Method for acetylation modification of biomass-based adsorption material for purification of catechin and use thereof

Abstract

The application discloses an acetylation modification method of a biomass-based adsorption material for tea catechin purification, which comprises the steps of alkali water pretreatment and acetylation. After the chemical acetylation molecular grafting reaction of biomass-based rice husk, peanut shell, walnut shell or bamboo powder and the like through the method, the cellulose and the lignin can synergistically play the enrichment and purification role. The application further discloses the use of the acetylation modification product of the biomass-based adsorption material, namely, the use in the purification and preparation of tea catechin.

Description

Technical Field

[0001] This invention belongs to the field of green preparation technology of active ingredients in tea, and more specifically, it relates to a method for preparing chromatographic adsorption materials using natural biomass resources such as rice husks, peanut shells, walnut shells and bamboo powder, and its application in the purification of tea catechins. Background Technology

[0002] Tea( Camellia sinensis L. (catechins) possess unique pharmacological effects such as anti-oxidation, UV protection, anti-aging, anti-tumor, anti-influenza virus and HIV virus, regulation of immune cell function, and protection of the cardiovascular system. They are currently used in new food ingredients, food additives, and cosmetics. However, the purification process of tea catechins often relies on non-renewable fossil resources. The adsorbent materials prepared from fossil raw materials cause "white pollution" to the environment after disposal; they may also contaminate the products prepared from them. This is mainly due to the presence of residual monomers, cross-linking agents, pore-forming agents, and dispersants (such as toluene, xylene, divinylbenzene, and benzene polymers) in fossil resin materials. Furthermore, detached fragments formed during resin aging can enter the product, causing secondary pollution. The regeneration of some resins is also challenging. Therefore, developing safe, efficient, low-toxicity, and inexpensive adsorbent materials for chromatographic separation will help promote the development and application of tea catechin products.

[0003] Biomass-based polymer materials derived from nature possess renewable, biocompatible, biodegradable, and biosafety properties. Developing new biomass resource materials for tea catechin purification will endow tea catechin products with higher specifications and green attributes. Furthermore, the development of biomass-based materials with excellent mechanical properties can further broaden their application scope and gradually replace currently widely used non-degradable fossil adsorption and separation materials, thereby contributing to the green and sustainable development of the tea industry and possessing significant social and economic value. Literature (e.g., Senji Sakanaka. A Novel Convenient Process To Obtain a Raw Decaffeinated Tea Polyphenol Fraction Using a Lignocellulose Column. J. Agric. Food Chem., 2003, 51, 3140-3143) and patents (e.g., application numbers: 200510060423.3, 200610049639.4) have reported that sawdust from processing fir, pine, willow, cypress, camphor, paulownia, tea twig powder, or other woody plant branches and wood, etc., after being successively soaked in alkaline solution and hydrochloric acid, utilizes the lignin in the lignin with abundant benzene ring structure to adsorb and separate tea polyphenols. The inventors mainly focus on the acetylation modification of cellulose media filler to achieve the enrichment and purification of tea catechins in tea polyphenols (patent application number: 202010073149.8).

[0004] Lignocellulose, primarily composed of cellulose, hemicellulose, and lignin linked by organic covalent bonds, possesses a three-dimensional network structure and is the most widely distributed and abundant organic compound on Earth. Plants and microorganisms in nature synthesize over a billion tons of cellulose or lignocellulose annually, making it an inexhaustible, green, and renewable resource. my country, a major agricultural country, has abundant and readily available biomass resources such as rice husks, peanut shells, walnut shells, and bamboo powder, which are easily processed into specific particle sizes and suitable for use as packing materials in atmospheric or medium-pressure chromatographic columns. These resources possess a hard, rigid, and resilient molecular framework. The cellulose, hemicellulose, and lignin contents in rice husks, peanut shells, walnut shells, and bamboo powder are approximately 35% (w / w), 20% (w / w), 20% (w / w); 25% (w / w), 36% (w / w), 25% (w / w); 26.4% (w / w), 25.9% (w / w), 50% (w / w); 40% (w / w), 15% (w / w), and 25% (w / w), respectively.

[0005] The purpose of soaking lignocellulose in a strongly alkaline aqueous solution is to dissolve its internal filler and release the three-dimensional network adsorption sites within the lignocellulose. However, biomass such as rice husks, peanut shells, walnut shells, and bamboo powder, after being soaked in conventional room-temperature alkaline solutions, fail to exert their adsorption function, mainly because their internal filler is not effectively released under low-temperature conditions. The inventors discovered that while appropriate high-temperature alkaline aqueous solution extraction partially degrades lignin, it plays a crucial role in enabling lignocellulose adsorption. On the other hand, biomass such as rice husks, peanut shells, walnut shells, and bamboo powder are rich in cellulose in addition to lignin. Cellulose itself does not have the characteristic of adsorbing catechins, but by acetylation modification, it can synergistically work with lignin to enrich and purify lignocellulose.

[0006] The acetylation process of cellulose is mainly divided into homogeneous and heterogeneous processes. The homogeneous mode requires high-purity cellulose as a liquefaction feedstock, and the acetylated cellulose prepared is mainly cellulose diacetate or cellulose triacetate, which has been widely used in spinning, cigarette filters, plastics, hollow fiber membranes, and pharmaceuticals. The heterogeneous mode has a lower acetylation synthesis efficiency, but it does not destroy the rigid skeleton of lignocellulose and is suitable for the preparation of packing materials for column chromatography. However, the type of catalyst and acetylation reaction parameters required in the heterogeneous mode are unknown. Summary of the Invention

[0007] To address the shortcomings of the existing technology, the present invention aims to provide a method for acetylation modification of biomass-based adsorbent materials. Another objective of the present invention is to provide the use of the aforementioned biomass-based adsorbent materials for the purification of tea catechins.

[0008] The acetylation modification method for biomass-based adsorbent materials for purifying tea catechins provided by this invention is achieved through the following steps: (a) Particle biomass was extracted by stirring with an alkaline aqueous solution at a certain temperature and then filtered to obtain alkaline-treated granular biomass. (b) Wash the alkaline-treated granular biomass from step (a) with water until neutral, and then dry it for later use. (c) The dried biomass from step (b) is mixed with glacial acetic acid, acetic anhydride and acid catalyst at a certain solid-liquid ratio and stirred in a water bath for reaction. (d) The reaction solution from step (c) is subjected to solid-liquid separation. The solid is washed with water to obtain the acetylated biomass-based adsorbent material.

[0009] Preferably, the alkaline aqueous solution in step (a) is a 0-10% potassium hydroxide aqueous solution or a 0-10% sodium hydroxide aqueous solution.

[0010] Preferably, the stirring and extraction in step (a) is carried out for 0.5-1.5 hours.

[0011] Preferably, the granular biomass mentioned in step (a) is rice husk, peanut shell, walnut shell or bamboo powder.

[0012] Preferably, the extraction temperature in step (a) is 60-90°C.

[0013] Preferably, the drying temperature in step (b) is 55-95°C.

[0014] Preferably, the acid catalyst in step (b) is a sulfuric acid solution containing 1-4% (v / v) in glacial acetic acid.

[0015] Preferably, the solid-liquid ratio of biomass: glacial acetic acid: acetic anhydride: acid catalyst in step (b) is 1g: 2-5mL: 1-2mL: 0.2-0.8mL.

[0016] Preferably, the water bath temperature in step (C) is 50-75°C.

[0017] Preferably, the water bath stirring reaction time in step (c) is 1-3 hours.

[0018] This invention relates to acetylated biomass materials prepared by the above method, which have the characteristics of adsorbing and purifying tea catechins. Beneficial effects

[0019] The biomass resources selected for this invention—rice husks, peanut shells, walnut shells, or bamboo powder—are widely available, inexpensive, abundant, and easily processed into specific particle sizes, making them suitable for atmospheric or medium-pressure chromatographic column packing materials with their hard and tough molecular framework. Furthermore, high-temperature alkaline water treatment combined with anhydride acetylation maximizes the potential for lignin and cellulose adsorption and separation of catechins from lignocellulose. Attached Figure Description

[0020] Figure 1 This is the HPLC chromatogram of the tea polyphenol control sample in Example 3. Detailed Implementation

[0021] The following examples are provided to facilitate a better understanding of the present invention, but do not limit the invention. All reagents used in the following examples, unless otherwise specified, are commercially available. Experimental methods, unless otherwise specified, are conventional methods.

[0022] The catechins referred to in this invention are derived from the Theaceae family ( Theaceae Camellia genus ( Camellia Tea tree (plant) Camellia SinensisIt was extracted from leaves. The terms EGCG and ECG are used to refer to epigallocatechin gallate and ECG, respectively. The analytical method for tea catechins is as follows: In the examples below, the determination of tea catechins EGCG and ECG was performed using reversed-phase high-performance liquid chromatography (RP-HPLC). HPLC used a two-phase gradient elution (1 mL / min), where the aqueous phase (A) was pure aqueous solution and the organic phase (B) was acetonitrile. The elution program was 0-5 min: a linear gradient from 90% A to 85% A; 5-15 min: a linear gradient from 85% A to 77% A; then 1 min: to 90% A; and equilibration for 5 min. The HPLC system was a Waters 1525 pump equipped with a column oven, a 2998 diode array detector, a 2707 autosampler, and Breeze control software. The chromatographic column was a Waters Symmetry C1000. 18 (4.6mm×150mm), column temperature 35℃. Detection wavelength 278nm, injection volume 5μL.

[0023] Example 1: Alkali pretreatment of biomass-based rice husks, peanut shells, walnut shells, and bamboo powder (1) Weigh 5 grams of 30-mesh rice husks, add 25 mL of pure water, extract by shaking in a 90°C water bath for 1.5 hours, then separate the solid and liquid, wash with water, and dry at 95°C for later use.

[0024] (2) Weigh 5 grams of 30-mesh rice husks, add 25 mL of 0.5% potassium hydroxide aqueous solution, extract by shaking in a 70°C water bath for 1.5 hours, separate the solid and liquid, wash with water until neutral, and dry at 95°C for later use.

[0025] (3) Weigh 5 grams of 30-mesh rice husks, add 25 mL of 1% potassium hydroxide aqueous solution, extract by shaking in a 70°C water bath for 1.5 hours, separate the solid and liquid, wash with water until neutral, and dry at 95°C for later use.

[0026] (4) Weigh 5 grams of 30-mesh rice husks, add 25 mL of 5% potassium hydroxide aqueous solution, extract by shaking in a 70°C water bath for 1 hour, separate the solid and liquid, wash with water until neutral, and dry at 95°C for later use.

[0027] (5) Weigh 5 grams of 30-mesh rice husks, add 25 mL of 10% sodium hydroxide aqueous solution, extract by shaking in a water bath at 60°C for 0.5 hours, separate the solid and liquid, wash with water until neutral, and dry at 95°C for later use.

[0028] (6) Weigh 5 grams of 100-mesh peanut shells, add 25 mL of 5% potassium hydroxide aqueous solution, extract by shaking in a 70°C water bath for 1 hour, separate the solid and liquid, wash with water until neutral, and dry at 80°C for later use.

[0029] (7) Weigh 5 grams of 0.2 mm walnut shells, add 25 mL of 5% potassium hydroxide aqueous solution, extract by shaking in a 70°C water bath for 1.5 hours, separate the solid and liquid, wash with water until neutral, and dry at 95°C for later use.

[0030] (8) Weigh 5 grams of 25-45 mesh bamboo powder, add 25 mL of pure water, extract by shaking in a 90°C water bath for 1.5 hours, separate the solid and liquid, wash with water until neutral, and dry at 55°C for later use.

[0031] (9) Weigh 5 grams of 25-45 mesh bamboo powder, add 25 mL of 5% potassium hydroxide aqueous solution, extract by shaking in a 70°C water bath for 1 hour, separate the solid and liquid, wash with water until neutral, and dry at 75°C for later use.

[0032] Example 2: Preparation of acetylated biomass-based adsorbent materials made from rice husks, peanut shells, walnut shells, and bamboo powder. (1) Weigh 1 gram of dried and pretreated biomass rice husk from Example 1, add 5 mL of glacial acetic acid, 2 mL of acetic anhydride, and 0.2 mL of catalyst (glacial acetic acid containing 4% (v / v) sulfuric acid solution), and shake in a water bath at 75°C for 3 hours. After solid-liquid separation and washing with water until neutral, set aside for later use.

[0033] (2) Weigh 1 gram of dried and pretreated biomass rice husks from Example 1 (2), add 5 mL of glacial acetic acid, 2 mL of acetic anhydride, and 0.2 mL of catalyst (glacial acetic acid containing 4% (v / v) sulfuric acid solution), and shake in a water bath at 75°C for 2.5 hours. After solid-liquid separation and washing with water until neutral, set aside for later use.

[0034] (3) Weigh 1 gram of dried and pretreated biomass rice husk from Example 1 (3), add 5 mL of glacial acetic acid, 2 mL of acetic anhydride, and 0.2 mL of catalyst (glacial acetic acid containing 4% (v / v) sulfuric acid solution), and shake in a water bath at 75°C for 2 hours. After solid-liquid separation and washing with water until neutral, set aside for later use.

[0035] (4) Weigh 1 gram of dried and pretreated biomass rice husk from Example 1 (4), add 5 mL of glacial acetic acid, 2 mL of acetic anhydride, and 0.2 mL of catalyst (glacial acetic acid containing 4% (v / v) sulfuric acid solution), and shake in a water bath at 70°C for 2 hours. After solid-liquid separation and washing with water until neutral, set aside for later use.

[0036] (5) Weigh 1 gram of dried and pretreated biomass rice husks from Example 1 (5), add 5 mL of glacial acetic acid, 2 mL of acetic anhydride, and 0.8 mL of catalyst (glacial acetic acid containing 1% (v / v) sulfuric acid solution), and shake in a water bath at 50°C for 1 hour. After solid-liquid separation and washing with water until neutral, set aside for later use.

[0037] (6) Weigh 1 gram of dried and pretreated biomass peanut shells from Example 1 (6), add 5 mL of glacial acetic acid, 2 mL of acetic anhydride, and 0.8 mL of catalyst (glacial acetic acid containing 4% (v / v) sulfuric acid solution), and shake in a water bath at 70°C for 3 hours. After solid-liquid separation and washing with water until neutral, set aside for later use.

[0038] (7) Weigh 1 gram of dried and pretreated biomass walnut shells from Example 1 (7), add 2 mL of glacial acetic acid, 1 mL of acetic anhydride, and 0.8 mL of catalyst (glacial acetic acid containing 4% (v / v) sulfuric acid solution), and shake in a water bath at 70°C for 3 hours. After solid-liquid separation and washing with water until neutral, set aside for later use.

[0039] (8) Weigh 1 gram of dried and pretreated biomass bamboo powder from Example 1 (8), add 5 mL of glacial acetic acid, 2 mL of acetic anhydride, and 0.2 mL of catalyst (glacial acetic acid containing 4% (v / v) sulfuric acid solution), and shake in a water bath at 70°C for 2 hours. After solid-liquid separation and washing with water until neutral, it is ready for use.

[0040] (9) Weigh 1 gram of dried and pretreated biomass bamboo powder from Example 1, add 5 mL of glacial acetic acid, 2 mL of acetic anhydride, and 0.2 mL of catalyst (glacial acetic acid containing 2.5% (v / v) sulfuric acid solution). After shaking and reacting in a water bath at 70°C for 1.5 hours, separate the solid and liquid and wash with water until neutral before use.

[0041] Example 3: Quantitative Characterization of Adsorption and Desorption of Tea Catechins by Acetylated Biomass-Based Adsorbents Preparation of 2% tea polyphenol aqueous solution: Accurately weigh 2 grams of commercially available 98% tea polyphenol powder (containing 40.36% (w / w) EGCG and 5.58% (w / w) ECG) into a beaker, add water to dissolve, then transfer to a 100mL volumetric flask, add 0.2mL glacial acetic acid, and dilute to the mark with water. Shake well and set aside.

[0042] Take 1 gram of each of the acetylated biomass-based adsorbent materials described in Examples 2 (1)-(9), and vertically load them into empty 10 mL syringe columns with sieve plates (pore size 10~100 μm) at the bottom using a wet loading method. Slowly add 5 mL of 2% tea polyphenol aqueous solution, controlling the flow rate. After loading, elute with water to 50 mL as the eluent. Then elute with 75% ethanol aqueous solution to 50 mL of eluent. Inject 5 μL of each eluent using HPLC. The specific results are shown in Table 1.

[0043] Table 1. Changes in the integral area of ​​EGCG and ECG in water and 70% ethanol aqueous solution eluents. Example EGCG (Integral Area) ECG (Integral Area) Example 3-(1) 2923377 / 1144945 374722 / 315348 Example 3-(2) 2892503 / 1176072 370542 / 319602 Example 3-(3) 2825742 / 1242580 362985 / 326982 Example 3-(4) 1271847 / 2649238 341516 / 351398 Example 3-(5) 1427987 / 2498318 253247 / 429857 Example 3-(6) 2549932 / 1367859 302451 / 356492 Example 3-(7) 3682969 / 235018 533274 / 126857 Example 3-(8) 1842983 / 2103805 144532 / 509283 Example 3-(9) 1499755.8 / 2449082 118250.94 / 542992 Using a 10-fold dilution of 2% tea polyphenol aqueous solution as a control, 5 μL of the sample was injected into the HPLC system. The integrated areas of EGCG and ECG were 3917932 and 660906, respectively. The area to the left of the " / " represents the HPLC integrated area of ​​each component in the water eluent, and the area to the right of the " / " represents the HPLC integrated area of ​​each component in the 75% ethanol eluent. Comparative Example 1

[0044] Take 1 gram of each of the alkaline pretreated and dried samples described in Examples 1 (1)-(9) and vertically wet-load them into empty 10 mL syringes with a sieve plate at the bottom (pore size 10~100 μm). Slowly add 5 mL of the 2% tea polyphenol aqueous solution prepared in Example 3, controlling the flow rate. After loading, elute with water to 50 mL as the eluent. Then elute with 75% ethanol aqueous solution to 50 mL of eluent. Inject 5 μL of each sample into the HPLC system. The specific results are shown in Table 2.

[0045] Table 2. Changes in the integral area of ​​EGCG and ECG in water and 70% ethanol aqueous solution eluents. Comparative Example EGCG (Integral Area) ECG (Integral Area) Comparative Example 1-(1) 3790849 / 205988 631369 / 45560 Comparative Example 1-(2) 3490928 / 464838 570565 / 103339 Comparative Example 1-(3) 3366275 / 550217 486035 / 173982 Comparative Example 1-(4) 1971847 / 1885261 271516 / 387716 Comparative Example 1-(5) 2285325 / 1658742 304294 / 353716 Comparative Examples 1-(6) 3511939 / 527526 506533 / 145451 Comparative Example 1-(7) 3856149 / 60942 641242 / 19592 Comparative Examples 1-(8) 3811427 / 101839 632547 / 27538 Comparative Example 1-(9) 3725148 / 189744 584153 / 76832 Using the 2% tea polyphenol aqueous solution diluted 10 times in Example 3 as a control, 5 μL was injected into the HPLC system. The integrated areas of EGCG and ECG were 3917932 and 660906, respectively. The part to the left of the " / " represents the HPLC integrated area of ​​each component in the water eluent, and the part to the right of the " / " represents the HPLC integrated area of ​​each component in the 75% ethanol eluent. Comparative Example 2

[0046] Take 1 gram of each of the alkaline pretreated and dried samples described in Examples 1 (4) and (8), add 5 mL of glacial acid, 2 mL of acetic anhydride, and 0.2 mL of sulfuric acid catalyst. After shaking and reacting in a 70°C water bath for 2 hours, the amount of water-washed flocculents increases, the density decreases, and most of the molecular skeleton is destroyed, affecting the atmospheric pressure column chromatography experiment. Comparative Example 3

[0047] Take 1 gram of rice husk, soak it in 5% potassium hydroxide alkaline solution at room temperature for 1 week, wash it with water until neutral, and pack it into a column, load the sample, elute it and detect it by HPLC according to the method described in Comparative Example 1. The integral areas of EGCG and ECG in the washing solution are 3745235 / 632357, respectively.

[0048] The HPLC analysis results of the eluents in Examples 3 and Comparative Examples 1 and 3 show that the method provided by this invention enables cellulose and lignin in biomass to synergistically enrich and purify catechins. In particular, the adsorption performance of rice husks is significantly enhanced after high-temperature alkaline treatment. Bamboo powder, without alkaline treatment, also exhibits strong catechin enrichment and purification after direct acetylation and grafting, which is related to its high cellulose and low lignin content. Walnut shells, after alkaline treatment and acetylation, show relatively weak adsorption of catechins by column chromatography, but the adsorption capacity is large over a long period of static adsorption. This may be because the hardness of the molecular network framework and the pore size of the walnut shell affect its dynamic adsorption of catechins.

Claims

1. An acetylation modification method for purifying tea catechins using biomass-based adsorbent materials, characterized in that, Includes the following steps: (a) Particle biomass was extracted by stirring with an alkaline aqueous solution at a certain temperature and then filtered to obtain alkaline-treated granular biomass. (b) Wash the alkaline-treated granular biomass from step (a) with water until neutral, and then dry it for later use. (c) The dried biomass from step (b) is mixed with acetic acid, acetic anhydride and acid catalyst at a certain solid-liquid ratio and reacted by stirring in a water bath. (d) The reaction solution from step (c) is subjected to solid-liquid separation. The solid is washed with water to obtain the acetylated biomass-based adsorbent material.

2. The method according to claim 1, wherein the alkaline aqueous solution in step (a) is a 0-10% potassium hydroxide aqueous solution or a 0-10% sodium hydroxide aqueous solution.

3. The method of claim 1, wherein the stirring extraction in step (a) is carried out for 0.5-1.5 hours.

4. The method as described in claim 1, wherein the granular biomass in step (a) is rice husk, peanut shell, walnut shell or bamboo powder.

5. The method of claim 1, wherein the extraction temperature in step (a) is 60-90°C.

6. The method of claim 1, wherein the drying temperature in step (b) is 55-95°C.

7. The method of claim 1, wherein the acid catalyst in step (b) is a solution of sulfuric acid and acetic acid containing 1-4% (v / v).

8. The method of claim 1, wherein the solid-liquid ratio of biomass: acetic acid: acetic anhydride: acid catalyst in step (b) is 1g: 2-5mL: 1-2mL: 0.2-0.8mL.

9. The method of claim 1, wherein the water bath temperature in step (c) is 50-75°C.

10. The method of claim 1, wherein the water bath stirring reaction time in step (c) is 1-3 hours.

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