NL2029007B1

NL2029007B1 - Extraction and purification method and application of plant-condensed tannin

Info

Publication number: NL2029007B1
Application number: NL2029007A
Authority: NL
Inventors: Huang Wen; Peng Kai; Chen Bing; Mo Wenyan
Original assignee: Inst Of Animal Science Guangdong Academy Of Agricultural Sciences
Priority date: 2021-01-21
Filing date: 2021-08-20
Publication date: 2022-07-28
Also published as: CN112876671B; CN112876671A

Abstract

The invention discloses an extraction and puriﬁcation method and application of plant condensed tannin. The extraction method conducts extraction on plant samples by means of a compound enzyme extractant, and the compound enzyme extractant contains laccase, cellulase, and pectinase. The invention ﬁrst proposes the use of an enzymatic hydrolysis method for extracting condensed tannin, and no organic solvent is used, so that the environmental pollution caused by organic solvents is avoided, the damage of plant cells caused by organic solvents is effectively avoided, and the yield loss caused by complexation between condensed tannin and substances such as protein and trace elements discharged from damaged plant cells is reduced. The yield and purity of condensed tannin prepared by the extraction method of the invention are far higher compared to the traditional method, so the extraction method has extremely high popularization value.

Description

EXTRACTION AND PURIFICATION METHOD AND APPLICATION OF PLANT- CONDENSED TANNIN BACKGROUND OF THE INVENTION

[0001] 1. Technical Field

[0002] The invention relates to the field of natural plant products, in particular to a plant-condensed tannin extraction and purification method and an application of plant-condensed tannin.

[0003] 2. Description of Related Art

[0004] Tannin is a complex polyphenolic derivative, and is the main component of a tannin extract for transforming rawhide into leather, thus also being called plant tannin. Tannin widely exists in the roots, stems, and leaves of plants. As a natural renewable resource, tannin has the advantages of high variety diversity, wide distribution, and large amount. Like cellulose, lignin, and hemicellulose, tannin is a substance with a high content in plants, and can react with protein, alkaloids, and polysaccharides. Based on chemical structure characteristics, tannin can be divided into hydrolyzable tannin and condensed tannin. Condensed tannin is generally derivatives of flavanols, with a molecular skeleton of C6:C3:C6. According to relative molecular mass, condensed tannin can be further divided into flavanol monomers and polymers. Traditionally, polymers with relative molecular mass above 500 Dalton (Da) are called condensed tannin.

[0005] In related arts, only the extraction methods of hydrolyzable tannin are disclosed, which are generally extraction methods using organic solvents such as acetone and ether. These methods are suitable for hydrolyzable tannin, which have low molecular weight or good water solubility, but have poor extraction effects for condensed tannin with high molecular weight (molecular weight between 1000 and 20,000 Da). Traditional organic solvent extraction methods can hardly effectively extract condensed tannin with high molecular weight from plant cells. In addition, under the action of organic solvents, cell structures are easily damaged, and the complexation of condensed tannin with protein and other macromolecules will further hinder the extraction process.

[0006] Therefore, an extraction and purification method for efficiently extracting plant-condensed tannin featuring easy operation and low cost is in urgent need, so as to obtain high-quality plant-condensed tannin and facilitate the further development and utilization of condensed tannin.

BRIEF SUMMARY OF THE INVENTION

[0007] The invention aims at solving at least one of the technical problems existing in the prior art. Therefore, the invention provides a condensed tannin extraction method, which can extract condensed tannin with high molecular weight from plants without using organic solvents, and can effectively protect the integrity of plant cells and reduce the yield loss caused by complexation between condensed tannin and substances such as protein and trace elements discharged from damaged plant cells.

[0008] In a first aspect of the invention, a condensed tannin extraction method is provided, which comprises the following steps:

[0009] (1) adding a compound enzyme extractant to a plant sample for extraction to obtain a crude extract; and

[0010] (2) dissolving the crude extract obtained in step (1) in a methanol solution, conducting filtration to remove impurities, passing through a cross-linked sephadex column, and conducting elution to obtain an eluent, that is, purified condensed tannin.

[0011] A specific embodiment of the invention has at least the following beneficial effects:

[0012] 1. The invention first proposes the use of the enzymatic hydrolysis method in preparing condensed tannin, and obtains the best combination of complex enzymes and the proportion of enzymes in each component through exploration, and the extraction rate far exceeds that of the traditional organic extraction method.

[0013] 2. According to the condensed tannin extraction method in the invention, no organic solvent is used, so that the environmental pollution caused by organic solvents is avoided, the damage of plant cells caused by organic solvents is effectively avoided, and the yield loss caused by complexation between condensed tannin and substances such as protein and trace elements discharged from damaged plant cells is reduced.

[0014] 3. The condensed tannin extracted by the condensed tannin extraction method of the invention has a yield of more than 3.0% and a purity of more than 85.85%, the polymerization degree of the extracted condensed tannin is 17-20, the mass ratio of procyanidin tannin to prodelphinidin tannin in the condensed tannin is 3.3-4.2, the content of an extended unit epicatechin is 62.3%-68.8%, the content of an extended unit epigallocatechin is 23.5%-28.0%, and all of the data is superior to those of the traditional chemical organic solvent extraction process.

[0015] According to the first aspect of the invention, in some embodiments, the compound enzyme extractant in the above step (1) contains laccase, cellulase, and pectinase.

[0016] In some preferred embodiments, the compound enzyme extractant contains laccase, cellulase, and pectinase.

[0017] According to the first aspect of the invention, in some preferred embodiments, the ratio of laccase to cellulase to pectinase is 1: (10-20): 2 by volume.

[0018] In some more preferred embodiments, the ratio of laccase to cellulase to pectinase is preferably 1: (10-15): 2 by volume.

[0019] In some more preferred embodiments, the added amount of laccase is 10 U/mL, cellulase is 150 U/mL, and pectinase is 20 U/mL.

[0020] The inventors found through various tests and screening that the single use of a certain enzyme (such as laccase, cellulase, or pectinase) or the combination of two enzymes could not achieve efficient extraction of condensed tannin, the yield of condensed tannin was not ideal, and only when laccase, cellulase, and pectinase were used together could the cell walls and lignin components of plant cells be effectively degraded, so that condensed tannin can be effectively released, thus improving the yield of condensed tannin.

[0021] According to the first aspect of the invention, in some embodiments, the plant sample in the above step (1) includes one or more of wine grape pomace, Stylosanthes, Dalea purpurea, or sainfoin. Of course, any plant containing condensed tannin can be used as the extraction sample.

[0022] According to the first aspect of the invention, in some embodiments, the plant sample in the above step (1) is crushed, and the particle size of the crushed plant sample is less than or equal to 1.0 mm.

[0023] According to the first aspect of the invention, in some embodiments, the extraction conditions of the compound enzyme extractant are as follows:

[0024] the extraction temperature is 40-50°C, the pH value is 4.5-5.5, and the solid-liquid ratio of the plant sample to the compound enzyme extractant is 1: (15-25) g/mL.

[0025] In some preferred embodiments, the extraction conditions of the compound enzyme extractant are as follows:

[0026] the extraction temperature is 45°C, the pH value is 5, and the solid-liquid ratio of the plant sample to the compound enzyme extractant is 1: 20 g/mL.

[0027] The inventors found that the extraction temperature, pH value, and solid- liquid ratio had significant effects on the extraction of condensed tannin using the complex enzyme extractant, and the extraction temperature, pH value, and solid-liquid ratio needed to be within a certain range to achieve efficient extraction of condensed tannin, otherwise, the yield of condensed tannin might decrease.

[0028] The inventors also found that the extraction duration of the complex enzyme extractant also affected the yield of condensed tannin, when the extraction duration was 30-60 min, the yield of condensed tannin was the highest, and especially when the extraction duration was stabilized at 40 min, the yield could reach 3.3%.

[0029] Of course, those skilled in the art can reasonably adjust the particle size of the extract to meet the extraction requirements according to the actual needs.

[0030] According to the first aspect of the invention, in some embodiments, the eluent obtained in the above step (2) contains an acetone solution and ascorbic acid, the volume percentage of the acetone solution is 45%-50%, and the volume percentage of the ascorbic acid is 0.1%-0.2%.

[0031] In some preferred embodiments, the volume percentage of the acetone solution is 50%, and the volume percentage of the ascorbic acid is 0.1%.

[0032] The addition of the ascorbic acid can effectively prevent the oxidation of condensed tannin, thus ensuring the stability of the yield and purity of condensed tannin.

[0033] According to the first aspect of the invention, in some embodiments, the column height of the cross-linked sephadex column in the above step (2) is 10-20 cm, and the flow rate of the eluent during elution is 1.0-3.0 mL/s.

[0034] In some preferred embodiments, the column height of the cross-linked sephadex column is 15 cm, and the flow rate of the eluent during elution is 2.0 mL/s.

[0035] According to the first aspect of the invention, in some embodiments, in the above step (2), passing through the cross-linked sephadex column is conducted repeatedly until the solution becomes colorless.

[0036] According to the first aspect of the invention, in some embodiments, in the above step (2), elution is conducted many times until the eluent becomes colorless.

[0037] Repeated column passing and elution are to ensure that all the condensed tannin in the crude extract can be adsorbed or precipitated by cross-linked sephadex, thus ensuring the yield.

[0038] A second aspect of the invention provides condensed tannin prepared by 5 the extraction method in the first aspect of the invention.

[0039] According to the second aspect of the invention, in some embodiments, the polymerization degree of the condensed tannin is 17-20;

[0040] the mass ratio of procyanidin tannin to prodelphinidin tannin in the condensed tannin is greater than 3.3, and in some preferred embodiments, the mass ratio IO of procyanidin tannin to prodelphinidin tannin in the condensed tannin is 3.3-4.2;

[0041] the content of an extended unit epicatechin in the condensed tannin is more than 62.3%, and in some preferred embodiments, the content of the extended unit epicatechin in the condensed tannin is 62.3%-68.8%;

[0042] the content of an extended unit epigallocatechin in the condensed tannin is more than 23.5%, and in some preferred embodiments, the content of the extended unit epigallocatechin in the condensed tannin is 23.5%-28.0%; and

[0043] the molecular weight of the condensed tannin is 1-20 kDa.

[0044] In some preferred embodiments, the purity of the condensed tannin is

96.5%. The polymerization degree of the condensed tannin is 17, the mass ratio of procyanidin tannin to prodelphinidin tannin in the condensed tannin is 3.3, the content of the extended unit epicatechin is 68.8%, and the content of the extended unit epigallocatechin is 28.0%.

[0045] A third aspect of the invention provides an application of condensed tannin prepared by the extraction method according to the first aspect of the invention in preparing animal feed.

[0046] According to the third aspect of the invention, in some embodiments, the above animal feed 1s aquatic animal feed.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0047] Fig. 1 is an NMR spectrum of condensed tannin in Embodiment 1 of the invention;

[0048] Fig. 2 shows the effects of different enzymes and their combinations on the yield of condensed tannin in the embodiments of the invention;

[0049] Fig. 3 shows the effects of different extraction temperatures on the yield of condensed tannin in the embodiments of the invention;

[0050] Fig. 4 shows the effects of different extraction durations on the yield of condensed tannin in the embodiments of the invention;

[0051] Fig. 5 shows the effects of different pH values on the yield of condensed tannin in the embodiments of the invention;

[0052] Fig. 6 shows the effects of different solid-liquid ratios on the yield of condensed tannin in the embodiments of the invention; and

[0053] Fig. 7 shows the effects of different enzyme ratios on the yield of condensed tannin in the embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0054] In order to make the invention purpose, technical solution, and technical effect of the invention clearer, the invention will be further described in detail with reference to specific embodiments. It should be understood that the specific embodiments described in this specification are only for explaining the invention, not for limiting the invention.

[0055] The experimental materials and reagents used, unless otherwise specified, are conventional consumables and reagents available from commercial sources.

[0056] Embodiment 1

[0057] A plant-condensed tannin extraction and purification method comprises the following steps:

[0058] (1) Freeze-drying a plant sample and crushing the plant sample until all particles pass through a 1.0 mm standard sieve.

[0059] (2) Adding an extracting solution (laccase 10 U/mL, cellulase 150 U/mL, pectinase 20 U/mL) for extraction which lasts 40 min, wherein the extraction temperature is 45°C, the pH value of the extracting solution is 5.0, and the solid-liquid ratio is 1:20 g/mL; and conducting vacuum filtration on the extracting solution, and conducting freeze-drying to obtain a crude extract.

[0060] (3) Dissolving the crude extract in an 80% methanol solution, conducting filtration to remove impurities, adding the filtrate into a cross-linked sephadex column, stirring thoroughly, standing for 30 min, conducting vacuum filtration repeatedly until a solution obtained by vacuum filtration is colorless, and discarding a filtrate.

[0061] (4) Rinsing the cross-linked sephadex column repeatedly with a 50% acetone solution (containing 0.1% ascorbic acid) until an eluent is colorless.

[0062] (5) Evaporating and concentrating the eluent (40°C, 30 min) and conducting freeze-drying to obtain high-purity plant-condensed tannin.

[0063] The plant sample in the step (1) includes one or more of wine grape pomace, Stvlosanthes, Dalea purpurea, or sainfoin.

[0064] The condensed tannin extracted from Embodiment 1 was detected by NMR. The NMR spectrum of the condensed tannin in Embodiment 1 is shown in Fig. I.

[0065] Embodiment 2

[0066] The method in Embodiment 1 was used to prepare condensed tannin. The difference between Embodiment 2 and Embodiment 1 is that the enzyme content in the extracting solution was adjusted in Embodiment 2 to 8 U/mL of laccase, 140 U/mL of cellulase and 15 U/mL of pectinase.

[0067] Embodiment 3

[0068] The method in Embodiment 1 was used to prepare condensed tannin. The difference between Embodiment 3 and Embodiment 1 is that the enzyme content in the extracting solution was adjusted in Embodiment 3 to 12 U/mL of laccase, 160 U/mL of cellulase and 25 U/mL of pectinase.

[0069] Comparative example |

[0070] A plant sample of the same mass was extracted by a conventional organic solvent extraction method.

[0071] The organic solvent extraction method adopted in this embodiment comprises the following steps:

[0072] (1) Freeze-drying a plant sample and crushing the plant sample until all particles pass through a 1.0 mm standard sieve.

[0073] (2) Adding an acetone solution for extraction and conducting filtration.

[0074] (3) Mixing a filtrate with an equal volume of ether solvent, standing, and discarding an upper organic phase solution and keeping a lower aqueous phase solution after the solution 1s layered.

[0075] (4) Repeating steps 2-3, and extracting filter residues 2-3 times.

[0076] (5) Collecting all of the aqueous solution, and conducting evaporation and concentration to separate acetone from the aqueous solution.

[0077] (6) Freeze-drying a concentrated solution to prepare a condensed tannin crude extract.

[0078] (7) Dissolving the crude extract in an ethanol solution, conducting filtration with filter paper to remove impurities, passing through a macroporous adsorption resin column, and repeatedly eluting the macroporous adsorption resin column with ethanol and acetone respectively until an eluent is colorless.

[0079] (8) Evaporating and concentrating the eluent and conducting freeze- drying to obtain purified plant-condensed tannin.

[0080] The plant sample in step (1) includes one or more of wine grape pomace, Swlosanthes, Dalea purpurea, or sainfoin.

[0081] Comparison of extraction effects

[0082] The plant-condensed tannins, extracted from Embodiments 1-3 and Comparative example 1, were compared. The purity and polymerization degree of the plant-condensed tannin, the ratio of procyanidin tannin to prodelphinidin tannin in the condensed tannin, the content of an extended unit epicatechin, and the content of an extended unit epigallocatechin were detected, and the yields of the condensed tannin obtained from Embodiments 1-3 and Comparative example 1 were calculated respectively.

[0083] The results are shown in Table 1.

[0084] Table 1 Comparison results of Embodiments 1-3 and Comparative Example 1

[0085] Item Embodiment | Embodiment 2 Embodiment 3 Comparative Example 1 Purity of condensed tannin %: 96.5%, 85.85%, 93.8%, 65.6% Polymerization degree of condensed tannin: 17, 20, 18, 26 Procyanidin tannin/prodelphinidin tannin: 4.2, 3.3,

3.7, 2.6 Content of extended unit epicatechin %: 68.8%, 62.3%, 64.4%, 60.5% Content of extended unit epigallocatechin %: 28.0%, 23.5%, 26.5%, 19.2% Yield: 3.5%, 2.2%,

3.0% <1%

[0086] It can be seen from Table 1 that the condensed tannin extracted by the methods in Embodiments 1-3 are significantly higher in purity, ratio of procyanidin tannin to prodelphinidin tannin, content of the extended unit epicatechin, content of the extended unit epigallocatechin, and yield than those obtained by the method in Comparative Example 1, and their polymerization degree is lower than that of the comparative example. The condensed tannin with a low polymerization degree also have a low molecular weight, so as to be more easily absorbed and utilized by animals to exert their biological activities. Therefore, the methods in the embodiments have advantages over traditional extraction technology, and can facilitate the extraction of condensed tannin from plant samples, and the molecular weight and composition ratio of the condensed tannin obtained are more beneficial to animal health. Besides, because no organic solution is used, environmental harm and cost are reduced. Therefore, the method is worth popularizing.

[0087] Influencing factors of extraction process

[0088] The effects of various extraction conditions on the extraction process were further studied by using the extraction method in Embodiment 1.

[0089] (1) Effect of different enzymes on the yield of condensed tannin:

[0090] The extraction method in Embodiment 1 was adopted, and the difference between this embodiment and the extraction method in Embodiment 1 is that different enzyme extractants (1. cellulase; 2. cellulase and pectinase; 3. cellulase and laccase; 4.

pectinase and laccase; 5. cellulase, pectinase, and laccase) were adopted.

[0091] The results are shown in Fig. 2.

[0092] It can be seen from Fig. 2 that after comparing the yield of condensed tannin, it can be found that the combination of cellulase, pectinase, and laccase can extract condensed tannin from plant samples to the greatest extent, which may be due to the fact that the combined enzymes can more fully degrade the cell walls and lignin components of plant cells, so that condensed tannin can be effectively released. Therefore, the combination of cellulase, pectinase, and laccase is the best extraction condition.

[0093] (2) Effect of different extraction temperatures on the yield of condensed tannin:

[0094] The extraction method in Embodiment 1 was adopted, and the difference between this embodiment and the extraction method in Embodiment 1 is that different extraction temperatures (35°C, 40°C, 45°C, 50°C, 55°C) were adopted.

[0095] The results are shown in Fig. 3.

[0096] It can be seen from Fig. 3 that after comparing the yield of condensed tannin, it can be found that the highest yield of condensed tannin is obtained when the extraction temperature 1s 45°C, which may be due to the degradation or structural damage of condensed tannin when the temperature exceeds 45°C. Therefore, 45°C is the best extraction condition.

[0097] (3) Effect of different extraction durations on the yield of condensed tannin:

[0098] The extraction method in Embodiment 1 was adopted, and the difference between this embodiment and the extraction method in Embodiment 1 is that different extraction durations (20 min, 40 min, 60 min, 80 min respectively) were adopted.

[0099] The results are shown in Fig. 4.

[00100] It can be seen from Fig. 4 that after comparing the yield of condensed tannin, it can be found that the highest yield of condensed tannin is obtained IO when the extraction duration is 40 min, which may be due to the degradation or structural damage of condensed tannin under the interference of temperature, light and other factors with the extension of extraction duration. Therefore, 40 min is the best extraction condition.

[00101] (4) Effect of different pH values on the yield of condensed tannin:

[00102] The extraction method in Embodiment 1 was adopted, and the difference between this embodiment and the extraction method in Embodiment 1 is that different pH values (3.5, 4.0, 4.5, 5.0, 5.5, 6.0) were adopted.

[00103] The results are shown in Fig. 5.

[00104] It can be seen from Fig. 5 that after comparing the yield of condensed tannin, it can be found that the yield of condensed tannin is the highest at pH

5.0, which may be due to the fact that lower or higher pH may affect the dissolution rate of condensed tannin, so pH 5.0 is the best extraction condition.

[00105] (5) Effect of different solid-liquid ratios on the yield of condensed tannin:

[00106] The extraction method in Embodiment 1 was adopted, and the difference between this embodiment and the extraction method in Embodiment 1 is that different solid-liquid ratios (1:10, 1:20, 1:30, 1:40) were adopted.

[00107] The results are shown in Fig. 6.

[00108] It can be seen from Fig. 6 that after comparing the yield of condensed tannin, it can be found that the yield of condensed tannin is the highest when the solid-liquid ratio is 1:20, which may be due to the fact that lower or higher solid- liquid ratios may affect the dissolution rate of condensed tannin, so the solid-liquid ratio of 1:20 is the best extraction condition.

[00109] (6) Effect of different enzyme ratios on the yield of condensed tannin:

[00110] The extraction method in Embodiment 1 was adopted, and the difference between this embodiment and the extraction method in Embodiment 1 is that different enzyme ratios (laccase: cellulase: pectinase is 1:5:2 or 1:10:2 or 1:15:2 or 1:20:2 or 1:25:2) were adopted.

[00111] The results are shown in Fig. 7.

[00112] It can be seen from Fig. 7 that after comparing the yield of condensed tannin, it can be found that different enzyme ratios have certain influence on the yield of condensed tannin, and the highest yield of condensed tannin is obtained when the ratio of laccase to cellulase to pectinase is 1:15:2, which may be due to the fact that the degree of coordination among the three enzymes is the highest within this range, and excessive enzymolysis will not occur due to the redundancy of a certain enzyme. Therefore, the ratio of laccase to cellulase to pectinase being 1:15:2 is the best extraction condition.

[00113] To sum up, the above embodiments show that all the extraction conditions adopted in the embodiments of the invention are necessary conditions obtained by screening, and only when extraction is conducted within this range can a higher yield of condensed tannin be achieved.

[00114] The above embodiments are preferred embodiments of the invention, but the embodiments of the invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the invention shall be equivalent substitutions and are included in the scope of protection of the invention.

Claims

Claims L Method for extracting condensed tannin, the method comprising the steps of: (1) adding a compound enzyme extractant to a plant sample for extraction to obtain a crude extract; and (2) dissolving the crude extract obtained in step (1) in a methanol solution, performing filtration to remove impurities, passing through a crosslinked sephadex column, and performing elution to obtain an eluent, that is say, purified condensed tannin; wherein the compound enzyme extractant comprises laccase, cellulase and pectinase.

Extraction method according to claim 1, characterized in that the ratio of laccase to cellulase to pectinase by volume is 1: (10-20): 2 .

Extraction method according to claim 1, characterized in that the ratio of laccase to cellulase to pectinase by volume is preferably 1:(10-15):2.

The extraction method according to claim 1, characterized in that the plant sample is crushed in step (1), and the particle size of the crushed plant sample is less than or equal to 1.0 mm.

Extraction method according to claim 1, characterized in that the eluent obtained in step (2) comprises an acetone solution and ascorbic acid, the percentage by volume of the acetone solution being preferably 45% - 50%, and the percentage by volume of the acetone solution ascorbic acid is preferably 0.1% - 0.2%.

An extraction method according to claim 1, characterized in that the column height of the crosslinked sephadex column in step (2) is 10-20 cm, and the flow rate of the eluent during elution is 1.0-3.0 mL/s.

-13-

The extraction method according to claim 1, characterized in that in step (2) the passage through the crosslinked sephadex column is carried out repeatedly until the solution becomes colorless.

Extraction method according to claim 1, characterized in that in step (2) elution is carried out many times until the eluent becomes colorless.

The condensed tannin prepared by the extraction method according to any one of claims 1 to 8, characterized in that the degree of polymerization of the condensed tannin is 17 to 20; the mass ratio of procyanidin tannin to prodelphinidin tannin in the condensed tannin is greater than 3.3, preferably 3.3 - 4.2; the content of an extended unit epicatechin in the condensed tannin is greater than 62.3%, preferably 62.3% - 68.8%; the content of an extended unit of epigallocatechin in the condensed tannin is greater than 23.5%, preferably 23.5% - 28.0%; and the molecular weight of the condensed tannin is 1-20 kDa.

Use of condensed tannin prepared by the extraction method according to any one of claims 1 to 8 in the manufacture of animal feed, wherein the animal feed is preferably aquatic animal feed.