Method for extracting tea saponin in oil tea cake by using enzymolysis coupling ultrasonic-assisted eutectic solvent and optimization method thereof

Abstract

The invention discloses a method for extracting tea saponin from oil tea cakes by using an enzymolysis-coupled ultrasonic-assisted deep-eutectic solvent and an optimization method of the method, and relates to the technical field of natural product extraction. According to the method, the tea saponin in the oil tea cake is extracted by combining an enzymolysis method, an ultrasonic-assisted method and a deep eutectic solvent, compared with a conventional extraction method, the content of the tea saponin extracted by the method is increased by 140%, and the method is novel, efficient, green and environment-friendly, avoids the use of a large amount of organic solvent, can effectively relieve the problem of environmental pollution, and is suitable for industrial production. And a new thought and a new method are provided for extraction of active components in natural products.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction, and specifically relates to a method for extracting tea saponin from tea oil cake by enzymatic hydrolysis coupled with ultrasound-assisted low eutectic solvent and an optimization method thereof, wherein tea saponin from tea oil cake is extracted by utilizing a low eutectic solvent. Background Art

[0002] Camellia oleifera Camellia oleifera Abel), also known as common tea, Wild camellia, mountain camellia, and single-seed camellia are an evergreen shrub or tree of the genus Camellia in the family Theaceae. Camellia grows in dense forests on high mountains or shallow hills, in mixed woods or bamboo forests on the edge of hillsides, scattered or concentrated, and sometimes forms large wild forests. Camellia oleifera is native to southern China and Vietnam, Laos, Myanmar, and Assam in India in Southeast Asia. It is distributed wild in Hunan, Jiangxi, Guangxi, Hainan and other provinces in China. The tea oil made from camellia oleifera is rich in nutrients, rich in oleic acid, linoleic acid, etc., and has a mellow taste. It is a high-quality edible oil; it is also an excellent industrial raw material and can be used in soap making, cosmetics, rubber and other industries. The roots, leaves, flowers, seeds, oil extracted from seeds, and the residues left after oil extraction can all be used as medicine, with the effects of clearing heat and detoxifying, cooling blood and stopping bleeding.

[0003] Tea saponin is a natural nonionic surfactant, with hydrophilic sugars and hydrophobic ligands in its molecules. Tea saponin is a pentacyclic triterpenoid glycoside compound extracted from Camellia Oleifera Abel, a plant of the Theaceae family. It is a mixture of oleanane-type pentacyclic triterpenoid saponins, and its basic structure includes three parts: sapogenin, sugars, and organic acids. It has multiple surface activities such as emulsification, dispersion, wetting, decontamination, and foaming, as well as multiple biological activities such as antibacterial, anti-inflammatory, and fish poisoning. It is widely used in daily chemical industry, pesticides, medicine, food, building materials, metallurgy and other fields. In recent years, countries have attached more and more importance to the extraction and application research of tea saponin.

[0004] The traditional conventional tea saponin extraction process uses chemical extraction methods, which requires the use of a large amount 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 type of excellent green solvent that has only emerged rapidly in recent years. Compared with traditional organic solvents, deep eutectic solvents have the advantages of low price, simple preparation, stable chemical properties, easy storage, non-toxicity and biodegradability. They are an ideal alternative to traditional organic solvents and green extraction solvents for natural products. Deep eutectic solvents are widely used in the extraction of natural products. Researchers have reported that they can be used to extract substances such as flavonoids, phenols, polysaccharides, proteins, anthocyanins and alkaloids. Summary of the invention

[0006] The present invention aims to provide a method for extracting tea saponin from tea oil cake by enzymatic hydrolysis coupled with ultrasound-assisted low eutectic solvent. The method can prepare high-purity tea saponin with a high yield, and can be safely and effectively used in the fields of food, agriculture, medicine, etc.

[0007] The objective of the present invention is achieved through the following technical solutions: On the one hand, the present invention provides a method for extracting tea saponin from tea cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent, comprising the following steps: S01, pre-treating camellia oil cake to obtain dry camellia oil cake powder; S02, preparing cellulase and pectinase enzyme solution, and using cellulase and pectinase to enzymolyze the tea oil cake powder; enzymolysis can destroy the fiber of the tea oil cake, making it easier to extract; S03, preparing a deep eutectic solvent; S04, extracting tea saponin from tea oil cake with the help of a low eutectic solvent to obtain a tea saponin extract from tea oil cake; S05. Determine the tea saponin content in the tea saponin extract of tea oil cake using the vanillin-sulfuric acid method; S06. The tea saponin extract is enriched by macroporous resin to obtain the camellia oleifera cake extract, and the tea saponin monomer is separated and purified by silica gel column, ODS column and thin layer chromatography.

[0008] Furthermore, in step S01, the camellia oil cake is dried, fully crushed using a grinder, passed through a 60-80 mesh sieve, and dried and stored.

[0009] Furthermore, in step S02, cellulase and pectinase are respectively dissolved in a PBS buffer solution with a pH of 5 to obtain an 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.

[0010] Further, in step S03, the low eutectic solvent is a choline-based low 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 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 and a certain proportion of water is added, and the mixture is heated and stirred at 80-85°C to synthesize a uniform and stable liquid.

[0011] Furthermore, in step S04, the specific process is: adding the low eutectic solvent to the tea cake after enzymatic hydrolysis, and then placing it in an ultrasonic cleaner, using the ultrasonic cleaner to assist in the extraction of tea saponin from the tea cake to obtain a crude tea saponin extract from the tea cake; finally, subjecting the crude tea saponin extract from the tea cake to high-speed centrifugation, and obtaining the supernatant, which is the tea saponin extract from the tea cake, which is stored at 2-8°C for standby use; wherein the ultrasonic extraction power is 240-260 W, the ultrasonic time is 2-3h, and the extraction material-liquid ratio is 1:20.

[0012] Furthermore, step S05 includes the following steps: Step 5.1, weigh 100 mg of tea saponin and dissolve it in 80% ethanol, dilute to 100 mL, and pipette 1.0, 2.0, 3.0, 4.0, and 5.0 mL into a 10 mL volumetric flask to dilute to volume, to obtain 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, which are stored at room temperature for later 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 for later use; weigh 0.8 g of vanillin, dilute to 10 mL with anhydrous ethanol, and prepare for use immediately; Step 5.3, take 0.5 mL of the above tea saponin standard solution into a stoppered test tube, add 0.5 mL of 8% vanillin solution, transfer the test tube into an ice-water mixture, add 4 mL of 77% concentrated sulfuric acid; after shaking, heat in a 60°C water bath for 20 min, and then cool in ice water for 10 min; use the blank as a control and measure the absorbance at a wavelength of 550 nm; Step 5.4, with tea saponin concentration as the horizontal axis and absorbance at 550 nm as the vertical axis, a tea saponin concentration standard curve was prepared to obtain the linear regression equation: y = 0.002x - 0.0622, R 2 = 0.9992; Step 5.5, diluting the tea saponin extract obtained from the camellia oleifera cake in step 4 by n times, where n is 5-10; using the solution instead of the tea saponin standard solution, and determining the absorbance of the diluted tea saponin extract at a wavelength of 550 nm according to the steps in step 5.3; Step 5.6, according to the linear regression equation of the tea saponin standard solution obtained in step 5.4, calculate the tea saponin content in the tea saponin extract of camellia oleifera cake, the formula is as follows: tea saponin content (mg / g) = [(y + 0.0622) × V ×n] / (0.002×m), wherein y is the absorbance value at a wavelength of 550 nm, V is the volume of the tea saponin extract of camellia oleifera cake (mL), and n is the dilution multiple of the tea saponin extract of camellia oleifera cake; 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 camellia oleifera cake powder (g).

[0013] The present invention also proposes an optimization method for extracting tea saponin from tea cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent, which is characterized by comprising the following steps: Step A: According to the method of extracting tea saponin from tea oil cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent as described above, 6 factors affecting the extraction rate of tea saponin were screened out, specifically: the type of deep eutectic solvent used for extraction, the molar ratio of hydrogen bond acceptors and hydrogen bond donors in the deep eutectic solvent, the ratio of 1,3-butanediol to 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 extraction material-liquid ratio; Step B: Taking the tea saponin content in the tea saponin extract of camellia oleifera cake as an indicator, a single factor experiment is performed based on the six factors screened out in step A to determine the optimal extraction process factor parameters.

[0014] Furthermore, through single factor experiments, the optimal extraction process conditions were obtained as follows: the molar ratio of hydrogen bond acceptor and hydrogen bond donor in the low eutectic solvent was 1:4, the molar ratio of 1,4-butanediol and 1,3-butanediol was 1:1, the extraction liquid ratio was 1:40 g / mL, the water content of the low eutectic solvent was 10%, and the cellulase accounted for 20% of the total enzyme solution composed of cellulase and pectinase.

[0015] The present invention also proposes a low eutectic solvent for extracting tea saponin from oil-tea cake, which is a choline-based low 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 ratio of 1:2:2, 10% of water is added, and the mixture is heated and stirred at 80-85°C to synthesize a uniform and stable liquid.

[0016] After the extraction process of the present invention, the tea saponin content of the tea cake extract is 28.26 mg / g (dry weight of tea cake powder).

[0017] The method for extracting tea saponin from oil-tea cake provided by the present invention uses a new green low eutectic solvent as an extractant, 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 amount of organic solvents used in traditional chemical extraction of tea saponin.

[0018] The method for extracting tea saponin from oil-tea cake provided by the present invention organically combines three extraction methods, namely, low eutectic solvent, enzymatic hydrolysis and ultrasound-assisted extraction, into the extraction of tea saponin from oil-tea cake. Compared with conventional extraction methods, the method is novel, green and environmentally friendly, has a high tea saponin extraction rate from oil-tea cake, low extraction cost, mild extraction conditions, a simple, stable and reliable extraction process, and low extraction energy consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the standard curve of tea saponin concentration.

[0020] Figure 2 Schematic diagram of the effect of the extraction solvent on the tea saponin content of the tea saponin extract of tea oil cake in the present invention.

[0021] Figure 3 Schematic diagram showing the effect of the ratio of 1,3-butanediol to 1,4-butanediol in DES on the tea saponin content in the tea saponin extract from camellia oleifera cake in the present invention.

[0022] Figure 4 Schematic diagram showing the effect of the molar ratio of the DES hydrogen bond acceptor to the hydrogen bond donor on the tea saponin content of the tea saponin extract from tea oil cake in the present invention.

[0023] Figure 5 Schematic diagram of the effect of DES water content on the tea saponin content of the tea saponin extract from tea oil cake in the present invention.

[0024] Figure 6 It is a schematic diagram showing the effect of the proportion of cellulase on the tea saponin content of the tea saponin extract from tea oil cake in the present invention.

[0025] Figure 7 Schematic diagram of the effect of the material-liquid ratio on the tea saponin content of the tea saponin extract of tea oil cake in the present invention. DETAILED DESCRIPTION

[0026] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited thereto: The oil tea of ​​the present invention is also known as common oil tea, Wild camellia oleifera is an evergreen shrub or tree of the genus Camellia in the family Theaceae. Camellia oleifera is native to southern China and is distributed wild in Hunan, Jiangxi, Guangxi, Hainan and other provinces.

[0027] Example 1: A method for extracting tea saponin from tea cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent, comprising the following steps: S01, pre-treating camellia oil cake to obtain dry camellia oil cake powder; S02, preparing cellulase and pectinase enzyme solutions, and enzymolyzing the tea oil cake powder with the cellulase and pectinase; S03, preparing a deep eutectic solvent; S04, extracting tea saponin from tea oil cake with the help of a low eutectic solvent to obtain a tea saponin extract from tea oil cake; S05. Determine the tea saponin content in the tea saponin extract of tea oil cake using the vanillin-sulfuric acid method; S06. The tea saponin extract is enriched by macroporous resin to obtain the camellia oleifera cake extract, and the tea saponin monomer is separated and purified by silica gel column, ODS column and thin layer chromatography.

[0028] The details are as follows: 1 Plant sample collection: Camellia oleifera cakes were sourced from Chun'an County, Hangzhou City, Zhejiang Province. Camellia oleifera cakes were placed in a portable high-speed universal grinder and ground at 25,000 rpm / min for 30 s to obtain camellia oleifera cake powder. The ground camellia oleifera cake powder was placed in a laboratory oven and dried at 60 °C. After drying, it was sealed in a bag and stored for later use.

[0029] 2 Enzyme solution preparation: Accurately weigh 0.25 g of cellulase (400 u / mg) and 0.20 g of pectinase (500 u / mg) and dissolve them in pH=5 phosphate buffer and dilute to 50 mL. 2000 u / mL of cellulase solution and pectinase solution are obtained respectively. The pH=5 phosphate buffer is prepared as follows: Accurately weigh 27.2 g of potassium dihydrogen phosphate powder and dissolve it in deionized water and dilute to 1000 mL, and adjust to pH=5 with sodium hydroxide solution.

[0030] 3. Preparation of low eutectic solvent: 13.96 g of choline chloride, 18.02 g of 1,4-butanediol and 18.02 g of 1,3-butanediol were weighed in a beaker (100 mL) with a magnetic stirring rotor according to a molar ratio of 1:2:2, and 1.00 mL of water was added. Subsequently, the beaker was placed in a constant temperature water bath with a magnetic stirrer, and heated and stirred at a constant temperature of 80°C until a clear, transparent, and uniform liquid was formed (about 1-1.5 h), which is the choline chloride-1,4-butanediol and 1,3-butanediol low eutectic solvent, which was cooled to room temperature and set aside.

[0031] 4 Preparation of tea saponin extract from oil-tea cake: Accurately weigh 0.10 g of oil-tea cake powder in a 10 mL centrifuge tube, add 0.2 mL of cellulase solution and 0.8 mL of pectinase solution, and enzymolyze in a 30 ℃ water bath for 3 h. Then add 4 mL of choline chloride-1,4-butanediol and 1,3-butanediol low eutectic solvent (the material-liquid ratio of extraction is 1:40, the material refers to the oil-tea cake powder, and the liquid refers to the low eutectic solvent), place the centrifuge tube in an ultrasonic cleaner, and use the ultrasonic cleaner to assist in the extraction of tea saponin from oil-tea cake to obtain a crude tea saponin extract from oil-tea cake. Finally, the crude tea saponin extract from oil-tea cake was centrifuged at 8000 rpm for 10 min, and the supernatant obtained was the tea saponin extract from oil-tea cake, which was stored at 2-8 ℃ for later use. Among them, the ultrasonic-assisted extraction conditions are: ultrasonic power 240 w, ultrasonic time 2 h.

[0032] 5 The tea saponin content in the tea saponin extract of tea oil cake was determined by the vanillin-sulfuric acid method. The specific process is as follows: (1) Preparation of tea saponin standard solution: accurately weigh 20 mg of tea saponin and dissolve it in 80% ethanol, dilute to 20 mL to obtain tea saponin standard solution. Pipette 1.0, 2.0, 3.0, 4.0, and 5.0 mL into a 10 mL volumetric flask and dilute to volume to obtain 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 at room temperature for later use. (2) Drawing of the tea saponin standard curve: Take 6 15mL test tubes, take 0.5mL of the tea saponin standard solution in step (1) above into the test tube, add 0.5mL of 8% vanillin solution, add 2.0ml of anhydrous ethanol solution, move the test tube into an ice-water mixture, and add 2.0mL of 77% sulfuric acid solution. After shaking, heat in a 60℃ water bath for 20 minutes, and then cool in ice water for 10 minutes. Measure the absorbance at a wavelength of 550 nm; at the same time, replace the tea saponin standard solution with 1.0mL of 80% ethanol, and perform a blank experiment according to the above method. Finally, use the tea saponin concentration as the horizontal axis and the absorbance value at a wavelength of 550 nm as the vertical axis to make a tea saponin concentration standard curve (such as Figure 1 As shown), we get the linear regression equation: y = 0.002x - 0.0622, and the correlation coefficient R 2 =0.9992, indicating that the tea saponin standard showed a good linear relationship in the range of 100.0-500.0 μg / mL.

[0033] Among them, 8% vanillin solution and 77% sulfuric acid solution are prepared as follows: Preparation of 8% vanillin solution: weigh 0.8 g vanillin and dissolve it in anhydrous ethanol, dilute to 10 mL to obtain 8% vanillin solution, store it at room temperature for later use, and use it immediately after preparation; Preparation of 77% sulfuric acid solution: Accurately weigh 77 mL of concentrated sulfuric acid and mix with 23 mL of deionized water to obtain 77% sulfuric acid solution, and store it at room temperature for later use; (3) Determination of the tea saponin content of the tea saponin extract from camellia oleifera cake: dilute the tea saponin extract from camellia oleifera cake obtained in step 4 by 10 times; replace the tea saponin standard solution in step 5 with 1.0 mL of the diluted tea saponin extract from camellia oleifera cake in step 4, measure the absorbance of the sample at a wavelength of 550 nm, and calculate the tea saponin content of the tea saponin extract from camellia oleifera cake according to the tea saponin standard linear regression equation in step 5 according to the following formula: Tea saponin content (mg / g tea cake powder dry weight) = [ ( y + 0.0622) × V × n ] / (0.002 × m ) = [ ( 0.503 + 0.0622) × 10 × 1 ] / (0.002 × 0.1 × 1000) = 28.26; Wherein, y is the absorbance value at a wavelength of 550 nm, V is the volume of tea saponin extract from camellia oleifera cake (mL), n is the dilution multiple of tea saponin extract from camellia oleifera cake; 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 camellia oleifera cake powder (g).

[0034] 6. The tea saponin extract was enriched by macroporous resin to obtain the tea cake extract, which was separated and purified by silica gel column, ODS column and thin layer chromatography to obtain the tea saponin monomer.

[0035] Example 2: A method for optimizing the process conditions of extracting tea saponin from tea cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent, the specific steps are as follows: 1 According to the aforementioned method of extracting tea saponin from camellia oleifera cake by enzymatic hydrolysis coupled with ultrasound-assisted low eutectic solvent, 6 factors affecting the tea saponin extraction rate were screened out, 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 the low eutectic solvent, the amount of water added when preparing the low eutectic solvent, the solid-liquid ratio of camellia oleifera cake powder and the low eutectic solvent, and the ratio of cellulase to pectinase. Taking the tea saponin content of tea saponin extract from camellia oleifera cake as the reference index, a single factor experiment was conducted, as follows: 1.1 According to the above-mentioned method of enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent extraction of tea saponin from camellia oleifera cake, 8 different deep eutectic solvents and two different concentrations of ethanol were selected for single-factor experiments on extraction solvents: the molar ratio of hydrogen bond acceptor to hydrogen bond donor was fixed at 1:2, the molar ratio of 1,3-butanediol to 1,4-butanediol was 1:1, the water addition amount during the preparation of the deep eutectic solvent was 20%, and the extraction material-liquid ratio was 1:50. g / mL, the proportion of cellulase in the total enzyme solution added with cellulase and pectinase was 50%, and the ultrasonic-assisted conditions remained unchanged. The tea saponin content of the tea saponin extract of camellia oil cake was determined when the extraction solvent was DES-1,3-butanediol, DES-1,4-butanediol, DES-triethylene glycol, DES-ethylene glycol, DES-propylene glycol, DES-urea, DES-triethylene glycol + 1,3-butanediol, DES-triethylene glycol + ethylene glycol, DES-triethylene glycol + 1,4-butanediol, DES-1,3-butanediol + 1,4-butanediol, 50% ethanol, and 80% ethanol.

[0036] Effect of extraction solvent on the tea saponin content of tea saponin extract from tea oil cake Figure 2 As shown, it can be seen that when the extraction solvent is choline chloride-1,3-butanediol and 1,4-butanediol, the tea saponin content of the tea saponin extract of camellia oil cake is higher.

[0037] 1.2 According to the aforementioned method of enzymatic hydrolysis coupled with ultrasound-assisted low eutectic solvent extraction of tea saponin from camellia oil cake, different proportions of 1,3-butanediol and 1,4-butanediol were selected for a single factor experiment: the low eutectic solvent was fixed as choline chloride-1,3-butanediol and 1,4-butanediol with a molar ratio of 1:2, the amount of water added during the preparation of the low eutectic solvent was 20%, the extraction material-liquid ratio was 1:50 g / mL, cellulase accounted for 60% of the total enzyme solution when cellulase and pectinase were added, and the ultrasonic-assisted conditions remained unchanged. The tea saponin content of the tea saponin extract from camellia oil cake was determined when the molar ratio of the low eutectic solvent 1,3-butanediol and 1,4-butanediol was 3:1, 2:1, 1:1, 1:2, and 1:3.

[0038] Effect of the molar ratio of 1,3-butanediol to 1,4-butanediol in the preparation of deep eutectic solvent on the content of tea saponin in tea cake extract Figure 3 As shown, it can be seen that with the increase of the molar ratio of 1,3-butanediol and 1,4-butanediol, the tea saponin content of the tea saponin extract from tea oil cake first increases and then decreases. When the molar ratio is 1:1, the tea saponin content of the tea saponin extract from tea oil cake reaches the maximum value. Subsequently, the tea saponin content of the tea saponin extract from tea oil cake begins to decrease. Therefore, the best low eutectic solvent is 1,3-butanediol and 1,4-butanediol with a molar ratio of 1:1.

[0039] 1.3 According to the above-mentioned method of enzymatic hydrolysis coupled with ultrasound-assisted low eutectic solvent extraction of camellia oil cake, 6 different molar ratios of choline chloride and hydrogen bond donors were selected for single factor experiment: the low eutectic solvent was fixed as choline chloride-1,3-butanediol and 1,4-butanediol, the molar ratio of 1,3-butanediol and 1,4-butanediol was 1:1, the amount of water added during the preparation of the low eutectic solvent was 20%, the extraction material-liquid ratio was 1:50 g / mL, cellulase and pectinase accounted for 60% of the total enzyme solution, and the ultrasonic-assisted conditions remained unchanged. The tea saponin content of the tea saponin extract of camellia oil cake was determined when the molar ratio of the low eutectic solvent choline chloride and hydrogen bond donor was 1:1, 1:2, 1:3, 1:4, 1:5, and 1:6.

[0040] Effect of the molar ratio of choline chloride to hydrogen bond donor in the preparation of deep eutectic solvent on the tea saponin content of tea saponin extract from camellia oleifera cake Figure 4 As shown, it can be seen that with the increase of the molar ratio of choline chloride to hydrogen bond donor, the tea saponin content of the tea saponin extract from tea oil cake first increases and then decreases. When the molar ratio is 1:4, the tea saponin content of the tea saponin extract from tea oil cake reaches the maximum value. Subsequently, the tea saponin content of the tea saponin extract from tea oil cake begins to decrease. Therefore, the best low eutectic solvent is choline chloride and (1,3-butanediol and 1,4-butanediol) with a molar ratio of 1:4.

[0041] 1.4 According to the aforementioned method of enzymatic hydrolysis coupled with ultrasound-assisted low eutectic solvent extraction of tea saponin from camellia oleifera cake, 7 levels were selected for a single-factor experiment on the amount of water added when preparing the low eutectic solvent. The low eutectic solvent was fixed to 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 material-liquid ratio was 1:50 g / mL, cellulase accounted for 60% of the total enzyme solution when cellulase and pectinase were added, and the ultrasonic-assisted conditions remained unchanged. The tea saponin content of the tea saponin extract from camellia oleifera cake was determined when the amount of water added when preparing the low eutectic solvent was 0%, 10%, 20%, 30%, 40%, 50%, and 60%.

[0042] Effect of water addition amount on tea saponin content of tea saponin extract in tea cake during preparation of deep eutectic solvent Figure 5 As shown, it can be seen that the tea saponin content of the tea saponin extract from tea cakes shows a decreasing trend with the increase of the amount of water added when preparing the low eutectic solvent. When the amount of water added is 10%, the tea saponin content of the tea saponin extract from tea cakes is the highest. Therefore, the amount of water added when preparing the best low eutectic solvent is 10%.

[0043] 1.5 According to the aforementioned method of enzymatic hydrolysis coupled with ultrasound-assisted low eutectic solvent extraction of tea saponin from camellia oleifera cake, 6 levels were selected for a single-factor experiment on the ratio of adding cellulase and pectinase, the low eutectic solvent was fixed to 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 amount of water added during the preparation of the low eutectic solvent was 20%, the extraction material-liquid ratio was 1:50 g / mL, and the ultrasonic-assisted conditions remained unchanged. The tea saponin content of the tea saponin extract from camellia oleifera cake was determined when the proportion of cellulase in the total enzyme solution of cellulase and pectinase was 0%, 20%, 40%, 60%, 80%, and 100%.

[0044] Effect of the proportion of cellulase in the total enzyme solution of cellulase and pectinase on the content of tea saponin in the tea saponin extract of camellia oil cake Figure 6 As shown, it can be seen that the tea saponin content of tea saponin extract from oil-tea cake shows a trend of first increasing and then decreasing with the increase of cellulase proportion. When the cellulase proportion in the total enzyme solution of cellulase and pectinase is 20%, the tea saponin content of tea saponin extract from oil-tea cake is the highest. Therefore, the optimal cellulase proportion is 20%.

[0045] 1.6 According to the aforementioned method of enzymatic hydrolysis coupled with ultrasound-assisted low eutectic solvent extraction of tea saponin from camellia oleifera cake, 8 levels were selected for a single-factor experiment on the solid-liquid ratio of extraction, and the low eutectic solvent was fixed to 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 amount of water added during the preparation of the low eutectic solvent was 10%, the proportion of cellulase in the total enzyme solution added with cellulase and pectinase was 20%, the ultrasonic-assisted conditions remained unchanged, and the tea saponin content of the tea saponin extract from camellia oleifera cake was determined when the solid-liquid ratio was 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, and 1:80.

[0046] Effect of extraction material-liquid ratio on the tea saponin content of tea saponin extract from tea oil cake Figure 7 As shown, it can be seen that the tea saponin content of the tea saponin extract from tea cake shows an increasing trend with the increase of the solid-liquid ratio. When the solid-liquid ratio is 1:40, the tea saponin content of the tea saponin extract from tea cake is high and the subsequent growth is slow. Therefore, the optimal solid-liquid ratio is 1:40.

[0047] 2 The optimal process conditions for extracting tea saponin from tea cake by ultrasound-assisted deep eutectic solvent are as follows: the molar ratio of hydrogen bond acceptor and hydrogen bond donor in DES is 1:4, the extraction material-liquid ratio is 1:40 g / mL, the cellulase and pectinase solution accounts for 20%, and the tea saponin content of tea cake extract is predicted to be 28.26 mg / g (dry weight of tea cake powder). This value is 140% higher than that of the traditional ethanol method, indicating that the process of extracting tea saponin from tea cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent is feasible and can be promoted for industrial application.

[0048] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for extracting tea saponin 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 enzymolyzing the tea oil cake powder with the cellulase and pectinase; S03, preparing a deep eutectic solvent; S04, extracting tea saponin from tea oil cake with the help of a low eutectic solvent to obtain a tea saponin extract from tea oil cake; S05. Determine the tea saponin content in the tea saponin extract of tea oil cake using the vanillin-sulfuric acid method; S06. The tea saponin extract is enriched by macroporous resin to obtain the camellia oleifera cake extract, and the tea saponin monomer is separated and purified by silica gel column, ODS column and thin layer chromatography.

2. The method for extracting tea saponin from tea cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent according to claim 1, characterized in that In step S01, the oil-tea 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 saponin from tea 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 at pH=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 2-3h.

4. The method for extracting tea saponin from tea cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent according to claim 1, characterized in that In step S03, the low eutectic solvent is a choline-based low eutectic solvent composed of a certain stoichiometric ratio of hydrogen bond acceptors and hydrogen bond donors, 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 and a certain proportion of water is added, and heated and stirred at 80-85°C to synthesize a uniform and stable liquid.

5. The method for extracting tea saponin from tea cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent according to claim 4, characterized in that In step S04, the specific process is: adding the low eutectic solvent to the tea cake after enzymatic hydrolysis, and then placing it in an ultrasonic cleaner, using the ultrasonic cleaner to assist in extracting tea saponin from the tea cake to obtain a crude tea saponin extract from the tea cake; finally, subjecting the crude tea saponin extract from the tea cake to high-speed centrifugation, and obtaining the supernatant, which is the tea saponin extract from the tea cake, which is stored at 2-8 °C for standby use; wherein, the ultrasonic extraction power is 240-260 W, the ultrasonic time is 2-3h, and the extraction material-liquid ratio is 1:

20.

6. The method for extracting tea saponin from tea cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent according to claim 5, characterized in that ,Step S05, includes the following steps: Step 5.1, weigh 100 mg of tea saponin and dissolve it in 80% ethanol, dilute to 100 mL, and pipette 1.0, 2.0, 3.0, 4.0, and 5.0 mL into a 10 mL volumetric flask to dilute to volume, to obtain 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, which are stored at room temperature for later 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 for later use; weigh 0.8 g of vanillin, dilute to 10 mL with anhydrous ethanol, and prepare for use immediately; Step 5.3, take 0.5 mL of the above tea saponin standard solution into a stoppered test tube, add 0.5 mL of 8% vanillin solution, transfer the test tube into an ice-water mixture, add 4 mL of 77% concentrated sulfuric acid; after shaking, heat in a 60°C water bath for 20 min, and then cool in ice water for 10 min; use the blank as a control and measure the absorbance at a wavelength of 550 nm; Step 5.4, with tea saponin concentration as the horizontal axis and absorbance at 550 nm as the vertical axis, a tea saponin concentration standard curve was prepared to obtain the linear regression equation: y = 0.002x - 0.0622, R 2 = 0.9992; Step 5.5, diluting the tea saponin extract obtained from the camellia oleifera cake in step 4 by n times, where n is 5-10; using the solution instead of the tea saponin standard solution, and determining the absorbance of the diluted tea saponin extract at a wavelength of 550 nm according to the steps in step 5.3; Step 5.6, according to the linear regression equation of the tea saponin standard solution obtained in step 5.4, calculate the tea saponin content in the tea saponin extract of camellia oleifera cake, the formula is as follows: tea saponin content (mg / g) = [(y + 0.0622) × V × n] / (0.002× m), wherein y is the absorbance value at a wavelength of 550 nm, V is the volume of the tea saponin extract of camellia oleifera cake (mL), and n is the dilution multiple of the tea saponin extract of camellia oleifera cake; 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 camellia oleifera cake powder (g).

7. An optimization method for extracting tea saponin from tea cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent, characterized in that: The steps include: Step A: According to the method for extracting tea saponin from tea oil cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent according to any one of claims 4 to 6, 6 factors affecting the extraction rate of tea saponin are screened out, specifically: the type of deep eutectic solvent used for extraction, the molar ratio of hydrogen bond acceptors and hydrogen bond donors in the deep eutectic solvent, the ratio of 1,3-butanediol to 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 extraction material-liquid ratio; Step B: Taking the tea saponin content in the tea saponin extract of camellia oleifera cake as an indicator, a single factor experiment is performed based on the six factors screened out in step A to determine the optimal extraction process factor parameters.

8. The optimization method of the method for extracting tea saponin from tea cake by enzymatic hydrolysis coupled with ultrasound-assisted deep eutectic solvent according to claim 7, characterized in that: The optimal extraction process conditions were obtained through single factor experiments: the molar ratio of hydrogen bond acceptor and hydrogen bond donor in the low eutectic solvent was 1:4, the molar ratio of 1,4-butanediol and 1,3-butanediol was 1:1, the extraction material-liquid ratio was 1:40 g / mL, the water content of the low eutectic solvent was 10%, and the proportion of cellulase in the cellulase and pectinase solution was 20%.

9. A low eutectic solvent for extracting tea saponin from tea oil cake, characterized in that: It is a choline-based low 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 ratio of 1:2:2, 10% of water is added, and the mixture is heated and stirred at 80-85°C to synthesize a uniform and stable liquid.

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