Method for extracting polyphenol from superfine powder of golden camellia leaves

Abstract

The invention discloses a method for extracting polyphenol from camellia nitidissima leaf superfine powder, which is characterized in that the two steps of superfine grinding and compound enzyme enzymolysis are synergistic to obviously dissolve out effective substances in the subsequent low-temperature ultrasonic-assisted alcohol extraction process, so that the polyphenol yield is obviously improved, and the problem of low polyphenol yield in the prior art is solved.

Description

Technical field:

[0001] The invention relates to a natural product extraction and separation technology, and in particular to a method for extracting polyphenols from golden camellia tea superfine powder. Background technology:

[0002] As a traditional tea beverage, Camellia chrysantha has many health benefits. In addition to showing obvious effects in regulating human blood lipids, blood sugar and enhancing the body's immunity, it can also improve various discomfort symptoms caused by hypertension, and can enhance liver and kidney vitality, prevent blood arteriosclerosis and inhibit tumor growth. Camellia chrysantha is rich in flavonoids, saponins, minerals and trace elements. However, at present, the extraction of phenolic acids in Camellia chrysantha is relatively complicated. The fiber structure in Camellia chrysantha hinders the dissolution rate of polyphenol active ingredients in alcohol extraction, and its products are also more diverse. At the same time, there are difficulties in identification and evaluation, and the extraction efficiency is not high, and resource utilization is not sufficient. Therefore, it is very important to explore the efficient extraction technology of phenolic acids in Camellia chrysantha and develop Camellia chrysantha products with practical application value.

[0003] By using fermentation or enzymatic hydrolysis to hydrolyze tea leaves, the cellulose structure of tea leaves can be destroyed in the process, and the natural products therein can be released to the outside, thereby achieving the goals of better increasing the content of nutrients and improving the quality and taste. CN 110613033 A discloses a method for extracting high tea polyphenol Jinhua black tea powder, which uses Jinhua black tea as raw material, firstly selects fermented Jinhua black tea as raw material, selects and removes impurities from the Jinhua black tea, performs preliminary cleaning, and then appropriately crushes it, adds 3-7 times the mass ratio of the raw material to soak it, adds a certain proportion of enzyme preparation after soaking, and then extracts it at 80-95℃, centrifuges it after coarse filtration, cools the clear liquid and performs secondary enzymatic hydrolysis, and after the second centrifugation, the clear liquid is finely filtered and concentrated under reduced pressure to obtain a concentrated solution, and spray-dried to obtain high tea polyphenol Jinhua black tea powder. However, the high-temperature extraction at 80-95℃ has the risk of damaging the polyphenol structure and thus losing its activity, which needs further improvement. Summary of the invention:

[0004] The invention aims to provide a method for extracting polyphenols from golden camellia tea superfine powder, which solves the problem of low polyphenol yield in the prior art.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for extracting polyphenols from golden camellia tea superfine powder, the method comprising the following steps:

[0007] 1) Ultrafine grinding: using a wall breaking machine to ultrafinely grind the dried golden camellia tea to obtain golden camellia tea ultrafine powder with a particle size of 500-1200 mesh, preferably 800-1200 mesh;

[0008] 2) compound enzyme enzymolysis: using the golden camellia tea superfine powder obtained in step 1) as a substrate, adding compound enzyme, using phosphate buffer to adjust the pH to 4-7, preferably 6-7, most preferably 7, mixing and stirring at 45-70°C, preferably 50-55°C, most preferably 50°C for 4-12h for enzymolysis treatment, and centrifuging after enzymolysis to obtain an enzymolysis supernatant; the compound enzyme is composed of cellulase, xylanase and pectinase in a ratio of (1.5-3): (26-29): (6-9); the amount of cellulase is 1.5-3IU / g;

[0009] 3) Ultrasonic-assisted alcohol extraction: add an equal volume of ethanol aqueous solution to the enzymatic hydrolysis supernatant obtained in step 2), extract at 30-70° C. for a period of time under the assistance of ultrasound, filter, pass through resin column chromatography, concentrate and evaporate, and finally freeze-dry to obtain a solid extract.

[0010] The specific steps of filtering, resin column chromatography, concentration and evaporation and finally freeze-drying to obtain a solid extract are as follows: filtering through a neutral quantitative filter paper, passing through an H103 resin column, eluting with an ethanol aqueous solution with a volume fraction of 40-80% after the sample is adsorbed, collecting the eluate and concentrating it under reduced pressure, passing the concentrate through a C18 solid phase extraction column, adding a washing solution to wash away impurities, and finally eluting with a methanol-2% acetic acid mixed solution, collecting the eluate, concentrating it under reduced pressure to a solid, and finally freeze-drying to obtain a golden camellia polyphenol extract.

[0011] Preferably, the mass fraction of the ethanol aqueous solution in step 3) is 40%-80%, more preferably 50%-70%, and most preferably 60%.

[0012] Preferably, in step 3), the ultrasonic-assisted extraction time is 30 to 120 min, preferably 60 to 90 min, and the ultrasonic power is 30 to 100 kHz, preferably 30 to 70 kHz, and most preferably 40 kHz.

[0013] The beneficial effects of the present invention are as follows:

[0014] 1) Through ultrafine grinding technology, the dissolution rate and accessibility of the active ingredients of Camellia chrysantha are increased, and the dissolution rate of polyphenols is improved;

[0015] 2) The micro powder of Camellia chrysantha is hydrolyzed by a composite enzyme composed of cellulase, xylanase and pectinase in a ratio of (1.5-3): (26-29): (6-9) to destroy the plant fiber components, so that the effective substances are more easily dissolved in the subsequent alcohol extraction process. At the same time, low-temperature ultrasound-assisted extraction at 30-70°C is used to reduce the damage of the polyphenol structure by high-temperature alcohol extraction, thereby increasing the yield of polyphenols.

[0016] 3) The synergistic effect of ultrafine grinding and compound enzyme hydrolysis enables the effective substances to be significantly dissolved in the subsequent low-temperature ultrasound-assisted alcohol extraction process, thereby significantly improving the yield of polyphenols. Specific implementation method:

[0017] The following is a further description of the present invention, rather than a limitation of the present invention.

[0018] The polyphenol separation and purification method in the following embodiment is as follows: the obtained alcohol extract is filtered through a neutral quantitative filter paper, and then passed through a H103 resin column. After the sample is adsorbed, it is eluted with a 40-80% ethanol aqueous solution by volume, and the eluate is collected and concentrated under reduced pressure. The concentrate is passed through a C18 solid phase extraction column, and an eluent is added to wash away impurities. Finally, a methanol-2% acetic acid mixed solution is used for elution, and the eluate is collected, concentrated under reduced pressure to a solid, and finally freeze-dried to obtain a golden camellia polyphenol extract.

[0019] The polyphenol content was detected by the Folin phenol method, that is, 2.5 mg of gallic acid reference substance was weighed and placed in a 25 mL volumetric flask, and the volume was adjusted to the mark with distilled water to obtain the reference substance stock solution. 0, 0.25, 0.5, 0.75, 1.0, and 1.25 mL of the stock solution were respectively placed in a 25 mL volumetric flask, 1.5 mL of Folin phenol reagent was added, and 1.5 mL of 10% Na 2 CO 3 The solution was diluted to volume with distilled water, reacted at 25°C for 1 h, and the absorbance at 765 nm was measured.

[0020] Embodiment 1:

[0021] 1) Ultrafine grinding: Use a wall-breaking machine to grind the golden flower tea leaves to 1200 mesh;

[0022] 2) Compound enzyme hydrolysis: weigh 100g of golden camellia tea powder and add compound enzyme hydrolysis, the compound enzyme used is composed of cellulase, xylanase and pectinase, the enzymatic hydrolysis conditions are 3IU / g, 28IU / g and 9IU / g of cellulase, xylanase and pectinase respectively, use phosphate buffer to adjust pH to 7, mix and stir at 50℃ for 8h, precipitate and separate the supernatant for standby use;

[0023] 3) Ultrasonic-assisted alcohol extraction: add an equal volume of ethanol to the separated supernatant, ultrasonic time 1h, ethanol concentration 60%, ultrasonic temperature 50°C, ultrasonic power 40kHz;

[0024] 4) The alcohol extract obtained in step 3) is filtered through a membrane, passed through a resin and column chromatography to obtain a mixed eluate, which is concentrated and dried to obtain 1.1 g / (100 g of Camellia chrysantha polyphenol solid extract. The polyphenol content in the solid extract is 91.2% as determined by the Folin phenol method.

[0025] Example 2

[0026] 1) Ultrafine grinding: Use a wall-breaking machine to grind the golden flower tea leaves to 800 mesh;

[0027] 2) Compound enzyme hydrolysis: weigh 100g of golden camellia tea powder and add compound enzyme hydrolysis, the compound enzyme used is composed of cellulase, xylanase and pectinase, the enzymatic hydrolysis conditions are cellulase, xylanase and pectinase 2.7IU / g, 27IU / g and 8IU / g respectively, use phosphate buffer to adjust pH to 6, mix and stir at 45℃ for 12h, precipitate and separate the supernatant for standby use;

[0028] 3) Ultrasonic assisted alcohol extraction: add an equal volume of ethanol to the separated supernatant, ultrasonic time 30 min, ethanol concentration 50%, ultrasonic temperature 60 ° C, ultrasonic power 30 kHz;

[0029] 4) The alcohol extract obtained in step 3) is filtered through a membrane, passed through a resin and column chromatography to obtain a mixed eluate, which is concentrated and dried to obtain a solid extract of polyphenols of Camellia chrysantha (0.3 g / (100 g Camellia chrysantha leaf powder). The polyphenol content in the solid extract is 64.7% as determined by the Folin phenol method.

[0030] Example 3

[0031] 1) Ultrafine grinding: Use a wall-breaking machine to grind the golden flower tea leaves to 500 mesh;

[0032] 2) Compound enzyme hydrolysis: weigh 100g of golden camellia tea powder and add compound enzyme hydrolysis, the compound enzyme used is composed of cellulase, xylanase and pectinase, the enzymatic hydrolysis conditions are cellulase, xylanase and pectinase 2IU / g, 26IU / g and 7IU / g respectively, use phosphate buffer to adjust pH to 5, mix and stir at 55℃ for 6h, precipitate and separate the supernatant for standby use;

[0033] 3) Ultrasonic assisted alcohol extraction: add ethanol of equal volume to the separated supernatant, ultrasonic time 90 min, ethanol concentration 40%, ultrasonic temperature 70 ° C, ultrasonic power 60 kHz;

[0034] 4) The alcohol extract obtained in step 3) is filtered through a membrane, passed through a resin and column chromatography to obtain a mixed eluate, which is concentrated and dried to obtain a solid extract of polyphenols of Camellia chrysantha (0.3 g / (100 g Camellia chrysantha leaf powder). The polyphenol content in the solid extract is 43.8% as determined by the Folin phenol method.

[0035] Example 4

[0036] 1) Ultrafine grinding: Use a wall-breaking machine to grind the golden flower tea leaves to 500 mesh;

[0037] 2) Compound enzyme hydrolysis: weigh 100g of golden camellia tea powder and add compound enzyme hydrolysis, the compound enzyme used is composed of cellulase, xylanase and pectinase, the enzymatic hydrolysis conditions are cellulase, xylanase and pectinase 1.5IU / g, 29IU / g and 6IU / g respectively, use phosphate buffer to adjust pH to 4, mix and stir at 60℃ for 4h, precipitate and separate the supernatant for standby use;

[0038] 3) Ultrasonic assisted alcohol extraction: add an equal volume of ethanol to the separated supernatant, ultrasonic time 120 min, ethanol concentration 70%, ultrasonic temperature 40 ° C, ultrasonic power 80 kHz;

[0039] 4) The alcohol extract obtained in step 3) is filtered through a membrane, passed through a resin and column chromatography to obtain a mixed eluate, which is concentrated and dried to obtain a solid extract of polyphenols of Camellia chrysantha (0.4 g / (100 g Camellia chrysantha leaf powder). The polyphenol content in the solid extract is 57.4% as detected by the Folin phenol method.

[0040] Example 5

[0041] 1) Ultrafine grinding: Use a wall-breaking machine to grind the golden flower tea leaves to 800 mesh;

[0042] 2) Compound enzyme hydrolysis: weigh 100g of golden camellia tea powder and add compound enzyme hydrolysis, the compound enzyme used is composed of cellulase, xylanase and pectinase, the enzymatic hydrolysis conditions are 3IU / g, 28IU / g and 9IU / g of cellulase, xylanase and pectinase respectively, use phosphate buffer to adjust pH to 7, mix and stir at 65℃ for 8h, precipitate and separate the supernatant for standby use;

[0043] 3) Ultrasonic assisted alcohol extraction: add ethanol of equal volume to the separated supernatant, ultrasonic time 60 min, ethanol concentration 80%, ultrasonic temperature 30 ° C, ultrasonic power 100 kHz;

[0044] 4) The alcohol extract obtained in step 3) is filtered through a membrane, passed through a resin and column chromatography to obtain a mixed eluate, which is concentrated and dried to obtain 0.8 g / (100 g of Camellia chrysantha polyphenol solid extract. The polyphenol content in the solid extract is 51.3% as determined by the Folin phenol method.

[0045] Example 6

[0046] 1) Ultrafine grinding: Use a wall-breaking machine to grind the golden flower tea leaves to 1200 mesh;

[0047] 2) Compound enzyme hydrolysis: weigh 100g of golden camellia tea powder and add compound enzyme hydrolysis, the compound enzyme used is composed of cellulase, xylanase and pectinase, the enzymatic hydrolysis conditions are cellulase, xylanase and pectinase 2.5IU / g, 27IU / g and 8IU / g respectively, use phosphate buffer to adjust pH to 6, mix and stir at 70℃ for 12h, precipitate and separate the supernatant for standby use;

[0048] 3) Ultrasonic assisted alcohol extraction: add an equal volume of ethanol to the separated supernatant, ultrasonic time 30 min, ethanol concentration 50%, ultrasonic temperature 50 ° C, ultrasonic power 70 kHz;

[0049] 4) The alcohol extract obtained in step 3) is filtered through a membrane, passed through a resin and column chromatography to obtain a mixed eluate, which is concentrated and dried to obtain 0.7 g / (100 g of Camellia chrysantha polyphenols solid extract. The polyphenol content in the solid extract is 68.9% as determined by the Folin phenol method.

[0050] Example 7

[0051] 1) Ultrafine grinding: Use a wall-breaking machine to grind the golden flower tea leaves to 500 mesh;

[0052] 2) Compound enzyme hydrolysis: weigh 100g of golden camellia tea powder and add compound enzyme hydrolysis, the compound enzyme used is composed of cellulase, xylanase and pectinase, the enzymatic hydrolysis conditions are 2IU / g, 26IU / g and 7IU / g of cellulase, xylanase and pectinase respectively, use phosphate buffer to adjust pH to 5, mix and stir at 45℃ for 6h, precipitate and separate the supernatant for standby use;

[0053] 3) Ultrasonic assisted alcohol extraction: add an equal volume of ethanol to the separated supernatant, ultrasonic time 90 min, ethanol concentration 40%, ultrasonic temperature 60 ° C, ultrasonic power 50 kHz;

[0054] 4) The alcohol extract obtained in step 3) is filtered through a membrane, passed through a resin and column chromatography to obtain a mixed eluate, which is concentrated and dried to obtain 0.5 g / (100 g of Camellia chrysantha polyphenol solid extract. The polyphenol content in the solid extract is 44.7% as determined by the Folin phenol method.

[0055] Example 8

[0056] 1) Ultrafine grinding: Use a wall-breaking machine to grind the golden flower tea leaves to 800 mesh;

[0057] 2) Compound enzyme hydrolysis: weigh 100g of golden camellia tea powder and add compound enzyme hydrolysis, the compound enzyme used is composed of cellulase, xylanase and pectinase, the enzymatic hydrolysis conditions are cellulase, xylanase and pectinase 1.5IU / g, 29IU / g and 6IU / g respectively, use phosphate buffer to adjust pH to 4, mix and stir at 55℃ for 4h, precipitate and separate the supernatant for standby use;

[0058] 3) Ultrasonic assisted alcohol extraction: add an equal volume of ethanol to the separated supernatant, ultrasonic time 120 min, ethanol concentration 70%, ultrasonic temperature 70 ° C, ultrasonic power 40 kHz;

[0059] 4) The alcohol extract obtained in step 3) is filtered through a membrane, passed through a resin and column chromatography to obtain a mixed eluate, which is concentrated and dried to obtain 0.8 g / (100 g of Camellia chrysantha polyphenol solid extract. The polyphenol content in the solid extract is 83.5% as detected by the Folin phenol method.

[0060] Example 9

[0061] 1) Ultrafine grinding: Use a wall-breaking machine to grind the golden flower tea leaves to 1200 mesh;

[0062] 2) Compound enzyme hydrolysis: weigh 100g of golden camellia tea powder and add compound enzyme hydrolysis, the compound enzyme used is composed of cellulase, xylanase and pectinase, the enzymatic hydrolysis conditions are 3IU / g, 28IU / g and 9IU / g of cellulase, xylanase and pectinase respectively, use phosphate buffer to adjust pH to 7, mix and stir at 60℃ for 8h, precipitate and separate the supernatant for standby use;

[0063] 3) Ultrasonic assisted alcohol extraction: add an equal volume of ethanol to the separated supernatant, ultrasonic time 60 min, ethanol concentration 60%, ultrasonic temperature 50 ° C, ultrasonic power 40 kHz;

[0064] 4) The alcohol extract obtained in step 3) is filtered through a membrane, passed through a resin and column chromatography to obtain a mixed eluate, which is concentrated and dried to obtain 0.9 g / (100 g of Camellia chrysantha polyphenol solid extract. The polyphenol content in the solid extract is 89.6% as determined by the Folin phenol method.

[0065] Comparative Example 1

[0066] 1) Compound enzyme hydrolysis: Weigh 100g of dried golden camellia tea leaves and add compound enzyme hydrolysis, the compound enzyme used is composed of cellulase, xylanase and pectinase, the enzymatic hydrolysis conditions are 3IU / g, 28IU / g and 9IU / g of cellulase, xylanase and pectinase respectively, use phosphate buffer to adjust pH to 7, mix and stir at 50℃ for 8h, precipitate and separate the supernatant for standby use;

[0067] 2) Ultrasonic assisted alcohol extraction: add an equal volume of ethanol to the separated supernatant, ultrasonic time 60 min, ethanol concentration 60%, ultrasonic temperature 50 ° C, ultrasonic power 40 kHz;

[0068] 3) The alcohol extract obtained in step 2) is filtered through a membrane, passed through a resin and column chromatography to obtain a mixed eluate, which is concentrated and dried to obtain a solid extract of polyphenols of Camellia chrysantha (0.3 g / (100 g Camellia chrysantha leaf powder). The polyphenol content in the solid extract is 64.4% as determined by the Folin phenol method.

[0069] Comparative Example 2

[0070] 1) Ultrafine grinding: Use a wall-breaking machine to grind the golden flower tea leaves to 1200 mesh;

[0071] 2) Ultrasonic-assisted alcohol extraction: 100 g of golden camellia tea powder was mixed in 500 mL of water, and then an equal volume of ethanol aqueous solution was added, the ultrasonic time was 60 min, the ethanol concentration was 60%, the ultrasonic temperature was 50 ° C, and the ultrasonic power was 40 kHz;

[0072] 3) The alcohol extract obtained in step 2) is filtered through a membrane, passed through a resin and column chromatography to obtain a mixed eluate, which is concentrated and dried to obtain 0.6 g / (100 g of Camellia chrysantha polyphenols solid extract. The polyphenol content in the solid extract is 47.3% as determined by the Folin phenol method.

[0073] Comparative Example 3

[0074] 1) Weigh 100 g of dried golden camellia tea leaves and mix them in 500 mL of water, then add an equal volume of ethanol aqueous solution, ultrasonically treat for 60 min, with an ethanol concentration of 60%, an ultrasonic temperature of 50°C, and an ultrasonic power of 40 kHz;

[0075] 2) The alcohol extract obtained in step 1) is filtered through a membrane, passed through a resin and column chromatography to obtain a mixed eluate, which is concentrated and dried to obtain a solid extract of polyphenols of Camellia chrysantha (0.3 g / (100 g Camellia chrysantha leaf powder). The polyphenol content in the solid extract is 25.8% as determined by the Folin phenol method.

[0076] From the comparison between Example 9 and Comparative Examples 1-3, it can be seen that the synergistic effect of the ultrafine grinding step and the complex enzyme hydrolysis step enables the effective substances to be significantly dissolved in the subsequent low-temperature ultrasound-assisted alcohol extraction process, thereby significantly improving the yield of polyphenols.

Claims

1. A method for extracting polyphenols from golden camellia tea superfine powder, characterized in that: The method comprises the following steps: 1) Ultrafine grinding: The dried golden camellia tea leaves are subjected to ultrafine grinding by a wall breaking machine to obtain golden camellia tea leaves ultrafine powder with a particle size of 500-1200 mesh; 2) compound enzyme hydrolysis: using the golden camellia tea superfine powder obtained in step 1) as a substrate, adding compound enzyme, adjusting the pH to 4-7 with phosphate buffer, mixing and stirring at 45-70° C. for 4-12 hours for enzymatic hydrolysis, and centrifuging to obtain an enzymatic hydrolysis supernatant after enzymatic hydrolysis; the compound enzyme is composed of cellulase, xylanase and pectinase in a ratio of (1.5-3): (26-29): (6-9); the amount of cellulase is 1.5-3 IU / g; 3) Ultrasonic-assisted alcohol extraction: add an equal volume of ethanol aqueous solution to the enzymatic hydrolysis supernatant obtained in step 2), extract at 30-70° C. for a period of time under the assistance of ultrasound, filter, pass through resin column chromatography, concentrate and evaporate, and finally freeze-dry to obtain a solid extract.

2. The method according to claim 1, characterized in that The method comprises the following steps: 1) Ultrafine grinding: The dried golden camellia tea leaves are subjected to ultrafine grinding by a wall breaking machine to obtain golden camellia tea leaves ultrafine powder with a particle size of 800-1200 meshes; 2) compound enzyme hydrolysis: using the golden camellia tea superfine powder obtained in step 1) as a substrate, adding compound enzyme, adjusting the pH to 6-7 with phosphate buffer, mixing and stirring at 50-55° C. for 4-12 hours for enzymatic hydrolysis, and centrifuging to obtain an enzymatic hydrolysis supernatant; the compound enzyme is composed of cellulase, xylanase and pectinase in a ratio of (1.5-3): (26-29): (6-9); the amount of cellulase is 1.5-3 IU / g; 3) Ultrasonic-assisted alcohol extraction: add an equal volume of ethanol aqueous solution to the enzymatic hydrolysis supernatant obtained in step 2), wherein the mass fraction of the ethanol aqueous solution is 40%-80%, extract under ultrasonic assistance at 30-70° C. for 30-120 min, filter, pass through resin column chromatography, concentrate and evaporate, and finally freeze-dry to obtain a solid extract.

3. The method according to claim 1 or 2, characterized in that: Step 3) The specific steps of filtering, passing through a resin column chromatography, concentrating and evaporating and finally freeze-drying to obtain a solid extract are as follows: filtering through a neutral quantitative filter paper, passing through a H103 resin column, and after the sample is adsorbed, eluting with a volume fraction of 40-80% ethanol aqueous solution, collecting the eluate and concentrating under reduced pressure, passing the concentrate through a C18 solid phase extraction column, adding an eluent to wash away impurities, and finally eluting with a methanol-2% acetic acid mixed solution, collecting the eluate, concentrating under reduced pressure to a solid, and finally freeze-drying to obtain a golden camellia polyphenol extract.

4. The method according to claim 1 or 2, characterized in that: In step 3), the mass fraction of the ethanol aqueous solution is 50%-70%.

5. The method according to claim 1 or 2, characterized in that: In step 3), the mass fraction of the ethanol aqueous solution is 60%.

6. The method according to claim 1 or 2, characterized in that: In step 3), the ultrasonic-assisted extraction time is 60-90 min, and the ultrasonic power is 30-100 kHz.

7. The method according to claim 1 or 2, characterized in that: In step 3), the ultrasonic assisted ultrasound power is 30-70kHz.

8. The method according to claim 1 or 2, characterized in that: In step 3), the ultrasonic power of the ultrasonic-assisted method is 40 kHz.