CN113105519A - Method for purifying tea saponin from tea seed cake - Google Patents
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- C07H1/08—Separation; Purification from natural products
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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Abstract
The invention discloses a method for purifying tea saponin from tea seed cake. The invention takes a sulfuric acid-vanillin method which is one of colorimetric methods as a concentration detection means, determines a standard curve of the tea saponin, has good linear correlation degree and can be used for concentration detection; performing single-factor experiments and orthogonal experiments, and determining the optimal extraction parameters under the premise of considering industrialization; purifying the tea saponin by adopting n-butanol and macroporous resin, and finally determining to purify by AB-8 macroporous resin and optimizing related parameters. The invention has the following beneficial effects: the method adopts the analytical means of liquid chromatography, mass spectrum, ultraviolet-visible spectrophotometry, infrared and the like to determine that the purity of the obtained tea saponin product reaches 90 percent, and the total recovery rate is 49.3 percent.
Description
(I) technical field
The invention relates to a method for purifying tea saponin from tea seed cake.
(II) background of the invention
The camellia oleifera is a perennial woody plant, belongs to the genus camellia of the family theaceae, and is a woody edible oil tree species unique to China. The components in the oil tea seed cake comprise moisture, fat, protein, crude fiber, saccharide, tea saponin, etc., wherein the content of the tea saponin is very high, about 12.8%. With the deep processing of the tea seed cake by tea-oil camellia processing enterprises, the price of the tea seed cake which is originally used by forest farmers only as a fertilizer field is increased in a saving way, and the price of the tea seed cake can be further increased along with the further fine processing of the tea saponin and the development of the whole tea saponin industry. The comprehensive utilization of the oil tea cakes can increase the income of vast forest farmers; the development of the oil tea dry cake comprehensive industry can also promote the lignification of the edible vegetable oil in China, and the oil tea dry cake comprehensive industry is a good matter benefiting the nation and the people for assisting the grain and oil safety and the farmland safety in China.
In recent years, tea saponin serving as a surfactant has great application potential in the aspects of water environment and soil remediation. In addition, the tea saponin also has wide application in the fields of daily chemicals, food, pesticides, medicines, buildings, printing and dyeing, environmental protection industries and the like.
At present, the extraction method of tea saponin comprises a water extraction method, an organic solvent method, an ultrasonic-assisted extraction method, a supercritical extraction method and the like. The organic solvent method is used for extracting tea saponin by using the property that the tea saponin is easily dissolved in organic solvents such as aqueous methanol, aqueous ethanol and the like. Ethanol and the like are mostly selected as an extracting agent due to the limitation of the toxicity of methanol. The organic solvent method has the advantages of low energy consumption, less dissolved impurities, light product color, high extraction rate, high purity, convenient powder production and easy further purification, and the product obtained by the method can be directly used as biochemical reagents and medical raw materials. The macroporous resin purification method can obtain the tea saponin with extremely high purity, the recovery rate is also higher, and the macroporous resin can be regenerated, recovered and recycled, so the macroporous resin method is a good method suitable for industrial operation.
Disclosure of the invention
Aiming at actual requirements, the invention aims to explore a process flow for extracting and purifying tea saponin from a camellia oleifera cake, optimize process parameters and provide a method for purifying tea saponin from the camellia oleifera cake.
The technical scheme adopted by the invention is as follows:
a method of purifying tea saponin from tea seed cake, the method comprising:
(1) tea cake pretreatment: insolating the tea seed cake for 2-3 days, and then crushing to 50-100 meshes;
(2) alcohol extraction and suction filtration: step (1), the tea seed cake powder is prepared by mixing the following raw materials in a ratio of 1: 4-20, adding 70-90 vol% ethanol solution, performing reflux extraction at 60-80 ℃ for 60-135 min, cooling the extract, and performing suction filtration to obtain tea saponin alcohol extract;
(3) and (3) macroporous resin purification: and (2) after the alcohol in the crude tea saponin extract is removed by rotary evaporation, preparing a 5-20 g/L aqueous solution of a crude tea saponin extract by using deionized water, loading the AB-8 macroporous adsorption resin into a column, loading the aqueous solution of the crude tea saponin extract into the column at the flow rate of 2/3-10/3 BV/h, passing the aqueous solution through the column, eluting the aqueous solution of the crude tea saponin extract by using deionized water, 20% ethanol and 80% ethanol respectively after adsorption saturation, collecting 80% ethanol eluent, and carrying out rotary evaporation and drying to obtain the purified tea saponin.
The invention uses a sulfuric acid-vanillin method which is one of colorimetric methods as a concentration detection means, determines a standard curve of the tea saponin, has good linear correlation degree, and can be used for concentration detection. And performing single-factor experiments and orthogonal experiments, and determining the optimal extraction parameters under the premise of considering industrialization. Purifying the tea saponin by adopting n-butanol and macroporous resin, and finally determining to purify by AB-8 macroporous resin and optimizing related parameters.
In the course of the above-mentioned process,
preferably, in the step (2), the ethanol concentration is 80%, and the feed-liquid ratio is 1: 12.
preferably, the reflux extraction temperature in the step (2) is 65 ℃, and the extraction time is 90 min.
Preferably, the concentration of the aqueous solution of the crude tea saponin extract in the step (4) is 15g/L, and the loading flow rate is 2 BV/h.
Preferably, the flow rate of deionized water elution in the step (4) is 3BV/h, the flow rate of 20% (v/v) ethanol elution is 3BV/h, and the flow rate of 80% (v/v) ethanol elution is 3 BV/h.
The beneficial effects of the invention are mainly reflected in that: the method adopts the analytical means of liquid chromatography, mass spectrum, ultraviolet-visible spectrophotometry, infrared and the like to determine that the purity of the obtained tea saponin product reaches 90 percent, and the total recovery rate is 49.3 percent.
(IV) description of the drawings
FIG. 1 is a process flow diagram of an embodiment of the present invention.
FIG. 2 is a standard curve diagram of tea saponin determined by the sulfuric acid-vanillin method.
FIG. 3 is the static adsorption and desorption of tea saponin by macroporous resin.
FIG. 4 is a static adsorption curve of a macroporous resin.
(V) detailed description of the preferred embodiments
For the purpose of enhancing understanding of the present invention, the present invention will be described in further detail with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention.
Example 1:
(1) and (3) insolating the tea seed cake for 3 days in the sun, and screening to obtain 60-mesh tea seed cake powder after crushing.
(2) Weighing 10g of tea saponin powder, adding 80 vol% ethanol into the oil tea camellia cake powder according to the material-to-liquid ratio of 1:4, performing hot reflux extraction at 80 ℃ for 120 minutes at the pH value of 10, and performing suction filtration after the extract is cooled to obtain the crude extract of tea saponin.
(3) Diluting the extractive solution with 80% (v/v) ethanol to appropriate concentration, developing with sulfuric acid-vanillin method, measuring absorbance, calculating tea saponin concentration in the filtrate according to standard curve, calculating tea saponin content in the extractive solution, and calculating extraction rate.
Example 2:
(1) and (3) insolating the tea seed cake for 3 days in the sun, and screening to obtain 60-mesh tea seed cake powder after crushing.
(2) Weighing 10g of tea saponin powder, adding 70 vol% ethanol into the oil tea camellia cake powder according to the material-to-liquid ratio of 1:12, performing hot reflux extraction at 80 ℃ for 120 minutes at a pH value of 10, and performing suction filtration after an extracting solution is cooled to obtain a tea saponin crude extract.
(3) Diluting the extractive solution with 80% ethanol to appropriate concentration, developing with sulfuric acid-vanillin method, measuring absorbance, calculating tea saponin concentration in the filtrate according to standard curve, and calculating tea saponin content in the extractive solution to obtain crude extraction rate.
Example 3:
(1) and (3) insolating the tea seed cake for 3 days in the sun, and screening to obtain 60-mesh tea seed cake powder after crushing.
(2) Weighing 10g of tea saponin powder, adding 80 vol% ethanol into the oil tea camellia cake powder according to the material-to-liquid ratio of 1:12, performing hot reflux extraction at 60 ℃ for 120 minutes at a pH value of 10, and performing suction filtration after an extracting solution is cooled to obtain a tea saponin crude extract.
(3) Diluting the extractive solution with 80% ethanol to appropriate concentration, developing with sulfuric acid-vanillin method, measuring absorbance, calculating tea saponin concentration in the filtrate according to standard curve, calculating tea saponin content in the extractive solution, and calculating extraction rate.
Example 4:
(1) and (3) insolating the tea seed cake for 3 days in the sun, and screening to obtain 60-mesh tea seed cake powder after crushing.
(2) Weighing 10g of tea saponin powder, adding 80 vol% ethanol into the oil tea camellia cake powder according to the material-liquid ratio of 1:12, performing hot reflux extraction at 80 ℃ for 60 minutes at the pH value of 10, cooling the extracting solution, and performing suction filtration to obtain a crude tea saponin extracting solution, wherein the volume of the liquid is obtained.
(3) Diluting the extractive solution with 80% ethanol to appropriate concentration, developing with sulfuric acid-vanillin method, measuring absorbance, calculating tea saponin concentration in the filtrate according to standard curve, calculating tea saponin content in the extractive solution, and calculating extraction rate.
Example 5:
(1) and (3) insolating the tea seed cake for 3 days in the sun, and screening to obtain 60-mesh tea seed cake powder after crushing.
(2) Weighing 10g of tea saponin powder, adding 80 vol% ethanol into the oil tea camellia cake powder according to the material-liquid ratio of 1:12, performing hot reflux extraction at 80 ℃ for 120 minutes at a pH value of 7, cooling the extracting solution, and performing suction filtration to obtain a crude tea saponin extracting solution, wherein the volume of the crude tea saponin extracting solution is measured.
(3) Diluting the extractive solution with 80% ethanol to appropriate concentration, developing with sulfuric acid-vanillin method, measuring absorbance, calculating tea saponin concentration in the filtrate according to standard curve, calculating tea saponin content in the extractive solution, and calculating extraction rate.
Comparative example 1:
compared with the example 1, the conditions are the same as the example 1 except that the feed-liquid ratio in the step (2) is changed to 1: 8.
Comparative example 2:
compared with the example 1, the conditions are the same as the example 1 except that the feed-liquid ratio in the step (2) is changed to 1: 12.
Comparative example 3:
compared with the example 1, the conditions are the same as the example 1 except that the feed-liquid ratio in the step (2) is changed to 1: 16.
Comparative example 4:
compared with the example 1, the conditions are the same as the example 1 except that the feed-liquid ratio in the step (2) is changed to 1: 20.
Comparative example 5:
the conditions were the same as in example 2 except that the ethanol volume concentration in step (2) was changed to 75% as compared with example 2.
Comparative example 6:
the conditions were the same as in example 2 except that the ethanol volume concentration in step (2) was changed to 85% as compared with example 2.
Comparative example 7:
the conditions were the same as in example 2 except that the ethanol volume concentration in step (2) was changed to 90% as compared with example 2.
Comparative example 8:
the conditions were the same as in example 3 except that the thermal reflux temperature in step (2) was changed to 65 ℃ as compared with example 3.
Comparative example 9:
the same conditions as in example 3 were used except that the thermal reflux temperature in step (2) was changed to 70 ℃ as compared with example 3.
Comparative example 10:
the conditions were the same as in example 3 except that the thermal reflux temperature in step (2) was changed to 75 ℃ as compared with example 3.
Comparative example 11:
the conditions were the same as in example 4 except that the thermal reflux time in step (2) was changed to 90 minutes as compared with example 4.
Comparative example 12:
the conditions were the same as in example 4 except that the thermal reflux time in step (2) was changed to 105 minutes as compared with example 4.
Comparative example 13:
the conditions were the same as in example 4 except that the thermal reflux time in step (2) was changed to 135 minutes as compared with example 4.
Comparative example 14:
the conditions were the same as in example 5 except that the pH in step (2) was changed to 7, as compared with example 5.
Comparative example 15
The conditions were the same as in example 5 except that the pH in step (2) was changed to 8 as compared with example 5.
Comparative example 16:
the conditions were the same as in example 5 except that the pH in step (2) was changed to 9 as compared with example 5.
Comparative example 17:
the conditions were the same as in example 5 except that the pH in step (2) was changed to 11 as compared with example 5.
Setting orthogonal experiment factor table according to each extraction factor, and adopting (L)9(34) Orthogonal experiment design table shows the influence of the feed-liquid ratio, the ethanol concentration, the extraction temperature and the extraction time on the yield of the tea saponin, and the orthogonal experiment factors are shown in table 1;
table 1: orthogonal experiment factor table
Designing L of each factor according to orthogonal experiment factor table9(34) Orthogonal design table, see table 2;
TABLE 2
The experimental results in Table 2 show that the yield of tea saponin obtained by the experimental combination with the serial number of 6 is the largest and is 33.0 percent, the optimal extraction process of the tea saponin in the tea tree oil meal comprises the steps of the solid-liquid ratio of 1:12, the ethanol concentration of 79 percent, the temperature of 61 ℃, the time of 90min and the crude extraction rate of the tea saponin of about 33.0 percent.
Example 6: macroporous resin purification and tea saponin preparation
MacroporeStatic adsorption and desorption experiments of the resin on the tea saponin: taking 50mL of 12.5g/L crude tea saponin water solution (the concentration is recorded as C)0) Placing in a conical flask, adding 5g AB-8 macroporous resin, oscillating, and measuring the concentration of tea saponin water solution (marked as C) after 24 hr1) And calculating the adsorption rate. Filtering the aqueous solution of tea saponin to obtain resin adsorbing tea saponin, shifting the resin back and forth on clean filter paper, and sucking off the residual aqueous solution of tea saponin on the surface; placing the resin in conical flask, adding 50mL 80% ethanol solution, shaking occasionally, and measuring tea saponin concentration (marked as C) after 24 hr2) And calculating the desorption rate. The calculation result is shown in figure 3, which can better guide the adsorption experiment.
Static adsorption experiment of macroporous resin: placing 50mL of the aqueous solution in a conical flask, adding 5g of the pretreated macroporous resin, oscillating at intervals, sucking a small amount of the aqueous solution of the tea saponin every half hour (or one hour) for the first 5.5h, sucking a small amount of the aqueous solution of the tea saponin after 24h, properly diluting the sucked solution, measuring the concentration, and calculating the adsorption rate, wherein the result is shown in figure 4. The curve can better guide the dynamic adsorption of the macroporous resin and design a dynamic adsorption experiment.
Macroporous resin purification and tea saponin preparation:
directly carrying out rotary evaporation on the crude tea saponin extract obtained in the example 1 to recover ethanol in the crude extract, and preparing the ethanol into a tea saponin aqueous solution with the concentration of 15g/L by using deionized water. Loading 30mL (20.3g) of macroporous resin into a chromatographic column with the diameter of 1.8cm, and passing a crude tea saponin aqueous solution with the volume of 2.22BV at the flow rate of 2BV/h through the resin; sequentially eluting with deionized water, 20% ethanol and 80% ethanol at flow rate of 3BV/h, collecting 80% ethanol eluate at volume of 3BV, rotary steaming, and oven drying to obtain light yellow solid powder 0.493g, wherein the purity of tea saponin is about 91.7%, and the total recovery rate of tea saponin is above 83.5%.
Claims (5)
1. A method of purifying tea saponin from tea seed cake, the method comprising:
(1) tea cake pretreatment: insolating the tea seed cake for 2-3 days, and then crushing to 50-100 meshes;
(2) alcohol extraction and suction filtration: step (1), the tea seed cake powder is prepared by mixing the following raw materials in a ratio of 1: 4-20, adding 70-90 vol% ethanol solution, performing reflux extraction at 60-80 ℃ for 60-135 min, cooling the extract, and performing suction filtration to obtain tea saponin alcohol extract;
(3) and (3) macroporous resin purification: and (2) after the alcohol in the crude tea saponin extract is removed by rotary evaporation, preparing a 5-20 g/L aqueous solution of a crude tea saponin extract by using deionized water, loading the AB-8 macroporous adsorption resin into a column, loading the aqueous solution of the crude tea saponin extract into the column at the flow rate of 2/3-10/3 BV/h, passing the aqueous solution through the column, eluting the aqueous solution of the crude tea saponin extract by using deionized water, 20% ethanol and 80% ethanol respectively after adsorption saturation, collecting 80% ethanol eluent, and carrying out rotary evaporation and drying to obtain the purified tea saponin.
2. The method according to claim 1, wherein the ethanol concentration in the step (2) is 80%, and the feed-liquid ratio is 1: 12.
3. the method according to claim 1, wherein the reflux extraction temperature in step (2) is 65 ℃ and the extraction time is 90 min.
4. The method of claim 1, wherein in step (4), the concentration of the aqueous solution of crude tea saponin extract is 15g/L, and the flow rate of the sample is 2 BV/h.
5. The method according to claim 1, wherein the flow rate of deionized water elution in step (4) is 3BV/h, the flow rate of 20% ethanol elution is 3BV/h, and the flow rate of 80% ethanol elution is 3 BV/h.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114656516A (en) * | 2022-02-25 | 2022-06-24 | 湖南粮食集团有限责任公司 | Method for extracting and purifying tea saponin from oil-tea camellia meal |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102949405A (en) * | 2012-06-29 | 2013-03-06 | 上海莱博生物科技有限公司 | Application of tea saponin and preparation thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102949405A (en) * | 2012-06-29 | 2013-03-06 | 上海莱博生物科技有限公司 | Application of tea saponin and preparation thereof |
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| 叶洲辰: ""不同产地油茶籽油和茶枯饼的化学成分及生物活性比较研究"", 《中国优秀硕士学位论文全文数据库(电子期刊) 工程科技Ⅰ辑》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114656516A (en) * | 2022-02-25 | 2022-06-24 | 湖南粮食集团有限责任公司 | Method for extracting and purifying tea saponin from oil-tea camellia meal |
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Application publication date: 20210713 |