阅读说明：本技术 一种获得含量大于99%没食子酸的制备方法 (Preparation method for obtaining gallic acid with content of more than 99% ) 是由 赵军 陆宝屹 李�杰 于 2021-01-26 设计创作，主要内容包括：本发明涉及没食子酸的制备方法,具体涉及一种获得含量大于99％没食子酸的制备方法。一种获得含量大于99％没食子酸的制备方法,是将五倍子粉碎、加水打浆之后加入第一酶酶解,所得第一酶解液进行闪式提取,分离出滤液,所述第一酶包括破壁酶,所得滤液浓缩、离心,收集离心液调节pH,所得溶液加入第二酶酶解,所得第二酶解液经过有机膜过滤、浓缩、结晶、干燥制得所需的没食子酸,所述第二酶由单宁水解酶和第三酶组成,所述第三酶为果胶酶、蛋白酶中的至少一种。本发明利用超微粉碎加生物酶破壁再用闪式提取替代原有的提取方法,常温提取,提取率高,操作简便,优选了复合酶酶解单宁,使酶解达到最大的效能。(The invention relates to a preparation method of gallic acid, in particular to a preparation method for obtaining gallic acid with the content of more than 99%. Crushing gallnut, adding water, pulping, adding a first enzyme for enzymolysis, carrying out flash extraction on the obtained first enzymolysis liquid, separating filtrate, wherein the first enzyme comprises a wall-breaking enzyme, concentrating and centrifuging the obtained filtrate, collecting a centrifugate, adjusting the pH value, adding a second enzyme for enzymolysis on the obtained solution, filtering, concentrating, crystallizing and drying the obtained second enzymolysis liquid through an organic membrane to obtain the required gallic acid, wherein the second enzyme comprises tannin hydrolase and a third enzyme, and the third enzyme is at least one of pectinase and protease. The invention utilizes ultramicro pulverization, biological enzyme wall breaking and flash extraction to replace the original extraction method, has the advantages of normal-temperature extraction, high extraction rate and simple and convenient operation, and optimizes the compound enzyme for tannin enzymolysis, so that the enzymolysis achieves the maximum efficiency.)

1. A preparation method for obtaining gallic acid with the content of more than 99 percent is characterized by comprising the following steps:

1) crushing gallnut, adding water, pulping, adding a first enzyme for enzymolysis, performing flash extraction on the obtained first enzymolysis liquid, and separating filtrate, wherein the first enzyme comprises a wall-breaking enzyme;

2) concentrating and centrifuging the filtrate obtained in the step 1), collecting a centrifugate and adjusting the pH value;

3) adding a second enzyme into the solution obtained in the step 2) for enzymolysis, filtering, concentrating, crystallizing and drying the obtained second enzymolysis solution by using an organic membrane to obtain the required gallic acid, wherein the second enzyme consists of tannin hydrolase and a third enzyme, and the third enzyme is at least one of pectinase and protease.

2. The preparation method for obtaining gallic acid with content higher than 99% according to claim 1, wherein the motor rotation speed of flash extraction is 5000-6000 r/min, the extraction times are 3-4 times, and the extraction time is 1 min/time.

3. The preparation method for obtaining gallic acid with content greater than 99% according to claim 1, wherein the mass-to-volume ratio of the gallnut and the first enzyme in step 1) is 100: 1 to 6.

4. The preparation method for obtaining gallic acid with content higher than 99% according to any one of claims 1-3, wherein the first enzyme enzymolysis temperature in step 1) is 40-45 ℃, the enzymolysis time is 1-5 hours, and the enzymolysis pH is 4.0-4.8.

5. The preparation method for obtaining gallic acid with content higher than 99% according to claim 1, wherein the enzymolysis temperature of the second enzyme in step 2) is 40-45 ℃, the enzymolysis time is 6-10 hours, and the enzymolysis pH is 5.2-5.5.

6. The preparation method for obtaining the gallic acid with the content more than 99% according to claim 5, wherein the enzymolysis temperature of the second enzyme is 43 ℃, the enzymolysis time is 8 hours, the enzymolysis pH is 5.3, and the adding amount of the second enzyme is 3% of the quality of the gallnut.

7. The method for preparing gallic acid with content higher than 99% according to claim 1, wherein the centrifugation in step 2) comprises a first centrifugation and a second centrifugation, wherein the first centrifugation is performed at 1400-1500 r/min for 40-50 min, and the second centrifugation is performed at 12000-15000 r/min for 1-1.5 h.

8. The preparation method for obtaining gallic acid with content higher than 99% according to claim 1, wherein the organic membrane filtration step comprises a first filtration and a second filtration, the first filtration uses organic membrane with molecular weight cut-off of 5000-7000, and the filtration pressure is 0.45-0.48 MPa; the second filtration uses an organic membrane for intercepting 300-600 molecular weight, and the filtration pressure is 1.8-2.0 MPa.

9. The method for preparing gallic acid with content higher than 99% according to claim 1, wherein the concentration is performed to obtain a thick paste with Baume degree of 8-10.

10. The method for preparing gallic acid with content higher than 99% according to claim 1, wherein the crystallization temperature is 6-15 deg.C and the crystallization time is 12-15 hr.

Technical Field

The invention relates to a preparation method of gallic acid, in particular to a preparation method for obtaining gallic acid with the content of more than 99%.

Background

Gallnut (gallainensis) is a forest by-product, also known as clam, and is an early gall formed by parasitism of the aphid gall (melaphischinensis (bell) Baker) on the leaves of Rhus chinensis (Rhuschingeinsis Mill.) of the family Rhus family, Rhus chinensis (Rhuschinganiimaxim.) or Rhus chinensis (Rhus punjabensis Stew. var. sinica (Diels) Rehd. et Wils.). China is a large producing country of the five-gall nut and accounts for 75% -90% of the world output. The gallnut is suitable for growing in warm and humid mountainous areas and hills, most areas of China are distributed, main production areas are concentrated in six provinces such as Hubei, Hunan, Guizhou, Sichuan, Shanxi and Yunnan, and the yield of the gallnut in the provinces accounts for more than 90 percent of the whole country. The five-gall nut is mainly classified into bellybutton, horn gall and flower gall because the aphid species are different from hosts and the appearance is different. Wherein the cerasus angustifolia contains about 65.5-67.5% of gallnut tannin, about 68.8-71.4% of belladonna and about 33.9-38.5% of gallnut. Tannic acid produced by using gallnut as a raw material and series products thereof are widely applied to the industries of medicine, food, tanning, metallurgy, printing and dyeing, electronics, cosmetics, national defense and the like. Along with the development of industry and agriculture, the demand for Chinese gall is larger and larger, and the phenomenon of no goods may occur in the future. In recent years, a large-area gallnut production base is established in many production areas, and the gallnut industry is vigorously developed to meet the requirements of industrial production.

At present, few manufacturers of gallic acid produced by a large-scale enzyme method in China mainly obtain the gallic acid by an acid-base method, and the defects are that the hydrolysis is incomplete, the color of the produced product is not white enough, a large amount of pollution is generated in the production process, and the quality of the product cannot reach high quality.

Disclosure of Invention

In order to solve the defects of the prior art, the preparation method for obtaining the gallic acid with the content of more than 99 percent is provided, the enzymatic hydrolysis is complete, the hydrolysis byproduct glucose can be used as an energy source and a carbon source to be metabolized by a biocatalyst, the effective utilization rate of resources is improved, the waste liquid is easy to treat, and the environmental protection is facilitated.

The purpose of the invention is realized by the following technical scheme:

a preparation method for obtaining gallic acid with content more than 99% comprises the following steps:

1) crushing gallnut, adding water, pulping, adding a first enzyme for enzymolysis, carrying out flash extraction on the obtained first enzymolysis liquid, and separating filtrate, wherein the first enzyme comprises a wall-breaking enzyme.

2) Concentrating and centrifuging the filtrate obtained in the step 1), collecting a centrifugate and adjusting the pH value;

3) adding a second enzyme into the solution obtained in the step 2) for enzymolysis, filtering, concentrating, crystallizing and drying the obtained second enzymolysis solution by using an organic membrane to obtain the required gallic acid, wherein the second enzyme consists of tannin hydrolase and a third enzyme, and the third enzyme is at least one of pectinase and protease.

Further, the rotating speed of a motor for flash extraction is 5000-6000 r/min, the extraction times are 3-4 times, and the extraction time is 1 minute/time.

Further, in g/mL, the mass-to-volume ratio of the gallnut to the first enzyme in the step 1) is 100: 1 to 6.

Further, in the step 1), the enzymolysis temperature of the first enzyme is 40-45 ℃, the enzymolysis time is 1-5 hours, and the enzymolysis pH is 4.0-4.8.

Further, in the step 2), the enzymolysis temperature of the second enzyme is 40-45 ℃, the enzymolysis time is 6-10 hours, and the enzymolysis pH is 5.2-5.5.

Furthermore, the enzymolysis temperature of the second enzyme is 43 ℃, the enzymolysis time is 8 hours, the enzymolysis pH is 5.3, and the adding amount of the second enzyme is 3% of the mass of the gallnut.

Further, the centrifugation in the step 2) comprises a first centrifugation and a second centrifugation, wherein the first centrifugation is performed at a rotating speed of 1400-1500 r/min for 40-50 minutes, and the second centrifugation is performed at a rotating speed of 12000-15000 r/min for 1-1.5 hours.

Further, the organic membrane filtration step comprises a first filtration and a second filtration, wherein the first filtration uses an organic membrane for intercepting 5000-7000 molecular weight, and the filtration pressure is 0.45-0.48 MPa; the second filtration uses an organic membrane for intercepting 300-600 molecular weight, and the filtration pressure is 1.8-2.0 MPa.

Further, concentrating to obtain thick paste with the Baume degree of 8-10.

Further, the crystallization temperature is 6-15 ℃, and the crystallization time is 12-15 hours.

The invention has the following technical effects:

1. the method has the advantages of high production efficiency, high speed and efficiency, normal-temperature extraction, high extraction rate, simple operation, energy conservation, consumption reduction, safety and reliability.

2. The compound enzyme is preferably used for tannin enzymolysis, so that the enzymolysis efficiency is maximized, the enzymolysis rate is more complete than that of the conventional single biological enzyme method, the production cost is reduced, and the production efficiency is increased.

3. The combination mode of enzymolysis and organic membrane is used for replacing the prior decolorization, and the method has the following advantages: the extraction time is reduced; the method is simple and convenient to operate, the energy consumption is low, the decolorized sample has 8% higher decolorization content than acid-base activated carbon, and finally the crystallized sample product is snow white, has little solvent residue, no pesticide residue and high quality.

4. Only water is used in the whole process, other organic reagents are not introduced, and the extraction process is pollution-free, environment-friendly and safe.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited to the scope of the examples. These examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. In addition, various modifications may occur to those skilled in the art upon reading the present disclosure, and such equivalent variations are within the scope of the present invention as defined in the appended claims.

Example 1

1) Pulverizing 100g of dried Galla chinensis to 100 mesh, adding 3 times of pure water, and pulping. Adjusting pH to 4.0 with 3% citric acid, adding 1mL of a first enzyme composed of 0.5mL of protease and 0.5mL of a wall-breaking enzyme comprising pectinase and cellulase, and performing enzymolysis at 40 deg.C for 1 hr. And putting the obtained first enzymolysis liquid into a flash extractor, using pure water as an extraction solvent, adding water with the volume being 12 times that of the first enzymolysis liquid, and extracting for 3 times at normal temperature for 3 minutes at the motor rotating speed of 5000r/min, wherein each time is one minute. After the extraction, the rest is carried out for 2 minutes, the filtrate is separated, and the filtrate obtained after 3 times of extraction is combined to obtain the total filtrate;

2) concentrating the total filtrate to 5 times of the weight of the Chinese gall raw material through a membrane, cooling the obtained concentrated solution to 25 ℃, firstly centrifuging for 40 minutes at a rotation speed of 1400r/min by using a butterfly centrifuge, then centrifuging for 1 hour at a rotation speed of 12000r/min by using a tubular centrifuge, discarding the centrifugal precipitate, adjusting the pH value of the centrifugate to 5.2 by using 10% w/w sodium hydroxide solution, and uniformly stirring to ensure that the pH value is not changed;

3) adding a second enzyme which is 3% of the gallnut by mass into the centrifugate after the pH is adjusted, wherein the second enzyme consists of tannin hydrolase and a third enzyme (pectinase) in a mass ratio of 1:1, performing enzymolysis for 6 hours at 40 ℃, and cooling the solution to normal temperature after the enzymolysis to obtain a second enzymolysis solution.

4) And (3) passing the second enzymolysis solution through an organic membrane, passing through an organic membrane with the molecular weight of 5000 (the membrane feeding pressure is 0.45MPa), and passing through an organic membrane with the molecular weight of 300 (the membrane feeding pressure is 1.8MPa) after the second enzymolysis solution is completely passed, so as to obtain the decolorized membrane passing solution.

5) Concentrating the membrane-passing solution under reduced pressure to obtain 8 Baume thick paste, stirring, crystallizing at 6 deg.C for 12 hr, filtering to obtain crystals, and drying to obtain 40.8g gallic acid product. The content was 99.1% by HPLC and the yield was 40.4%.

Example 2

1) Pulverizing 100g of dried Galla chinensis to 100 mesh, adding 3 times of pure water, and pulping. Adjusting pH to 4.0 with 3% citric acid, adding 2mL of a first enzyme composed of 0.8mL of protease and 1.2mL of a wall-breaking enzyme comprising pectinase, cellulase and hemicellulase, and performing enzymolysis at 40 deg.C for 1 hr. And putting the obtained first enzymolysis liquid into a flash extractor, using pure water as an extraction solvent, adding water with the volume being 12 times that of the first enzymolysis liquid, and extracting for 3 times at normal temperature for 3 minutes at the motor rotating speed of 5000r/min, wherein each time is one minute. After the extraction, the rest is carried out for 2 minutes, the filtrate is separated, and the filtrate obtained after 3 times of extraction is combined to obtain the total filtrate;

2) concentrating the total filtrate to 5 times of the amount of the gallnut raw material through a membrane, cooling the obtained concentrated solution to 25 ℃, firstly centrifuging for 40 minutes at 1470r/min of a butterfly centrifuge, then centrifuging for 1 hour at 14000r/min of a tubular centrifuge, discarding the centrifugal precipitate, adjusting the pH of the centrifugate to 5.3 by using 10% w/w potassium hydroxide solution, and uniformly stirring to ensure that the pH value is not changed;

3) adding a second enzyme which is 3% of the gallnut by mass into the centrifugate after the pH is adjusted, wherein the second enzyme consists of tannin hydrolase and a third enzyme (pectinase and protease in a mass ratio of 1: 1) in a mass ratio of 1:1, carrying out enzymolysis for 8 hours at 43 ℃, and cooling the solution to normal temperature after the enzymolysis to obtain a second enzymolysis solution.

4) And (3) passing the second enzymolysis solution through an organic membrane, namely passing the second enzymolysis solution through an organic membrane with the molecular weight of 6000 (the membrane feeding pressure is 0.45MPa), and then passing the second enzymolysis solution through an organic membrane with the molecular weight of 500 (the membrane feeding pressure is 1.8MPa) after the second enzymolysis solution is completely passed, so as to obtain the decolorized membrane passing solution.

5) Concentrating the membrane-passing solution under reduced pressure to obtain 8 Baume thick paste, stirring, crystallizing at 6 deg.C for 12 hr, vacuum filtering to obtain crystal, and drying to obtain 42.5g gallic acid product. The content was 99.3% by HPLC and the yield was 42.2%.

Example 3

1) Taking 100g of dried Chinese gall, crushing to 150 meshes, and adding 45 times of pure water for pulping. Adjusting pH to 4.5 with 4% citric acid, adding 6mL of a first enzyme consisting of 3mL of protease and 3mL of a wall-breaking enzyme comprising pectinase, cellulase and hemicellulase, and performing enzymolysis at 42 deg.C for 3 hr. And putting the obtained first enzymolysis liquid into a flash extractor, using pure water as an extraction solvent, adding 13 times of water by volume of the first enzymolysis liquid, and extracting for 4 times at normal temperature for one minute at the motor rotating speed of 5500r/min for 4 minutes each time. After the extraction, the rest is carried out for 2 minutes, the filtrate is separated, and the filtrates obtained after 4 times of extraction are combined to obtain the total filtrate;

2) membrane-concentrating the total filtrate to 5 times of the amount of the gallnut raw material, cooling the obtained concentrated solution to 25 ℃, centrifuging for 45 minutes at the rotating speed of 1480r/min by a butterfly centrifuge, centrifuging for 1.2 hours at the rotating speed of 13000r/min by a tubular centrifuge, discarding the centrifugal precipitate, adjusting the pH of the centrifugate to 5.4 by 10% w/w calcium hydroxide solution, and uniformly stirring to keep the pH value unchanged;

3) adding a second enzyme with the mass of 4% of Chinese gall into the centrifugate after the pH is adjusted, wherein the second enzyme consists of tannin hydrolase and a third enzyme (protease) in a mass ratio of 1:0.5, carrying out enzymolysis for 8 hours at 42 ℃, and cooling the solution to the normal temperature after the enzymolysis to obtain a second enzymolysis solution.

4) And (3) passing the second enzymolysis solution through an organic membrane, firstly passing through an organic membrane with 6500 molecular weight (the membrane feeding pressure is 0.47MPa), and then passing through an organic membrane with 400 molecular weight (the membrane feeding pressure is 1.9MPa) after the second enzymolysis solution is completely passed, so as to obtain the decolorized membrane passing solution.

5) Concentrating the membrane-passing solution under reduced pressure to obtain a thick paste with a Baume degree of 9, stirring uniformly, crystallizing at 10 ℃ for 13 hours, and filtering to obtain crystals, and drying to obtain 43.2g of gallic acid product. The content was 99.2% by HPLC and the yield was 42.8%.

Example 4

1) Pulverizing 100g of dried Galla chinensis to 200 meshes, and pulping with 5 times of pure water. Adjusting pH to 4.8 with 5% citric acid, adding 1.2mL of a first enzyme consisting of 0.4mL of protease and 0.8mL of a wall-breaking enzyme comprising pectinase, cellulase and hemicellulase, and performing enzymolysis at 45 deg.C for 5 hours. And putting the obtained first enzymolysis liquid into a flash extractor, using pure water as an extraction solvent, adding water with the volume 15 times that of the first enzymolysis liquid, and extracting for 4 times at normal temperature for one minute each time for 4 minutes under the condition that the motor rotating speed is 6000 r/min. After the extraction, the rest is carried out for 2 minutes, the filtrate is separated, and the filtrates obtained after 4 times of extraction are combined to obtain the total filtrate;

2) concentrating the total filtrate to 5 times of the amount of the gallnut raw material through a membrane, cooling the obtained concentrated solution to 25 ℃, firstly centrifuging for 50 minutes at the rotating speed of 1500r/min by using a butterfly centrifuge, then centrifuging for 1.5 hours at the rotating speed of 15000r/min by using a tubular centrifuge, discarding the centrifugal precipitate, adjusting the pH value of the centrifugate to 5.5 by using 10% w/w sodium hydroxide solution, and uniformly stirring to ensure that the pH value is not changed;

3) adding a second enzyme which is 5% of the gallnut by mass into the centrifugate after the pH is adjusted, wherein the second enzyme consists of tannin hydrolase and a third enzyme (protease) in a mass ratio of 1:2, performing enzymolysis for 10 hours at 45 ℃, and cooling the solution to normal temperature after the enzymolysis to obtain a second enzymolysis solution.

4) And (3) passing the second enzymolysis solution through an organic membrane, passing through an organic membrane with the molecular weight of 7000 (the membrane feeding pressure is 0.48MPa), and passing through an organic membrane with the molecular weight of 600 (the membrane feeding pressure is 2.0MPa) after the second enzymolysis solution is completely passed, so as to obtain the decolorized membrane passing solution.

5) Concentrating the membrane-passing solution under reduced pressure to obtain 10 Baume thick paste, stirring, crystallizing at 15 deg.C for 15 hr, filtering to obtain crystals, and drying to obtain 41.7g gallic acid product. The content was 99.1% by HPLC and the yield was 41.3%.

Comparative example 1

The same gallic acid preparation method as in example 2 is adopted, except that step 1) does not contain a first enzymatic hydrolysis step, and step 1) is specifically as follows:

pulverizing 100g of dried Galla chinensis to 100 mesh, adding 3 times of pure water, and pulping. Adjusting pH to 4.0 with 3% citric acid, placing the obtained solution into a flash extractor, adding pure water as extraction solvent, adding 12 times of water, extracting at motor speed of 5000r/min at room temperature for 3 min for 3 times, each time for one min. After extraction, the rest is carried out for 2 minutes, the filtrate is separated, and the filtrate obtained after 3 times of extraction is combined to obtain the total filtrate.

This comparative example finally yielded 35.8g of gallic acid product. The content was 81.9% by HPLC and the yield was 29.3%.

Comparative example 2

The same gallic acid preparation method as in example 2 was adopted, except that the second enzymatic hydrolysis treatment step of step 3) was not included, and the solution obtained after pH adjustment in step 2) was directly subjected to organic membrane filtration in step 4).

This comparative example finally yielded 24.8g gallic acid product. The content was 67.5% by HPLC and the yield was 16.7%.

Comparative example 3

The same gallic acid preparation method as in example 2 was used, except that the second enzyme used in step 3) was tannin hydrolase, and step 3) was specifically as follows:

adding tannin hydrolase with the mass of 3% of Chinese gall into the centrifugate after the pH is adjusted, carrying out enzymolysis for 8 hours at the temperature of 43 ℃, and cooling the solution to the normal temperature after the enzymolysis to obtain a second enzymolysis solution.

This comparative example finally yielded 32.2g gallic acid product. The content was 90.58% by HPLC, and the yield was 29.2%.

The method adopts the two-step enzyme method to prepare the gallic acid, has the advantages of high treatment effect of single enzymolysis, energy saving, consumption saving and environmental protection, and is an ideal method for replacing the traditional chemical treatment.