Method for extraction and fractionation of coffee chlorogenic acids by enzymatic method

Abstract

This invention discloses an enzymatic extraction and fractional purification method for chlorogenic acid from coffee, belonging to the field of natural product extraction technology. The method employs a two-stage synergistic enzymatic hydrolysis strategy using cellulase, pectinase, and ferulic acid esterase. In the first stage, the cell wall skeleton is destroyed to release free chlorogenic acid. In the second stage, ester bonds are directionally cleaved to release bound chlorogenic acid. The enzymatic hydrolysate is then eluted with a four-stage polar gradient through a macroporous adsorption resin to achieve fractional purification. This method can simultaneously obtain products of three purity levels, with the pure chlorogenic acid component achieving a purity of over 95% and a total extraction yield of over 75%.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction and separation purification technology, specifically involving a method for efficient extraction and fractional purification of coffee chlorogenic acid by using a two-stage synergistic hydrolysis of a complex enzyme combined with polar gradient elution of macroporous adsorption resin. Background Technology

[0002] Chlorogenic acid is a class of polyphenolic compounds formed by the esterification condensation of caffeic acid and quinic acid. It is widely found in plant tissues such as green coffee beans, honeysuckle, eucommia leaves, and sunflower seeds. In green coffee beans, the chlorogenic acid content can reach 6%–10% on a dry basis, making it one of the most economically valuable raw material sources for industrial extraction of chlorogenic acid. Chlorogenic acid possesses significant antioxidant, anti-inflammatory, glucose and lipid metabolism regulating, and neuroprotective biological activities, and its market demand continues to grow in functional foods, natural preservatives, cosmetic raw materials, and pharmaceutical intermediates.

[0003] Existing methods for extracting chlorogenic acid mainly include organic solvent extraction, supercritical fluid extraction, and water extraction. Organic solvent extraction uses methanol or ethanol aqueous solutions as the extraction solvent. While this method offers high extraction efficiency, it carries the risk of solvent residue, and the large amount of organic solvent used increases production costs and environmental impact. For example, Chinese invention patent CN103497106B discloses a method for extracting chlorogenic acid from green coffee beans. This method involves extraction with sodium sulfite aqueous solution followed by purification using XAD-16 and LX-28 macroporous resins in series. Although a product with 90% purity can be obtained, the yield is only 3.5%, and the process is cumbersome with a large amount of organic solvent elution. Another example is Chinese invention patent CN113336755B, which discloses a method for separating and purifying trigonelline, chlorogenic acid, and caffeine. This method uses a two-step chromatography process combining polyamide resin and macroporous adsorption resin, but its core purpose is the simultaneous separation of the three components rather than maximizing the purity of chlorogenic acid, and it does not involve an enzyme-assisted extraction step.

[0004] In enzyme-assisted extraction, existing literature reports the use of cellulase and pectinase for the auxiliary extraction of plant polyphenols. The principle is that these enzymes can hydrolyze the cellulose and pectin backbone in plant cell walls, promoting the diffusion and release of intracellular active ingredients into the extraction solvent. Domestic and international scholars have applied this technology to the extraction of chlorogenic acid or polyphenols from raw materials such as honeysuckle, eucommia leaves, and grape skins, achieving some improvement. However, the enzyme systems used in the above studies are all cell wall degrading enzymes, whose mechanism of action is limited to breaking the glycosidic bond backbone of cellulose and pectin. They lack the ability to release phenolic compounds that are covalently bonded to cell wall polysaccharides. The cell wall structure of green coffee beans contains a large amount of chlorogenic acid covalently bonded to the side chains of arabinoxylan—this bound chlorogenic acid accounts for 15%–30% of the total chlorogenic acid content in green coffee beans. Traditional cell wall degrading enzymes cannot effectively release this component. Currently, there is no known technical solution to introduce ferulic acid esterase into the coffee chlorogenic acid extraction process to directionally cleave the ester bonds between chlorogenic acid and cell wall polysaccharides. Therefore, how to achieve efficient release of both free and bound chlorogenic acid in an all-aqueous system and obtain high-purity, high-yield chlorogenic acid products in subsequent purification processes remains a technical problem that urgently needs to be solved in this field.

[0005] In terms of purification processes, existing technologies generally employ macroporous adsorption resins for the adsorption and separation of chlorogenic acid. However, most processes only include a single concentration of ethanol elution step, resulting in products with limited purity and grade, making it difficult to simultaneously meet the diverse market demands of food additives (50%–80% content), cosmetic raw materials (80%–95% content), and pharmaceutical intermediates (over 95% content). Furthermore, existing enzyme-assisted extraction processes typically employ prolonged boiling water baths or high-pressure steam treatment during enzyme inactivation. Prolonged exposure to high temperatures can easily lead to intramolecular acyl migration reactions in chlorogenic acid, converting the thermodynamically unstable 5-caffeoylquinic acid to 3-caffeoylquinic acid and 4-caffeoylquinic acid, reducing the proportion of the main isomer and the bioactivity of the product. While supercritical fluid extraction can avoid organic solvent residues, its high equipment investment and operating costs create technical barriers to industrial scale-up, and its selectivity for moderately polar compounds like chlorogenic acid is inferior to chromatographic methods. Water extraction is simple to operate and safe, but due to the limited ability of water to penetrate plant cell walls, the extraction yield is generally low. The reported yield of chlorogenic acid by water extraction is usually no more than 45%. Summary of the Invention

[0006] To address the technical problems in existing coffee chlorogenic acid extraction processes, such as high risk of organic solvent residue, insufficient release of bound chlorogenic acid leading to limited overall yield, and single grade of purified product failing to meet the needs of different application scenarios, the present invention aims to provide an enzymatic extraction and fractional purification method for coffee chlorogenic acid. This method achieves efficient extraction of chlorogenic acid under all-aqueous conditions and obtains products with multiple purity grades through polar gradient elution technology.

[0007] The technical solution of this invention employs a two-stage synergistic enzymatic hydrolysis strategy using cell wall degrading enzymes and ferulic acid esterases. In the first stage, cellulase and pectinase disrupt the cellulose-pectin network framework of coffee cell walls, allowing for the full dissolution of free chlorogenic acid. In the second stage, ferulic acid esterase is added to directionally cleave the ester bonds between chlorogenic acid and arabinoxylan, releasing bound chlorogenic acid into its free state. The reaction conditions for both stages are set according to the optimal temperature and pH of each enzyme system, ensuring that each enzyme functions within its optimal catalytic efficiency window and avoiding any compromise that could lead to a decrease in the catalytic performance of any enzyme. After enzymatic hydrolysis, a process design that couples rapid enzyme inactivation with hot extraction further enhances the dissolution efficiency of chlorogenic acid by inactivating residual enzyme activity and utilizing high temperature to strengthen the mass transfer process. In the purification stage, this invention employs a four-stage polar gradient elution process using a moderately polar macroporous adsorption resin. Based on the differences in adsorption-desorption behavior of chlorogenic acid isomers and coexisting impurities at different ethanol concentrations, it achieves graded collection of low-purity components, high-purity components, and refined components, meeting the application requirements of different purity levels in food additives, cosmetic raw materials, and pharmaceutical intermediates.

[0008] Compared with existing technologies, this invention has the following advantages: First, the two-stage enzymatic hydrolysis strategy achieves synergistic and efficient release of free and bound chlorogenic acid under all-aqueous conditions, with a total extraction yield of over 75% based on dry coffee raw materials, which is 18-25 percentage points higher than that of a single wall-degrading enzyme system. Second, the introduction of ferulic acid esterase increases the release rate of bound chlorogenic acid from less than 40% in traditional processes to over 85%, while avoiding the use of organic solvents and eliminating the risk of solvent residue in the product. Third, the four-stage polar gradient elution process can simultaneously obtain chlorogenic acid products of three purity levels in a single chromatography operation, with the purity of the refined component reaching over 95%. Fourth, the coupled design of rapid enzyme inactivation and hot extraction shortens the high-temperature exposure time, effectively inhibiting the isomerization and oxidative degradation of chlorogenic acid under alkaline or high-temperature conditions, and maintaining the proportion of 5-caffeoylquinic acid in the total chlorogenic acid at over 60%. Fifth, the entire extraction process uses water as the sole solvent, and the purification stage uses only food-grade ethanol as the eluent. The process is green and environmentally friendly, and the final product has no toxic or harmful solvent residues, meeting the requirements of food safety and pharmaceutical production quality management standards. Sixth, the method of this invention demonstrates good applicability and robustness to raw materials from different sources, such as Arabica green coffee beans, Robusta green coffee beans, and coffee grounds, which is conducive to industrial promotion and application and the high-value utilization of coffee processing by-products. Attached Figure Description

[0009] Figure 1 This is a schematic diagram of the process flow for the enzymatic extraction and fractionation purification method of coffee chlorogenic acid according to the present invention.

[0010] Figure 2 This is a scanning electron microscope image of the cell wall of green coffee bean powder before enzymatic hydrolysis.

[0011] Figure 3 This is a scanning electron microscope image of the cell wall of green coffee bean residue after two-stage enzymatic hydrolysis.

[0012] Figure 4 This is a kinetic curve showing the cumulative release of chlorogenic acid over time during the two-stage enzymatic hydrolysis process.

[0013] Figure 5 This is a chromatogram showing the change in absorbance of the eluent at 325 nm with elution volume during polar gradient elution of HPD-600 macroporous adsorption resin.

[0014] Figure 6 The bar chart shows the comparison of the total extraction yield of chlorogenic acid and the purity of the purified components in each example and the comparative example. Detailed Implementation

[0015] The technical solution of the present invention will be described in detail below with reference to specific embodiments. These embodiments are only used to illustrate the technical concept of the present invention and do not constitute a limitation on the scope of protection of the present invention. Unless otherwise stated, all reagents used in the following embodiments are of analytical grade, all water is deionized water, all percentage concentrations are mass percentages, and enzyme activity units U are defined as the amount of enzyme required to catalyze the hydrolysis of the corresponding substrate to release 1 μmol of reducing sugar per minute under optimal conditions for each enzyme.

[0016] Example 1: This example uses green Arabica coffee beans from Yunnan as raw material to illustrate in detail the complete operation process of the enzymatic extraction and fractionation purification of coffee chlorogenic acid according to the present invention, such as... Figure 1 As shown, the process includes five core stages in sequence: raw material crushing, two-stage enzymatic hydrolysis, rapid enzyme inactivation and hot extraction, microfiltration separation, and macroporous resin fractionation purification.

[0017] Dry Arabica green coffee beans are ground in a grinder and passed through 40-mesh and 100-mesh standard sieves to collect powder with a particle size of 0.15~0.38 mm for later use. 100 g of green coffee bean powder is weighed and placed in a 1000 mL glass reactor. 1200 mL of deionized water is added at a feed-to-liquid mass-to-volume ratio of 1:12. The mixture is stirred with a glass stirring rod to form a homogeneous suspension.

[0018] The first stage of enzymatic hydrolysis was performed as follows: Cellulase preparation (derived from *Trichoderma reesei*, enzyme activity 15000 U / g) and pectinase preparation (derived from *Aspergillus niger*, enzyme activity 8000 U / g) were weighed separately. Based on a cellulase to pectinase activity ratio of 2:1.5, the amount of cellulase added was 2.67 g (corresponding to approximately 400 U / g of cellulase contribution in dry material), and the amount of pectinase added was 2.81 g (corresponding to approximately 300 U / g of pectinase contribution in dry material), resulting in a total cellulase activity of 700 U / g of dry material. Both enzyme preparations were dissolved separately in a small amount of deionized water, combined, and added to the reactor at once. The pH of the system was adjusted to 4.8 using 0.1 mol / L citrate-sodium citrate buffer. The reactor was placed in a constant-temperature water bath shaker, with the temperature set at 48°C, the stirring speed at 80 r / min, and the enzymatic hydrolysis reaction time at 2 h. During enzymatic hydrolysis, cellulase degrades highly crystalline cellulose microfibrils into soluble oligosaccharide fragments by hydrolyzing the β-1,4-glycosidic bonds in the cellulose chains, while pectinase acts on the α-1,4-galacturonic acid bonds in pectin, causing the pectin network in the intercellular layer to depolymerize. The synergistic effect of these two enzymes leads to the gradual disintegration of the coffee cell wall structure from the outermost layer to the innermost layer. Free chlorogenic acid, physically encapsulated by the cell wall, rapidly dissolves into the aqueous phase due to a significant reduction in diffusion resistance. Figure 2As shown, scanning electron microscopy images of green coffee bean powder before enzymatic hydrolysis reveal a dense and smooth cell wall surface, with cellulose microfibrils and pectin matrix tightly interwoven to form a continuous barrier layer.

[0019] The second-stage enzymatic hydrolysis is as follows: After the first-stage enzymatic hydrolysis, without solid-liquid separation, 0.40 g of ferulic acid esterase (derived from Aspergillus niger, enzyme activity 250 U / g) is added directly to the reaction system (corresponding to an enzyme activity of approximately 100 U / g dry material) is added. The pH of the system is adjusted to 5.5 using a 0.5 mol / L sodium bicarbonate solution, while the water bath temperature is lowered to 40°C. The stirring speed is maintained at 80 r / min, and the enzymatic hydrolysis reaction continues for 1 h. Ferulic acid esterase belongs to the carbohydrate esterase family and can specifically recognize and cleave the ester bond between hydroxycinnamic acid compounds (including chlorogenic acid and ferulic acid) and the arabinofuranose residues on the arabinoxylan side chain. With the cell wall skeleton significantly disrupted in the first stage, the ferulic acid esterase introduced in the second stage can more fully access the exposed arabinoxylan-chlorogenic acid bond sites, releasing bound chlorogenic acid into the aqueous phase in a free state. The two stages of enzymatic hydrolysis were designed with different temperatures and pH levels. The slightly acidic and higher temperature conditions in the first stage favored the catalytic activity of cellulase and pectinase, while the slightly acidic and lower temperature conditions in the second stage matched the optimal activity window of ferulic acid esterase, while avoiding the oxidative degradation of chlorogenic acid at higher temperatures. Figure 3 As shown, scanning electron microscopy images of coffee residue after two-stage enzymatic hydrolysis reveal significant structural differences—numerous irregular pores and cracks appear on the cell wall surface, cellulose microfibril fragments are scattered on the matrix surface, the originally continuous barrier layer has been completely destroyed, and intracellular material release channels are clearly visible. Figure 4 As shown, the cumulative release of chlorogenic acid during the two-stage enzymatic hydrolysis process exhibits a two-stage growth curve: during the first stage of enzymatic hydrolysis (0~120 min), the release rate of chlorogenic acid is relatively fast, and the cumulative release reaches about 68% of the total release by 120 min, mainly due to the dissolution of free chlorogenic acid; after the addition of ferulic acid esterase in the second stage (120~180 min), the release curve shows a second upward inflection point, and the cumulative release reaches about 96% of the total release by 180 min, with the increase in this stage mainly coming from the directional release of bound chlorogenic acid.

[0020] The enzyme inactivation and extraction procedures are as follows: After the second stage of enzymatic hydrolysis, the reactor was directly transferred to a preheated steam heater, and the system temperature was rapidly raised to 95°C at a heating rate of no less than 15°C / min, and maintained for 3 minutes to complete the enzyme inactivation process. After enzyme inactivation, without cooling, the temperature was directly adjusted to 80°C and maintained for extraction for 40 minutes. During this extraction stage, the high temperature conditions enhanced the solvation ability of water molecules and the diffusion coefficient of chlorogenic acid molecules, which facilitated the further transfer of chlorogenic acid remaining inside the cell wall fragments into the liquid phase. After extraction, the suspension was passed through a 0.45 μm polyethersulfone microfiltration membrane while hot for solid-liquid separation. Approximately 1050 mL of clear filtrate was collected. The filtrate was dark brown, and the concentration of chlorogenic acid in it was determined to be 7.8 g / L.

[0021] The fractional purification procedure is as follows: Take HPD-600 medium polarity macroporous adsorption resin (specific surface area 620 m²) 2 500 mL of a 40 mm inner diameter glass chromatography column (with an average pore size of 9.2 nm) was packed into the column, with a resin bed height of approximately 400 mm. Before use, the column was soaked in 2 BV of 95% ethanol for 12 h, followed by rinsing with 3 BV of deionized water until the eluent had no alcohol odor. The filtrate was adjusted to pH 3.2 with 0.5 mol / L hydrochloric acid, and then loaded at a flow rate of 2.5 BV / h. During loading, the absorbance of the eluent at 325 nm was monitored. When the absorbance reached 5% of the loaded solution, it was considered to have reached breakthrough, and loading was stopped. After loading, the following elution operations were performed: first, the column was washed with 2.5 BV of pure water at a flow rate of 3 BV / h to remove water-soluble impurities such as sugars, amino acids, organic acids, and inorganic salts. This washing solution was discarded. Then, elute with 15% ethanol solution at 3.5 BV, flow rate 2.5 BV / h, and collect the eluent. This is the low-purity fraction, mainly containing chlorogenic acid and some co-soluble impurities of caffeic acid, ferulic acid, and catechins. Continue eluting with 45% ethanol solution at 4 BV, flow rate 2 BV / h, and collect the eluent. This is the high-purity fraction, in which chlorogenic acid is highly enriched. Finally, elute with 70% ethanol solution at 2.5 BV, flow rate 2 BV / h, and collect the eluent. This is the purified fraction, containing a less polar dicaffeoylquinic acid isomer and residual high-purity chlorogenic acid. Figure 5 As shown, the absorbance at 325 nm during the elution process exhibits three distinct elution peaks as the elution volume changes: the first broadened peak (approximately 1.5–4.5 BV) appears during the 15% ethanol elution stage, the highest and narrowest main peak (approximately 5–8 BV) appears during the 45% ethanol elution stage, and the third moderate-intensity elution peak (approximately 9–11 BV) appears during the 70% ethanol elution stage. The baseline separation between the three peaks is good, confirming that the polar gradient elution strategy can effectively achieve the fractional collection of chlorogenic acid components with different polarities.

[0022] The drying and finished product preparation procedures are as follows: The high-purity component eluent was placed in a rotary evaporator and concentrated under reduced pressure at 45°C and a vacuum of -0.08 MPa to recover ethanol, until the solid content was approximately 20%. The concentrate was then transferred to a spray dryer with an inlet air temperature of 140°C, an outlet air temperature of 65°C, a feed rate of 15 mL / min, and an atomization pressure of 0.2 MPa. Drying yielded 58.2 g of a pale yellow to light brown powder. High-performance liquid chromatography (HPLC) analysis showed that the total chlorogenic acid content in the high-purity component powder was 88.6%, of which 5-caffeoylquinic acid accounted for 63.5% of the total chlorogenic acid. The purified component underwent the same concentration and drying process to obtain 12.7 g of powder with a total chlorogenic acid content of 96.2%. After concentration and drying, the low-purity component yielded 15.3 g of powder with a total chlorogenic acid content of 38.2%. Although this component has low purity, it is rich in polyphenols such as chlorogenic acid, caffeic acid, and ferulic acid, and can be used directly as a food-grade antioxidant mixture for oil anti-oxidation or food preservation. Based on 100 g of green coffee beans, the total chlorogenic acid extraction yield of the high-purity and refined components was 78.3%. Gas chromatography analysis showed that the residues of methanol, ethyl acetate, and dichloromethane were all below the detection limit, and the ethanol residue was 0.02%, far below the limit stipulated by the national food safety standards. The product has a uniform color, good powder flowability, and solubility tests showed a solubility greater than 50 g / L in deionized water at 25°C, making it suitable for subsequent formulation processing.

[0023] Example 2: This example uses Robusta coffee grounds from Vietnam as raw material to investigate the applicability of the method of the present invention to coffee raw materials from different sources. The Robusta coffee grounds are a byproduct of commercial coffee extraction production lines, with a moisture content of approximately 55%. They are dried with hot air at 60°C until the moisture content is below 8%, then pulverized and passed through a 60-mesh standard sieve to collect powder with a particle size not greater than 0.25 mm.

[0024] Weigh 150 g of coffee grounds powder and place it in a 2000 mL reactor. Add 1500 mL of deionized water at a feed-to-liquid mass-to-volume ratio of 1:10 and mix well. The first-stage enzymatic hydrolysis conditions are as follows: cellulase addition corresponding to an enzyme activity of 300 U / g dry material, pectinase addition corresponding to an enzyme activity of 225 U / g dry material, total cell wall degradation enzyme activity of 525 U / g dry material, system pH adjusted to 4.6, temperature 50°C, stirring speed 100 r / min, and hydrolysis time 1.5 h. Since the cell walls of coffee grounds are damaged to some extent during hot water extraction, the hydrolysis time required compared to green coffee beans can be appropriately shortened.

[0025] The second-stage enzymatic hydrolysis conditions were as follows: ferulic acid esterase was added at an amount corresponding to an enzyme activity of 80 U / g dry material; the pH of the system was adjusted to 5.3; the temperature was lowered to 38°C; and the hydrolysis time was 1.5 h. While the proportion of bound chlorogenic acid in coffee grounds is lower than in green coffee beans, approximately 10%–18% of chlorogenic acid remains linked to residual cell wall polysaccharides via ester bonds. The addition of ferulic acid esterase effectively releases this portion of chlorogenic acid.

[0026] The enzyme inactivation conditions were the same as in Example 1. After enzyme inactivation, the mixture was extracted at 75°C for 60 min, and the filtrate of approximately 1300 mL was collected after filtration through a 0.45 μm microfiltration membrane. The chlorogenic acid concentration was determined to be 4.2 g / L. The fractional purification operation used the same HPD-600 resin and four-stage polar gradient elution program as in Example 1, with the loading flow rate adjusted to 3 BV / h. After purification and spray drying, the total chlorogenic acid content in the high-purity component powder was 82.4%, and the total chlorogenic acid content in the refined component powder was 95.8%. Based on 150 g of dry coffee grounds, the total chlorogenic acid extraction yield was 71.6%, indicating that the method of this invention also has good applicability to coffee grounds raw materials. It is worth noting that coffee grounds, as a major byproduct of coffee beverage processing, have a global annual production exceeding 6 million tons, still containing 1.5%–3.5% residual chlorogenic acid on a dry basis. Using the method of this invention for extraction and utilization not only achieves resource utilization and high-value transformation of waste but also significantly reduces raw material costs. In Example 2, the proportion of 5-caffeoylquinic acid in the purified component was 61.2% of the total chlorogenic acid, slightly lower than 63.5% in Example 1. This is presumably because some of the 5-caffeoylquinic acid had already undergone thermal conversion to other isomers during the initial hot water extraction process of the coffee grounds.

[0027] Example 3: In this example, Arabica coffee beans from Hainan and Robusta coffee grounds were mixed at a mass ratio of 1:1 as raw materials. The extraction effect of the method of the present invention under mixed raw material conditions was investigated, and the boundary conditions of enzymatic hydrolysis parameters were explored.

[0028] Weigh 75 g of Arabica green coffee bean powder and 75 g of Robusta coffee grounds powder, mix them thoroughly, and place them in a 2000 mL reactor. Add 2250 mL of deionized water at a feed-to-liquid mass-to-volume ratio of 1:15 and stir thoroughly. The first-stage enzymatic hydrolysis conditions were: a cellulase to pectinase activity ratio of 2:1.5, a total cellulase activity of 400 U / g dry material, a pH adjusted to 5.0, a temperature of 46°C, a stirring speed of 60 r / min, and a hydrolysis time of 2 h. The second-stage enzymatic hydrolysis conditions were: ferulic acid esterase addition of 50 U / g dry material, a pH adjusted to 5.8, a temperature of 42°C, and a hydrolysis time of 0.5 h.

[0029] Enzyme inactivation was performed by heating at 92°C for 5 min, followed by extraction at 85°C for 30 min. Approximately 2000 mL of the filtrate was collected after microfiltration, and the chlorogenic acid concentration was determined to be 3.9 g / L. Fractional purification followed the same procedure as in Example 1, with a loading flow rate of 1.5 BV / h. After purification and drying, the total chlorogenic acid content of the high-purity fraction was 79.5%, and the total chlorogenic acid content of the refined fraction was 95.1%, resulting in a total chlorogenic acid extraction yield of 68.2%. These results demonstrate that even under conditions where the enzymatic hydrolysis parameters are at their lower limits, the method of this invention can still achieve satisfactory extraction yields and product purity, confirming the robustness of the technical solution. Compared to Example 1, the total extraction yield of Example 3 decreased by approximately 10 percentage points, mainly because the release efficiency of bound chlorogenic acid decreased after the ferulic acid esterase addition was reduced from 100 U / g to 50 U / g. Furthermore, while the high liquid-to-solid ratio of 1:15 is beneficial for mass transfer, it also results in a low concentration of chlorogenic acid in the extract (3.9 g / L), increasing energy consumption in the subsequent concentration stage. Based on the results of Examples 1-3, the recommended operating conditions for industrial production are a liquid-to-solid ratio of 1:12, a total cell wall degrading enzyme activity of 600-700 U / g dry material, and a ferulic acid esterase addition of 80-100 U / g dry material.

[0030] Comparative Example 1 This comparative example uses the same green coffee beans and the same material-to-liquid ratio as Example 1, but the enzymatic hydrolysis process only uses a single-stage enzymatic hydrolysis treatment with cellulase and pectinase, without the addition of ferulic acid esterase. Specifically, the amount of cellulase and pectinase added and their enzyme activity ratio are the same as in the first stage of Example 1; the total cellulase activity is 700 U / g dry material; pH is 4.8; temperature is 48°C; hydrolysis time is 3 h (extended by 1 h compared to Example 1 to compensate for the lack of a second stage); and stirring speed is 80 r / min. After hydrolysis, enzyme inactivation, extraction, filtration, and fractionation purification are performed under the same conditions as in Example 1. The results show that the total chlorogenic acid content of the high-purity component is 84.1%, the total chlorogenic acid content of the refined component is 93.7%, and the total chlorogenic acid extraction yield is 57.8%. Compared with Example 1, the total extraction yield decreased by 20.5 percentage points, indicating that ferulic acid esterase has an irreplaceable contribution to the release of bound chlorogenic acid. Further alkaline hydrolysis analysis of the residue after enzymatic hydrolysis in Comparative Example 1 revealed that approximately 1.85 g / 100 g dry weight of bound chlorogenic acid remained, while the amount of bound chlorogenic acid remaining in the residue of Example 1 was only 0.42 g / 100 g dry weight. This comparative data directly demonstrates the efficient cleavage and release capacity of ferulic acid esterase for ester-linked chlorogenic acid from a material balance perspective. Furthermore, in Comparative Example 1, the total enzymatic hydrolysis time was extended to 3 h to compensate for the lack of a second-stage enzymatic hydrolysis, but the extended time did not significantly improve the yield. This indicates that the substrates of the cell wall degrading enzyme (cellulose and pectin) were essentially hydrolyzed within 2 h, and further extending the reaction time could not replace the specific ester bond cleavage function of ferulic acid esterase.

[0031] Comparative Example 2 This comparative example uses the same green coffee bean raw material as Example 1, but without any enzymatic hydrolysis, it directly employs hot water extraction. Specifically, 100 g of green coffee bean powder is weighed and added to 1200 mL of deionized water, and extracted in an 80°C water bath for 3 hours, stirring every 30 minutes. After extraction, the filtrate is collected by filtration. After the filtrate undergoes the same fractional purification process as in Example 1, the total chlorogenic acid content of the high-purity component is 80.5%, and the total chlorogenic acid content of the refined component is 91.2%, resulting in a total chlorogenic acid extraction yield of only 42.3%. Compared to Example 1, the total extraction yield decreased by 36.0 percentage points, and the purity of the refined component also decreased significantly, indicating that without enzymatic hydrolysis, a large amount of chlorogenic acid remains trapped by the cell wall structure and cannot be effectively dissolved. The residual chlorogenic acid content in the residue of Comparative Example 2 was determined to be 4.12 g / 100 g dry basis by alkaline hydrolysis, which is much higher than 0.42 g / 100 g dry basis in Example 1, further confirming the decisive role of enzymatic hydrolysis in the complete release of chlorogenic acid.

[0032] Comparative Example 3 This comparative example used the same green coffee beans and two-stage enzymatic hydrolysis as Example 1, but the purification step only involved a single-step elution with 50% ethanol, without fractionation. Specifically, the enzymatic hydrolysis, enzyme inactivation, and extraction were identical to those in Example 1. After loading the filtrate onto an HPD-600 resin column, it was washed with 2.5 BV of pure water to remove impurities, followed by a one-step elution with 5 BV of 50% ethanol solution. All eluents were combined, concentrated, and dried. The results showed that the total chlorogenic acid content in the obtained powder was 72.3%, and the total chlorogenic acid extraction yield was 76.8%. Although the total yield was close to that of Example 1, the product purity was only 72.3%, far lower than the 88.6% of the high-purity component and the 96.2% of the refined component in Example 1, indicating that fractionation elution is crucial for obtaining a high-purity product. Single-concentration elution results in the mixed collection of components with different polarities. The chlorogenic acid main peak is co-eluted with small molecule phenolic acid impurities such as caffeic acid, protocatechuic acid, and ferulic acid, as well as dicaffeoylquinic acid isomers, which cannot be separated by subsequent simple concentration and drying operations. Therefore, polarity gradient fractionation elution is a necessary technical means to obtain high-purity chlorogenic acid products.

[0033] Comparative Example 4 This comparative example uses the same raw materials and enzymatic hydrolysis system as Example 1, but replaces the two-stage enzymatic hydrolysis with a one-step enzymatic hydrolysis involving the simultaneous addition of three enzymes. Specifically, cellulase, pectinase, and ferulic acid esterase are added to the reaction system simultaneously, with the total enzyme activity remaining the same as the combined total of the two stages in Example 1. The pH is uniformly set at 5.0, the temperature at 45°C, and the hydrolysis time at 3 hours. The results show that the total chlorogenic acid extraction yield is 63.5%, a decrease of 14.8 percentage points compared to Example 1. The reason for this is that the catalytic efficiency of cellulase and pectinase decreases under pH 5.0 and 45°C conditions compared to their optimal conditions, while ferulic acid esterase is also not at its optimal activity under the same conditions. More importantly, when ferulic acid esterase is added simultaneously before the cell wall skeleton is fully disintegrated, insufficient substrate accessibility leads to a large amount of ferulic acid esterase failing to reach the deep ester bond sites and becoming inactive. This result confirms the rationality and necessity of the two-stage stepwise enzymatic hydrolysis strategy of this invention.

[0034] To further verify the advantages of the method of the present invention in terms of storage stability, an accelerated stability test was conducted on the purified component powder of Example 1. The powder was sealed in an aluminum foil bag and stored in a constant temperature and humidity chamber at 40°C and 75% relative humidity for 90 days. Samples were taken every 30 days to detect the total chlorogenic acid content and the proportion of 5-caffeoylquinic acid. The results showed that after 30 days of storage, the total chlorogenic acid content was 95.8% (initially 96.2%), after 60 days it was 95.3%, and after 90 days it was 94.7%, with a decrease of only 1.5 percentage points. The proportion of 5-caffeoylquinic acid decreased slowly from 63.5% to 61.8% during the 90-day storage period, a decrease of 1.7 percentage points, indicating that the chlorogenic acid product prepared by the method of the present invention has good chemical stability and storage tolerance. As a control, the total chlorogenic acid content of the hot water extract in Comparative Example 2 decreased from 91.2% to 86.5% after 90 days of storage under the same accelerated conditions, a decrease of 4.7 percentage points. It is speculated that the polysaccharide and protein impurities remaining in the hot water extract underwent Maillard reaction or oxidative condensation reaction with chlorogenic acid under high temperature and high humidity conditions.

[0035] The determination of chlorogenic acid content in all the above examples and comparative examples was performed using high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: an Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm); mobile phase A was 0.1% formic acid aqueous solution; mobile phase B was acetonitrile; the gradient elution program was as follows: from 0 to 5 min, phase B linearly increased from 5% to 15%; from 5 to 20 min, phase B linearly increased from 15% to 35%; from 20 to 25 min, phase B linearly increased from 35% to 50%; from 25 to 28 min, phase B decreased from 50% back to 5% and equilibrated to 30 min. The detection wavelength was 325 nm, the column temperature was 30°C, the injection volume was 10 μL, and the flow rate was 1.0 mL / min. Quantification was performed using a standard curve plotted with 5-caffeoylquinic acid standard (purity ≥98%, Sigma-Aldrich). The total chlorogenic acid content was calculated by summing the peak areas of the six isomers: 5-caffeoylquinic acid, 3-caffeoylquinic acid, 4-caffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid. The extraction yield was calculated as follows: the extraction yield equals the total mass of chlorogenic acid in the product divided by the theoretical total mass of chlorogenic acid in the raw material, multiplied by 100%. The theoretical total mass of chlorogenic acid in the raw material was obtained by combining the extracts after three ultrasonic-assisted extractions of equal amounts of raw material with methanol-water (volume ratio 7:3) at 60°C.

[0036] The method for determining the release rate of bound chlorogenic acid was as follows: equal amounts of material residue before and after enzymatic hydrolysis were respectively subjected to alkaline hydrolysis with 2 mol / L sodium hydroxide solution at room temperature for 4 h. After neutralization, the chlorogenic acid released by alkaline hydrolysis was extracted with methanol-water. The release rate of bound chlorogenic acid was equal to the difference between the amount of chlorogenic acid released by alkaline hydrolysis of the residue before enzymatic hydrolysis and the amount of chlorogenic acid released by alkaline hydrolysis of the residue after enzymatic hydrolysis, divided by the amount of chlorogenic acid released by alkaline hydrolysis of the residue before enzymatic hydrolysis, and then multiplied by 100%. In Example 1, the release rate of bound chlorogenic acid was 32.4% after the first stage of enzymatic hydrolysis, and reached 87.6% after both stages of enzymatic hydrolysis, demonstrating that ferulic acid esterase has a significant synergistic effect on the release of ester-linked chlorogenic acid.

[0037] Antioxidant activity was evaluated using the DPPH radical scavenging method and the ABTS cationic radical scavenging method. Sample solutions with a concentration of 0.1 mg / mL were prepared using the purified component, and the radical scavenging rates in both systems were determined according to standard procedures. The results showed that the DPPH radical scavenging rate of the purified component in Example 1 was 92.8%, and the ABTS cationic radical scavenging rate was 95.4%, both higher than the corresponding values ​​of the hot water extract product in Comparative Example 2 (DPPH scavenging rate 85.3% and ABTS scavenging rate 88.7%), indicating that enzymatic extraction can better preserve the biological activity of chlorogenic acid.

[0038] The experimental results are summarized and analyzed as follows:

[0039] The data in the table above clearly shows that Examples 1-3, employing the complete process route of the present invention—two-stage enzymatic hydrolysis plus four-stage polar gradient elution—achieved a total chlorogenic acid extraction yield of over 68%, a purity of over 95% for the purified components, and a proportion of 5-caffeoylquinic acid in the total chlorogenic acid of over 60%. Figure 6 As shown, the extraction yield and purity of each embodiment were significantly better than those of the comparative examples. Comparative Example 1 omitted the ferulic acid esterase stage, and the total extraction yield decreased by 20.5 percentage points compared to Example 1, demonstrating that the targeted release of bound chlorogenic acid is the key to improving the total yield. Comparative Example 2 did not use any enzymatic hydrolysis, and the total extraction yield was only 42.3%, further confirming the necessity of enzyme-assisted cell wall disruption for the efficient release of chlorogenic acid from coffee cell walls. Comparative Example 3 used a single concentration of ethanol for one-step elution. Although the total yield was close to that of Example 1, the product purity was only 72.3%, which could not meet the requirements for high-purity applications. Comparative Example 4 simultaneously added three enzymes for one-step enzymatic hydrolysis, and the total yield decreased by 14.8 percentage points compared to Example 1. This was because the difference in the optimal conditions of the enzyme system and insufficient substrate accessibility led to the ineffective consumption of ferulic acid esterase, verifying the necessity of the two-stage stepwise strategy.

[0040] The core innovation of this invention lies in constructing a phased synergistic enzymatic hydrolysis strategy of cell wall degrading enzyme and ferulic acid esterase in the time dimension. Its underlying mechanism can be understood from the following three levels.

[0041] From the perspective of cell wall structure disintegration, the cell wall of green coffee beans is composed of a dense three-dimensional network of three major polysaccharides: cellulose microfilaments, hemicellulose (mainly arabinoxylan), and pectin. The synergistic action of cellulase and pectinase in the first stage disrupts the integrity of the cellulose-pectin backbone, creating numerous cracks and channels in the cell wall. The key output of this stage is not only the rapid dissolution of free chlorogenic acid, but also the exposure of the arabinoxylan-chlorogenic ester cross-linking sites previously embedded in the backbone, providing the structural prerequisite for substrate accessibility for ferulic acid esterase in the second stage.

[0042] From the perspective of ester bond cleavage, ferulic acid esterase belongs to Group 8 carbohydrate esterases. Its active site contains the classic serine-histidine-aspartic acid catalytic triplet, enabling selective hydrolysis of the ester bond between the arabinoxylan side chain and hydroxycinnamic acid via a nucleophilic attack mechanism. In the coffee cell wall, chlorogenic acid is mainly linked to the polysaccharide backbone via an ester bond formed by the carboxyl group of the caffeoyl group and the hydroxyl group at the C-5 position of the arabinofuranose residue. Ferulic acid esterase precisely recognizes this ester bond and catalyzes its hydrolysis, releasing free chlorogenic acid and deacylated arabinose residues. The substrate specificity of this process ensures that the chlorogenic acid molecule does not undergo isomerization or degradation during release, thus maintaining a high proportion of 5-caffeoylquinic acid in the product.

[0043] From a fractional purification perspective, the adsorption mechanism of chlorogenic acid isomers by HPD-600 macroporous adsorption resin is mainly based on hydrophobic interactions and hydrogen bonding. Under loading conditions of pH 3.0–3.5, the carboxyl group (pKa approximately 3.6) in the chlorogenic acid molecule is in a partially protonated state, enhancing the overall hydrophobicity of the molecule and facilitating the establishment of hydrophobic interactions between it and the styrene-divinylbenzene backbone of the resin. As the ethanol concentration in the eluent increases, the polarity of the water-ethanol mixed solvent decreases progressively, competitively desorbing components with different hydrophobicities from the resin surface in sequence. Low-concentration ethanol first elutes the more polar monocaffeoylquinic acid isomer and co-soluble impurities; medium-concentration ethanol selectively enriches the main peak 5-caffeoylquinic acid; and high-concentration ethanol elutes the more strongly adsorbed dicaffeoylquinic acid derivatives. The differences in elution behavior among these three types of isomers stem from the varying contributions of the number and spatial configuration of caffeoyl groups in their molecules to the hydrophobic interaction. This molecular-level mechanism provides the physicochemical basis for the fractional purification process of this invention. In summary, this invention employs a four-pronged synergistic design: wall-degrading enzymes to remove physical barriers, ferulic acid esterases to cleave chemical bonds, rapid enzyme inactivation to inhibit thermal degradation, and gradient elution to achieve precise fractionation. Under all-aqueous green process conditions, it achieves the comprehensive and efficient release of coffee chlorogenic acid from both its free and bound states, and simultaneously prepares products of multiple purity levels in a single chromatography operation. This technical solution breaks the existing process's trade-off between extraction yield and product purity, providing a practical and feasible technical path for the industrial-scale high-value utilization of coffee-derived chlorogenic acid.

[0044] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A method for enzymatic extraction and fractional purification of chlorogenic acid from coffee, characterized in that, Includes the following steps: The coffee raw material was pulverized to a particle size of 0.15~0.45 mm. Deionized water was added as a solvent at a mass-to-volume ratio of 1:10~1:

15. After mixing, a two-stage enzymatic hydrolysis treatment was carried out. The first stage of enzymatic hydrolysis was the cell wall degradation stage. A mixed enzyme solution of cellulase and pectinase was added to the material system, wherein the enzyme activity ratio of cellulase to pectinase was 2:1.5, and the total enzyme addition was 400~800 U / g dry material based on enzyme activity. The pH of the system was adjusted to 4.6~5.0, and enzymatic hydrolysis was carried out at 46~50°C for 1.5~2 h, with continuous stirring at a speed of 60~100 r / min during the process. The second stage of enzymatic hydrolysis is the directional cleavage of ester bonds. Ferulic acid esterase is added to the first-stage hydrolysis system at a dosage of 50-150 U / g dry material. The pH of the system is adjusted to 5.3-5.8, and the temperature is lowered to 38-42°C. Enzymatic hydrolysis continues for 0.5-1.5 h. After hydrolysis, the system is heated at 92-98°C for 3-5 min for rapid enzyme inactivation. While still hot, it is extracted at 75-85°C for 30-60 min, and then filtered through a 0.45 μm microfiltration membrane to collect the filtrate. The filtrate is loaded onto a pretreated medium-polarity macroporous adsorption resin column at a flow rate of 1.5-3.5 BV / h. After adsorption saturation, the column is washed sequentially with 2-3 BV of pure water, eluted with 3-4 BV of 15% ethanol solution to collect the low-purity fraction, eluted with 3-5 BV of 45% ethanol solution to collect the high-purity fraction, and eluted with 2-3 BV of 70% ethanol solution. BV elution was used to collect the purified components; the high-purity components and the purified components were concentrated under reduced pressure to recover ethanol until the solid content was 15%~25%; the concentrate was spray-dried to prepare chlorogenic acid powder, with the inlet air temperature of spray drying being 130~150°C and the outlet air temperature being 60~70°C.

2. The method according to claim 1, characterized in that, The coffee raw material is selected from one or more of Arabica green coffee beans, Robusta green coffee beans, or coffee grounds.

3. The method according to claim 1, characterized in that, The cellulase is a cellulase preparation derived from Trichoderma reesei, with an enzyme activity of not less than 10,000 U / g; the pectinase is a pectinase preparation derived from Aspergillus niger, with an enzyme activity of not less than 5,000 U / g.

4. The method according to claim 1, characterized in that, The ferulic acid esterase is a ferulic acid esterase preparation derived from Aspergillus niger or Thermophilus pyriformis, with an enzyme activity of not less than 200 U / g, an optimal operating temperature of 35~45°C, and an optimal operating pH of 5.0~6.

0.

5. The method according to claim 1, characterized in that, The moderately polar macroporous adsorption resin is HPD-600 type resin, with a specific surface area of ​​550~700 m². 2 / g, with an average pore size of 8~10 nm, is activated by rinsing with 2 BV of 95% ethanol and 3 BV of deionized water before use.

6. The method according to claim 1, characterized in that, The rapid enzyme inactivation process uses direct steam heating with a heating rate of no less than 15°C / min. After enzyme inactivation, the process is immediately transferred to the extraction step, and the entire transfer time from enzyme inactivation to extraction does not exceed 2 minutes.

7. The method according to claim 1, characterized in that, Before loading the filtrate, the pH is adjusted to 3.0-3.5 to enhance the hydrophobic interaction between the phenolic hydroxyl groups in the chlorogenic acid molecules and the resin skeleton, thereby increasing the adsorption capacity.

8. The method according to claim 1, characterized in that, The low-purity fraction eluted with 15% ethanol contains 25% to 45% chlorogenic acid by mass, the high-purity fraction eluted with 45% ethanol contains 75% to 92% chlorogenic acid by mass, and the purified fraction eluted with 70% ethanol contains not less than 95% chlorogenic acid by mass.

9. The method according to claim 1, characterized in that, During the spray drying process, 5% to 10% by mass of maltodextrin is added to the concentrate as a wall material to improve the powder's flowability and storage stability.

10. The method according to claim 1, characterized in that, The chlorogenic acid powder prepared by the method has a total chlorogenic acid content of not less than 95%, of which 5-caffeoylquinic acid accounts for not less than 60% of the total chlorogenic acid, and the total extraction yield of chlorogenic acid based on dry coffee raw materials is not less than 75%.

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