracting tea saponins from camellia oleifera cake using an enzymatic hydrolysis-coupled ultrasound-assisted deep eutectic solvent and an optimization method thereof, relating to the technical field of natural product extraction. The present invention utilizes a method combining enzymatic hydrolysis, ultrasound-assisted extraction, and a deep eutectic solvent to extract tea saponins from camellia oleifera cake. The tea saponins extracted using this method are 140% higher than those extracted using conventional extraction methods. This novel, efficient, and environmentally friendly method avoids the extensive use of organic solvents, effectively alleviating environmental pollution issues and providing new ideas and methods for extracting active ingredients from natural products.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural product extraction, and specifically relates to a method for extracting tea saponin from camellia oleifera cake by using an enzymatic decomposition coupled with ultrasound-assisted deep eutectic solvent and an optimization method thereof. The tea saponin from camellia oleifera cake is extracted by using a deep eutectic solvent. Background Art

[0002] Camellia oleifera Camellia oleifera Abel), also known as common tea, Wild camellia, mountain camellia, or single-seed camellia is an evergreen shrub or tree of the genus Camellia in the family Theaceae. Camellia grows in dense forests on high mountains or low hills, in mixed woods or bamboo forests along hillsides, and in scattered or concentrated distribution, sometimes forming large, wild forests. Camellia oleifera is native to southern China and Southeast Asia, including Vietnam, Laos, Myanmar, and Assam in India. It also grows wild in Hunan, Jiangxi, Guangxi, and Hainan provinces in China. Camellia oleifera oil is highly nutritious, rich in oleic and linoleic acids, and has a mellow flavor, making it a high-quality edible oil. It is also an excellent industrial raw material for use in soap, cosmetics, rubber, and other industries. The roots, leaves, flowers, and seeds of the tree, as well as the oil extracted from the seeds and the residual residue after oil extraction, are all used medicinally, with benefits such as clearing heat and detoxifying, cooling blood, and stopping bleeding.

[0003] Tea saponin is a natural, nonionic surfactant composed of a hydrophilic saccharide and a hydrophobic ligand. Extracted from Camellia oleifera (Abel), a plant of the Theaceae family, tea saponin is a pentacyclic triterpenoid glycoside compound. It is a mixture of oleanane-type pentacyclic triterpenoid saponins, with a basic structure consisting of a sapogenin, a saccharide, and an organic acid. It exhibits diverse surface activities, including emulsification, dispersing, wetting, detergency, and foaming, as well as various biological activities, including antibacterial, anti-inflammatory, and fish poison removal. It is widely used in daily chemical products, pesticides, pharmaceuticals, food, building materials, and metallurgy. In recent years, research on the extraction and application of tea saponin has received increasing attention worldwide.

[0004] The traditional conventional tea saponin extraction process uses chemical extraction methods, which involve the use of large amounts of organic solvents. The large-scale use of organic solvents will bring a series of problems such as high cost, high risk, high harm and high pollution.

[0005] Deep eutectic solvents (DES) are a class of high-quality green solvents that have rapidly emerged in recent years. Compared with traditional organic solvents, deep eutectic solvents offer advantages such as low cost, simple preparation, stable chemical properties, easy storage, non-toxicity, and biodegradability. They are ideal alternatives to traditional organic solvents and green solvents for the extraction of natural products. Deep eutectic solvents have been widely used in the extraction of natural products, and researchers have reported their use in extracting flavonoids, phenols, polysaccharides, proteins, anthocyanins, and alkaloids. Summary of the Invention

[0006] The application aims to provide a method for extracting tea saponin from oil tea cake by coupling enzymolysis with ultrasonic-assisted deep eutectic solvent.

[0007] The application aims to achieve the above-mentioned purpose through the following technical solutions.

[0008] In one aspect, the application provides a method for extracting tea saponin from oil tea cake by coupling enzymolysis with ultrasonic-assisted deep eutectic solvent, which comprises the following steps:

[0009] S01, pretreating the oil tea cake to obtain dry oil tea cake powder;

[0010] S02, preparing an enzyme solution of cellulase and pectinase, and performing enzymolysis on the oil tea cake powder by using the cellulase and pectinase; the enzymolysis can destroy the fibers of the oil tea cake, thereby facilitating extraction;

[0011] S03, preparing a deep eutectic solvent;

[0012] S04, extracting tea saponin from the oil tea cake by using ultrasonic-assisted deep eutectic solvent to obtain an oil tea cake tea saponin extraction solution;

[0013] S05, determining the content of tea saponin in the oil tea cake tea saponin extraction solution by using vanillin-sulfuric acid method;

[0014] S06, enriching the tea saponin extraction solution by using a macroporous resin to obtain an oil tea cake extract, and separating and purifying the oil tea cake extract by using a silica gel column, an ODS column and thin layer chromatography to obtain tea saponin monomers.

[0015] Further, in step S01, the oil tea cake is dried, fully crushed by using a crusher, and then sieved through a 60-80 mesh sieve for dry storage.

[0016] Further, in step S02, the cellulase and the pectinase are respectively dissolved in a PBS buffer with a pH of 5 to obtain an enzyme solution, and the cellulase accounts for 20% of the total enzyme solution of the cellulase and the pectinase, and the enzymolysis time is 2-3 hours.

[0017] Further, in step S03, the deep eutectic solvent is a choline-based deep eutectic solvent composed of a hydrogen bond acceptor and a hydrogen bond donor in a certain stoichiometric ratio, the hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is 1,4-butanediol and 1,3-butanediol; specifically, choline chloride, 1,4-butanediol and 1,3-butanediol are mixed in a certain proportion, a certain proportion of water is added, and the mixture is heated and stirred at 80-85 DEG C until a uniform and stable liquid is obtained.

[0018] Further, in step S04, the specific process is: the eutectic solvent is added to the oil tea cake meal after enzymatic hydrolysis, and then placed in an ultrasonic cleaning instrument. The ultrasonic cleaning instrument is used to assist in extracting the oil tea cake meal saponin to obtain an oil tea cake meal saponin crude extract. Finally, the oil tea cake meal saponin crude extract is subjected to high-speed centrifugal treatment, and the supernatant obtained is the oil tea cake meal saponin extract solution, which is stored at 2-8 ℃ for use. The ultrasonic extraction power is 240-260 W, the ultrasonic time is 2-3 h, and the extraction liquid-solid ratio is 1:20.

[0019] Further, in step S05, the following steps are included:

[0020] Step 5.1, 100 mg of tea saponin is weighed and dissolved with 80% ethanol, and the volume is made to 100 mL. 1.0, 2.0, 3.0, 4.0, and 5.0 mL are respectively taken into 10 mL volumetric flasks and made to volume, i.e. tea saponin standard series solutions with concentrations of 100.0 μg / mL, 200.0 μg / mL, 300.0 μg / mL, 400.0 μg / mL, and 500.0 μg / mL are obtained, which are stored at room temperature for use;

[0021] Step 5.2, 77% sulfuric acid solution and 8% vanillin solution are prepared: 77 mL of concentrated sulfuric acid and 23 mL of deionized water are mixed and stored at room temperature for use; 0.8 g of vanillin is weighed and made to 10 mL with anhydrous ethanol, and it is prepared immediately for use;

[0022] Step 5.3, 0.5 mL of the above tea saponin standard solution is taken in a stoppered test tube, 0.5 mL of 8% vanillin solution is added, the test tube is moved into an ice water mixture, and 4 mL of 77% concentrated sulfuric acid is added; after shaking, it is heated in a 60°C water bath for 20 min, and then placed in ice water for 10 min; compared with the blank, the absorbance is measured at a wavelength of 550 nm;

[0023] Step 5.4, a tea saponin concentration standard curve is made with tea saponin concentration as the abscissa and the absorbance at 550 nm wavelength as the ordinate, and a linear regression equation y = 0.002x - 0.0622 is obtained, R 2 = 0.9992;

[0024] Step 5.5, the oil tea cake meal tea saponin extract solution obtained in step 4 is diluted n times, n is 5-10; the solution is used instead of the tea saponin standard solution, and the absorbance value of the diluted tea saponin extract solution at 550 nm wavelength is measured according to the steps of step 5.3;

[0025] Step 5.6, according to the linear regression equation of the tea saponin standard solution obtained in step 5.4, the content of tea saponin in oil tea cake tea saponin extract is calculated, the formula is as follows: tea saponin content (mg / g) = [(y + 0.0622) x V x n] / (0.002 x m), wherein, y is the absorbance value at 550 nm wavelength, V is the volume of oil tea cake tea saponin extract solution (mL), n is the dilution multiple of oil tea cake tea saponin extract solution; 0.0622 is the intercept of the linear regression equation, 0.002 is the slope of the equation, m is the dry weight of oil tea cake powder (g).

[0026] The application also provides an optimization method of the method for extracting tea saponin from oil tea cake by coupling enzymolysis and ultrasonic-assisted deep eutectic solvent, characterized by comprising the following steps:

[0027] Step A: According to the method for extracting tea saponin from oil tea cake by coupling enzymolysis and ultrasonic-assisted deep eutectic solvent, six factors affecting the extraction rate of tea saponin are screened, specifically: the type of deep eutectic solvent used for extraction, the molar ratio of hydrogen bond acceptor and hydrogen bond donor in the deep eutectic solvent, the ratio of 1,3-butanediol and 1,4-butanediol, the water content of the deep eutectic solvent, the proportion of cellulase in the total enzyme solution of cellulase and pectinase, and the solid-liquid ratio.

[0028] Step B: Taking the content of tea saponin in the oil tea cake tea saponin extract as an index, single-factor experiments are carried out according to the six factors screened in step A to determine the optimal extraction process factor parameters.

[0029] Further, the optimal extraction process conditions obtained through single-factor experiments are: the molar ratio of hydrogen bond acceptor and hydrogen bond donor in the deep eutectic solvent is 1:4, the molar ratio of 1,4-butanediol and 1,3-butanediol is 1:1, the solid-liquid ratio is 1:40 g / mL, the water content of the deep eutectic solvent is 10%, and the proportion of cellulase in the total enzyme solution composed of cellulase and pectinase is 20%.

[0030] The application also provides a deep eutectic solvent for extracting tea saponin from oil tea cake, which is a choline deep eutectic solvent composed of hydrogen bond acceptors and hydrogen bond donors in a certain stoichiometric ratio, the hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is 1,4-butanediol and 1,3-butanediol, specifically, choline chloride, 1,4-butanediol and 1,3-butanediol are mixed in a certain ratio of 1:2:2, 10% water is added, and the mixture is heated and stirred at 80-85℃ until a uniform and stable liquid is obtained.

[0031] Through the extraction process of the application, the tea saponin content of the oil tea cake extract is 28.26 mg / g (dry weight of oil tea cake powder).

[0032] The oil-tea camellia cake tea saponin extraction method provided by the application uses a novel green low-eutectic solvent as an extraction agent, avoids the use of traditional organic solvents, and fundamentally solves a series of problems such as high cost, high risk, high harm, and high pollution caused by the large use of organic solvents in traditional chemical extraction of tea saponin.

[0033] The oil-tea camellia cake tea saponin extraction method provided by the application organically combines low-eutectic solvent, enzymatic hydrolysis, and ultrasonic-assisted extraction into the extraction of oil-tea camellia cake tea saponin, and compared with conventional extraction methods, the method is novel, green, and environmentally friendly, has high oil-tea camellia cake tea saponin extraction rate, low extraction cost, mild extraction conditions, simple and stable and reliable extraction process, and low extraction energy consumption. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The tea saponin concentration standard curve.

[0035] Figure 2 The figure is a schematic diagram of the influence of the extraction solvent on the tea saponin content of the oil-tea camellia cake tea saponin extraction liquid in the application.

[0036] Figure 3 The figure is a schematic diagram of the influence of the ratio of 1,3 butanediol and 1,4 butanediol of DES on the tea saponin content of the oil-tea camellia cake tea saponin extraction liquid in the application.

[0037] Figure 4 The figure is a schematic diagram of the influence of the molar ratio of the hydrogen bond acceptor and the hydrogen bond donor of the DES on the tea saponin content of the oil-tea camellia cake tea saponin extraction liquid in the application.

[0038] Figure 5 The figure is a schematic diagram of the influence of the water content of the DES on the tea saponin content of the oil-tea camellia cake tea saponin extraction liquid in the application.

[0039] Figure 6 The figure is a schematic diagram of the influence of the cellulase proportion on the tea saponin content of the oil-tea camellia cake tea saponin extraction liquid in the application.

[0040] Figure 7 The figure is a schematic diagram of the influence of the extraction liquid ratio on the tea saponin content of the oil-tea camellia cake tea saponin extraction liquid in the application. DETAILED DESCRIPTION

[0041] The following reference to the drawings of the specification introduces a plurality of preferred embodiments of the application, so that the technical content is more clear and convenient to understand. The application can be embodied by many different forms of embodiments, and the protection scope of the application is not limited to this:

[0042] The oil tea described in the application, alias ordinary oil tea, Camellia oleifera Abel, etc. is a kind of evergreen shrubs or trees of Theaceae Camellia. Camellia oleifera is native to southern China, and has wild distribution in Hunan, Jiangxi, Guangxi, Hainan and other provinces.

[0043] Embodiment 1: a method for extracting tea saponin from oil tea cake by coupling enzymatic hydrolysis with ultrasonic-assisted deep eutectic solvent, specifically comprising the following steps:

[0044] S01, pretreatment of oil tea cake to obtain dry oil tea cake powder;

[0045] S02, configuring enzyme solution of cellulase and pectinase, and using cellulase and pectinase to hydrolyze the oil tea cake powder;

[0046] S03, preparing deep eutectic solvent;

[0047] S04, extracting tea saponin from oil tea cake by ultrasonic-assisted deep eutectic solvent to obtain tea saponin extract solution from oil tea cake;

[0048] S05, using vanillin-sulfuric acid method to determine the content of tea saponin in the tea saponin extract solution from oil tea cake;

[0049] S06, using macroporous resin to enrich the tea saponin extract solution to obtain oil tea cake extract, and using silica gel column, ODS column and thin layer chromatography to separate and purify the tea saponin monomer.

[0050] Specifically as follows:

[0051] 1. Plant sample collection: the oil tea cake was collected from Chun'an County, Hangzhou City, Zhejiang Province, and was ground in a portable high-speed universal grinder at a speed of 25000 rpm / min for 30 s to obtain oil tea cake powder. The ground oil tea cake powder was dried in a laboratory oven at 60 ℃ and then sealed in a bag for storage.

[0052] 2. Enzyme solution preparation: 0.25 g of cellulase (400 u / mg) and 0.20 g of pectinase (500 u / mg) were accurately weighed and dissolved in pH=5 phosphate buffer solution, and then diluted to 50 mL. The cellulase solution and pectinase solution were obtained at 2000 u / mL, respectively. The pH=5 phosphate buffer solution was prepared as follows: 27.2 g of potassium dihydrogen phosphate powder was accurately weighed and dissolved in deionized water, and then diluted to 1000 mL. Sodium hydroxide solution was used to adjust the pH to 5.

[0053] 3 Deep eutectic solvent configuration: 13.96 g of choline chloride and 18.02 g of 1,4-butanediol and 18.02 g of 1,3-butanediol were weighed according to a 1:2:2 molar ratio in a beaker (100 mL) with a magnetic stirrer rotor built-in, and 1.00 mL of water was added. Subsequently, the beaker was placed in a thermostatic water bath magnetic stirrer and heated at 80°C until a clear, transparent, homogeneous liquid was formed (about 1-1.5 h), which was the choline chloride-1,4-butanediol and 1,3-butanediol deep eutectic solvent, and was used after cooling to room temperature.

[0054] 4 Preparation of tea saponin extract from oil tea cake meal: 0.10 g of oil tea cake meal powder was accurately weighed in a 10 mL centrifuge tube, 0.2 mL of cellulase solution and 0.8 mL of pectinase solution were added, and enzyme hydrolysis was carried out in a 30°C water bath for 3 h. Then 4 mL of choline chloride-1,4-butanediol and 1,3-butanediol deep eutectic solvent (extraction liquid ratio of 1:40, material refers to oil tea cake meal powder, liquid refers to deep eutectic solvent) was added, and the centrifuge tube was placed in an ultrasonic cleaning instrument. The ultrasonic cleaning instrument was used to assist in the extraction of oil tea cake meal tea saponin, and a crude oil tea cake meal tea saponin extract was obtained. Finally, the crude oil tea cake meal tea saponin extract was centrifuged at 8000 rpm for 10 min, and the supernatant obtained was the oil tea cake meal tea saponin extract, which was stored at 2-8°C for use. The ultrasonic assisted extraction conditions were: ultrasonic power 240 w, ultrasonic time 2 h.

[0055] 5 The content of tea saponin in the oil tea cake meal tea saponin extract was determined by vanillin-sulfuric acid method, and the specific process was as follows:

[0056] (1) Preparation of tea saponin standard solution: 20 mg of tea saponin was accurately weighed and dissolved in 80% ethanol, and then diluted to 20 mL to obtain a tea saponin standard solution. 1.0, 2.0, 3.0, 4.0, and 5.0 mL of the solution were respectively taken and diluted to 10 mL to obtain tea saponin standard series solutions with concentrations of 100.0 μg / mL, 200.0 μg / mL, 300.0 μg / mL, 400.0 μg / mL, and 500.0 μg / mL, which were stored at room temperature for use;

[0057] (2) Preparation of tea saponin standard curve: Take 6 15 mL test tubes, and add 0.5 mL of tea saponin standard solution prepared in step (1) into each test tube, then add 0.5 mL of 8% vanillin solution, 2.0 mL of anhydrous ethanol solution, and 2.0 mL of 77% sulfuric acid solution into each test tube. Shake well, and then heat in a 60°C water bath for 20 min, and then cool in ice water for 10 min. Measure the absorbance at 550 nm; meanwhile, perform a blank test according to the above method by replacing the tea saponin standard solution with 1.0 mL of 80% ethanol. Finally, prepare a tea saponin concentration standard curve (as shown in FIG. 1) with the tea saponin concentration as the abscissa and the absorbance value at 550 nm as the ordinate, and obtain the linear regression equation: y = 0.002x - 0.0622, with a correlation coefficient R Figure 1 2 = 0.9992, indicating that the tea saponin standard has a good linear relationship in the range of 100.0-500.0 μg / mL.

[0058] The 8% vanillin solution and the 77% sulfuric acid solution are prepared according to the following methods.

[0059] Preparation of 8% vanillin solution: weigh 0.8 g of vanillin, dissolve in anhydrous ethanol, and dilute to 10 mL to obtain 8% vanillin solution, which is stored at room temperature and used immediately after preparation;

[0060] Preparation of 77% sulfuric acid solution: accurately weigh 77 mL of concentrated sulfuric acid, mix with 23 mL of deionized water, and obtain 77% sulfuric acid solution, which is stored at room temperature and used immediately after preparation;

[0061] (3) Determination of tea saponin content in oil-tea camellia cake tea saponin extract: dilute the oil-tea camellia cake tea saponin extract obtained in step 4 by 10 times; replace the tea saponin standard solution in step 5 with 1.0 mL of the diluted oil-tea camellia cake tea saponin extract, and measure the absorbance value of the sample at 550 nm, and then calculate the tea saponin content of the oil-tea camellia cake tea saponin extract according to the linear regression equation of the tea saponin standard in step 5 according to the following formula:

[0062] Tea saponin content (mg / g of oil-tea camellia cake powder dry weight) = [(y + 0.0622) x V x n] / (0.002 x m) = [(0.503 + 0.0622) x 10 x 1] / (0.002 x 0.1 x 1000) = 28.26;

[0063] ​Wherein, y is the absorbance value at 550 nm wavelength, V is the volume of tea saponin extract solution (mL), n is the dilution multiple of tea saponin extract solution; 0.0622 is the intercept of the linear regression equation, 0.002 is the slope of the equation, and m is the dry weight of oil-tea camellia cake powder (g).

[0064] 6. The oil-tea camellia cake extract is obtained by enriching tea saponin extract solution with macroporous resin, and the tea saponin monomer is obtained by separating and purifying the oil-tea camellia cake extract with silica gel column, ODS column and thin layer chromatography.

[0065] Example 2: A method for optimizing the process conditions of enzymatic hydrolysis coupled with ultrasonic-assisted deep eutectic solvent extraction of tea saponin from oil-tea camellia cake, and the specific steps are as follows:

[0066] 1. According to the method for extracting tea saponin from oil-tea camellia cake by enzymatic hydrolysis coupled with ultrasonic-assisted deep eutectic solvent, six factors affecting the extraction rate of tea saponin are screened, including the selection of extraction solvent, the molar ratio of 1,3-butanediol and 1,4-butanediol, the ratio of HBA and HBD when preparing deep eutectic solvent, the amount of water added when preparing deep eutectic solvent, the solid-liquid ratio of oil-tea camellia cake powder and deep eutectic solvent, and the ratio of cellulase to pectinase. Taking the tea saponin content of oil-tea camellia cake tea saponin extract solution as the reference index, single factor experiment is carried out, which is as follows:

[0067] 1.1 According to the method for extracting tea saponin from oil-tea camellia cake by enzymatic hydrolysis coupled with ultrasonic-assisted deep eutectic solvent, eight different deep eutectic solvents and two different concentrations of ethanol are selected for single factor experiment of extraction solvent: the molar ratio of hydrogen bond acceptor-hydrogen bond donor is fixed at 1:2, the molar ratio of 1,3-butanediol and 1,4-butanediol is 1:1, the amount of water added when preparing deep eutectic solvent is 20%, the solid-liquid ratio of extraction is 1:50 g / mL, the ratio of cellulase to total enzyme liquid is 50% when adding cellulase and pectinase, and the ultrasonic-assisted conditions remain unchanged. The tea saponin content of oil-tea camellia cake tea saponin extract solution is measured when the extraction solvent is DES-1,3-butanediol, DES-1,4-butanediol, DES-glycol, DES-glycerol, DES-urea, DES-glycol+1,3-butanediol, DES-glycol+ethanol, DES-glycol+1,4-butanediol, DES-1,3-butanediol+1,4-butanediol, 50% ethanol, and 80% ethanol.

[0068] The effect of extraction solvent on the tea saponin content of oil-tea camellia cake tea saponin extract solution is shown in Figure 2 As can be seen from the table, the tea saponin content of oil-tea camellia cake tea saponin extract solution is higher when the extraction solvent is choline chloride-1,3-butanediol and 1,4-butanediol.

[0069] 1.2 According to the foregoing, a method for extracting tea saponin from oil tea cake by coupling enzymolysis with ultrasonic-assisted deep eutectic solvent, different proportions of 1,3-butanediol and 1,4-butanediol are selected for single factor experiment: the deep eutectic solvent is choline chloride-1,3-butanediol and 1,4-butanediol with a molar ratio of 1:2, the water addition amount during preparation of the deep eutectic solvent is 20%, the extraction solid-liquid ratio is 1:50 g / mL, the proportion of cellulase in the total enzyme solution of cellulase and pectinase added is 60%, the conditions of ultrasonic assistance are unchanged, the molar ratio of deep eutectic solvent 1,3-butanediol and 1,4-butanediol is 3:1, 2:1, 1:1, 1:2, 1:3, and the tea saponin content of the oil tea cake tea saponin extract is determined.

[0070] The effect of the molar ratio of 1,3-butanediol and 1,4-butanediol during preparation of the deep eutectic solvent on the tea saponin content of the oil tea cake tea saponin extract is shown in Figure 3 As can be seen from the figure, with the increase of the molar ratio of 1,3-butanediol and 1,4-butanediol, the tea saponin content of the oil tea cake tea saponin extract showed a trend of first increasing and then decreasing, and when the molar ratio was 1:1, the tea saponin content of the oil tea cake tea saponin extract reached the maximum value. Subsequently, the tea saponin content of the oil tea cake tea saponin extract began to decrease. Therefore, the optimal deep eutectic solvent is 1,3-butanediol and 1,4-butanediol with a molar ratio of 1:1.

[0071] 1.3 According to the foregoing, a method for extracting tea saponin from oil tea cake by coupling enzymolysis with ultrasonic-assisted deep eutectic solvent, six different molar ratios of choline chloride and hydrogen bond donors are selected for single factor experiment: the deep eutectic solvent is choline chloride-1,3-butanediol and 1,4-butanediol, the molar ratio of 1,3-butanediol and 1,4-butanediol is 1:1, the water addition amount during preparation of the deep eutectic solvent is 20%, the extraction solid-liquid ratio is 1:50 g / mL, the proportion of cellulase in the total enzyme solution of cellulase and pectinase added is 60%, the conditions of ultrasonic assistance are unchanged, the molar ratio of deep eutectic solvent choline chloride and hydrogen bond donor is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, and the tea saponin content of the oil tea cake tea saponin extract is determined.

[0072] The effect of the molar ratio of choline chloride and hydrogen bond donor during preparation of the deep eutectic solvent on the tea saponin content of the oil tea cake tea saponin extract is shown in Figure 4 As can be seen from the figure, with the increase of the molar ratio of choline chloride and hydrogen bond donor, the tea saponin content of the oil tea cake tea saponin extract showed a trend of first increasing and then decreasing, and when the molar ratio was 1:4, the tea saponin content of the oil tea cake tea saponin extract reached the maximum value. Subsequently, the tea saponin content of the oil tea cake tea saponin extract began to decrease. Therefore, the optimal deep eutectic solvent is choline chloride with a molar ratio of 1:4 to (1,3-butanediol and 1,4-butanediol).

[0073] 1.4 According to the foregoing, a method for extracting tea saponin from oil tea cake by coupling enzymolysis with ultrasonic-assisted deep eutectic solvent, single-factor experiments of water addition amount in deep eutectic solvent preparation were carried out at 7 levels, the deep eutectic solvent was choline chloride-1, 3-butanediol and 1, 4-butanediol with a molar ratio of 1:4, the molar ratio of 1, 3-butanediol and 1, 4-butanediol was 1:1, the extraction solid-liquid ratio was 1:50 g / mL, the proportion of cellulase in the total enzyme solution of cellulase and pectinase was 60%, and the conditions of ultrasonic assistance were unchanged. The tea saponin content of oil tea cake tea saponin extract was determined when the water addition amount in deep eutectic solvent preparation was 0%, 10%, 20%, 30%, 40%, 50%, and 60%.

[0074] The effect of water addition amount in deep eutectic solvent preparation on the tea saponin content of oil tea cake tea saponin extract is shown in Figure 5 As can be seen from the figure, the tea saponin content of oil tea cake tea saponin extract showed a decreasing trend with the increase of water addition amount in deep eutectic solvent preparation, and the tea saponin content of oil tea cake tea saponin extract was the highest when the water addition amount was 10%. Therefore, the optimal water addition amount in deep eutectic solvent preparation was 10%.

[0075] 1.5 According to the foregoing, a method for extracting tea saponin from oil tea cake by coupling enzymolysis with ultrasonic-assisted deep eutectic solvent, single-factor experiments of the proportion of cellulase and pectinase were carried out at 6 levels, the deep eutectic solvent was choline chloride-1, 3-butanediol and 1, 4-butanediol with a molar ratio of 1:2, the molar ratio of 1, 3-butanediol and 1, 4-butanediol was 1:1, the water addition amount in deep eutectic solvent preparation was 20%, and the extraction solid-liquid ratio was 1:50 g / mL. The conditions of ultrasonic assistance were unchanged. The tea saponin content of oil tea cake tea saponin extract was determined when the proportion of cellulase in the total enzyme solution of cellulase and pectinase was 0%, 20%, 40%, 60%, 80%, and 100%.

[0076] The effect of the proportion of cellulase in the total enzyme solution of cellulase and pectinase on the tea saponin content of oil tea cake tea saponin extract is shown in Figure 6 As can be seen from the figure, the tea saponin content of oil tea cake tea saponin extract showed a decreasing trend with the increase of cellulase proportion, and the tea saponin content of oil tea cake tea saponin extract was the highest when the proportion of cellulase in the total enzyme solution of cellulase and pectinase was 20%. Therefore, the optimal proportion of cellulase was 20%.

[0077] 1.6 According to the foregoing, a method for the enzymatic decoupling of ultrasonic-assisted deep eutectic solvent extraction of tea saponin from oil tea cake, eight levels are selected for single factor experiment of extraction liquid ratio, the deep eutectic solvent is choline chloride-1, 3-butanediol and 1, 4-butanediol with a molar ratio of 1:4, the molar ratio of 1, 3-butanediol and 1, 4-butanediol is 1:1, the water content is 10% when the deep eutectic solvent is prepared, the proportion of cellulase in the total enzyme solution of cellulase and pectinase is 20%, the ultrasonic-assisted conditions are unchanged, and the tea saponin content of the oil tea cake tea saponin extract is determined when the solid-liquid ratio is 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70 and 1:80.

[0078] The effect of extraction liquid ratio on the tea saponin content of the oil tea cake tea saponin extract is shown in Figure 7 As can be seen from the above, the tea saponin content of the oil tea cake tea saponin extract increases with the increase of the solid-liquid ratio, and the tea saponin content of the oil tea cake tea saponin extract is higher when the solid-liquid ratio is 1:40 and the subsequent growth is slow. Therefore, the optimal solid-liquid ratio is 1:40.

[0079] 2 The optimal process conditions for ultrasonic-assisted deep eutectic solvent extraction of tea saponin from oil tea cake are as follows: the molar ratio of hydrogen bond acceptor and hydrogen bond donor in DES is 1:4, the extraction solid-liquid ratio is 1:40 g / mL, and the proportion of cellulase and pectinase solution is 20%, the predicted value of the tea saponin content of the oil tea cake tea saponin extract is 28.26 mg / g (dry weight of oil tea cake powder). This value is 140% higher than that of the traditional ethanol method, indicating that the enzymatic decoupling of ultrasonic-assisted deep eutectic solvent extraction of tea saponin from oil tea cake is feasible and can be popularized in industrial application.

[0080] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for extracting tea saponins from tea cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent, characterized in that , including the following steps: S01, pre-treating camellia oil cake to obtain dry camellia oil cake powder; S02, preparing cellulase and pectinase enzyme solutions, and enzymatically hydrolyzing the camellia oil cake powder using the cellulase and pectinase; S03, preparing a deep eutectic solvent; S04, extracting tea saponins from camellia oil cake with the aid of a deep eutectic solvent to obtain a tea saponin extract from camellia oil cake; S05. Determine the tea saponin content in the tea saponin extract of camellia oil cake using the vanillin-sulfuric acid method; S06. Enriching the tea saponin extract using a macroporous resin to obtain a camellia oil cake extract, and separating and purifying the extract using a silica gel column, an ODS column, and thin layer chromatography to obtain a tea saponin monomer; In step S03, the deep eutectic solvent is a choline-based deep eutectic solvent composed of a hydrogen bond acceptor and a hydrogen bond donor in a certain stoichiometric ratio, wherein the hydrogen bond acceptor is choline chloride, and the hydrogen bond donors are 1,4-butanediol and 1,3-butanediol. Specifically, choline chloride, 1,4-butanediol, and 1,3-butanediol are mixed in a certain proportion, a certain proportion of water is added, and the mixture is heated and stirred at 80-85°C to form a uniform and stable liquid.

2. The method for extracting tea saponins from camellia oil cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent according to claim 1, characterized in that In step S01, the oil-tea camellia cake is dried, fully crushed using a grinder, passed through a 60-70 mesh sieve, and dried and stored.

3. The method for extracting tea saponins from camellia oil cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent according to claim 1, characterized in that In step S02, cellulase and pectinase are respectively dissolved in PBS buffer with a pH of 5 to obtain enzyme solution, wherein the cellulase accounts for 20% of the total enzyme solution of cellulase and pectinase, and the enzymolysis time is 2-3 hours.

4. The method for extracting tea saponins from camellia oil cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent according to claim 1, characterized in that In step S04, the specific process is as follows: adding the low eutectic solvent to the enzymatically hydrolyzed camellia cake, and then placing it in an ultrasonic cleaner to perform auxiliary extraction of tea saponins from the camellia cake to obtain a crude tea saponin extract from the camellia cake; finally, subjecting the crude tea saponin extract from the camellia cake to high-speed centrifugation to obtain a supernatant, which is the tea saponin extract from the camellia cake, and storing it at 2-8°C for later use; wherein, the ultrasonic extraction power is 240-260 W, the ultrasonic time is 2-3 hours, and the extraction material-liquid ratio is 1:

20.

5. The method for extracting tea saponins from camellia oil cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent according to claim 4, characterized in that ,Step S05, includes the following steps: In step 5.1, weigh 100 mg of tea saponin and dissolve it in 80% ethanol. Dose 1.0, 2.0, 3.0, 4.0, and 5.0 mL of the solution into a 10-mL volumetric flask and dilute to volume. This yields a series of tea saponin standard solutions with concentrations of 100.0 μg / mL, 200.0 μg / mL, 300.0 μg / mL, 400.0 μg / mL, and 500.0 μg / mL. Store these solutions at room temperature until use. Step 5.2, prepare 77% sulfuric acid solution and 8% vanillin solution: Weigh 77 mL of concentrated sulfuric acid and mix with 23 mL of deionized water, and store at room temperature until ready to use. Weigh 0.8 g of vanillin and dilute to 10 mL with anhydrous ethanol. Prepare immediately. In step 5.3, place 0.5 mL of the above tea saponin standard solution in a stoppered test tube, add 0.5 mL of 8% vanillin solution, transfer the test tube to an ice-water mixture, and add 4 mL of 77% concentrated sulfuric acid. Shake well, heat in a 60°C water bath for 20 min, and then cool in ice water for 10 min. Measure the absorbance at 550 nm using a blank as a control. Step 5.4: Prepare a tea saponin concentration standard curve with tea saponin concentration as the horizontal axis and absorbance at 550 nm as the vertical axis. The linear regression equation is: y = 0.002x - 0.0622, R 2 = 0.9992; Step 5.5, diluting the tea saponin extract from the camellia oil cake obtained in step S04 by n times, where n is 5-10; using this solution instead of the tea saponin standard solution, and measuring the absorbance of the diluted tea saponin extract at a wavelength of 550 nm according to the steps of step 5.3; Step 5.6, calculating the tea saponin content in the tea saponin extract of camellia oleifera cake according to the linear regression equation of the tea saponin standard solution obtained in step 5.4.

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