Preparation method of curcumin and curcumin

Abstract

The invention belongs to the technical field of natural product extraction, and particularly relates to a curcumin preparation method and curcumin, the preparation method comprises the following steps: (1) turmeric pretreatment; (2) primary flash extraction; (3) performing variable-frequency ultrasonic enzymolysis; (4) performing secondary flash extraction and enzyme deactivation; (5) macroporous resin purification; and (6) concentrating and drying. The method has the beneficial effects that through the cooperation of two-stage flash extraction and variable-frequency ultrasonic enzymolysis, the effective extraction (containing wall breaking and enzymolysis) time is shortened to 20-30 minutes from 3-4 hours in the prior art. The data of the embodiment shows that the extraction yield of the curcumin reaches 5.42-5.45%, and the method is short in process time and low in energy consumption; the enzyme dosage is small; the action time is short; an ethanol-water system is adopted, so that safety and low toxicity are realized; the use of strong acid, strong base or surfactant is avoided, the product is purer, and the post-treatment is simple.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction technology, specifically relating to a method for preparing curcumin and curcumin itself. Background Technology

[0002] Turmeric is a plant belonging to the genus Curcuma in the family Zingiberaceae. Curcuma longa L Turmeric, the dried rhizome of turmeric, is a perennial herb mainly distributed in Asia, primarily in China and India. Studies have shown that turmeric possesses a wide range of pharmacological activities, including antitumor, anti-inflammatory, antioxidant, antiviral, and immunomodulatory effects. The main active ingredient in turmeric is curcumin, which can kill cancer cells and inhibit their proliferation, exhibiting good anticancer effects. However, the content of curcumin in turmeric is relatively low (usually about 3-6%), and it is encased in a dense cell wall and intercellular matrix composed of cellulose, hemicellulose, pectin, lignin, and lipids, resulting in low extraction efficiency, complex processing, and high energy consumption.

[0003] Existing methods for curcumin extraction mainly include: Organic solvent extraction: Ethanol, acetone, and other solvents are commonly used for reflux or immersion extraction. This method is simple to operate, but the extraction rate is low, solvent consumption is high, residues are easily left, and high-temperature, long-term extraction may lead to curcumin degradation.

[0004] Enzyme-assisted extraction: This method utilizes cellulase, pectinase, and other enzymes to disrupt cell walls. For example, Chinese invention patent application CN108147955A discloses a "flash extraction + compound enzymatic hydrolysis" process. However, its enzymatic hydrolysis step is an independent static process (3 hours), and it uses amylase and papain, which are not highly targeted to the main components of the turmeric cell wall. After enzymatic hydrolysis, high-temperature inactivation of the enzyme (usually above 85°C) is still required, which may destroy heat-sensitive curcumin. Subsequent purification requires complex crystallization steps.

[0005] Ultrasonic-assisted extraction: This method utilizes ultrasonic cavitation and mechanical effects to promote mass transfer. For example, Chinese invention patent application CN113387785A discloses "compound enzymatic hydrolysis + continuous flow ultrasonic extraction". However, it relies on continuous solvent flow and ultrasound, does not use high-speed mechanical shearing for physical cell disruption, and the enzymatic hydrolysis time is still as long as 3-4 hours, resulting in a long overall process time.

[0006] Flash extraction: This method uses high-speed shear force to instantly break down cells. However, when used alone, it often results in incomplete cell wall disruption and has limited impact on the deep cell wall structure.

[0007] In summary, the existing technologies have the following problems: (1) Single physical cell wall disruption (such as flash extraction) or biological enzymatic hydrolysis is difficult to completely destroy the complex cell wall structure of turmeric. (2) Static enzymatic hydrolysis is time-consuming and has low mass transfer efficiency; the enzyme system selection is not targeted at the optimal composition of the turmeric cell wall. (3) Multiple steps are connected in series, resulting in a long total time; the enzyme inactivation and purification steps are complex. (4) High-temperature enzyme inactivation or long-term extraction may lead to curcumin degradation.

[0008] Therefore, developing an integrated process that can efficiently, rapidly, and gently extract high-purity, highly active curcumin from turmeric has significant industrial value. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention aims to provide a highly efficient method for preparing curcumin. By creatively integrating two-stage flash extraction with variable frequency ultrasound coupled with enzymatic hydrolysis and designing a specific process sequence, the method achieves synergistic effects of cell wall disruption, enzymatic hydrolysis, enzyme inactivation, and extraction, thereby shortening the extraction time, increasing the extraction rate and product purity, and protecting the bioactivity of curcumin.

[0010] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: a method for preparing curcumin, comprising the following steps: (1) Turmeric pretreatment: After pulverizing turmeric, it is rinsed with a mixed solvent of petroleum ether and n-hexane, centrifuged and dried to obtain defatted turmeric powder; (2) First-stage flash extraction: The defatted turmeric powder is placed in a flash extractor and ethanol is added for high-speed shear extraction; (3) Variable frequency ultrasonic enzymatic hydrolysis: The material after primary flash extraction is subjected to primary low frequency ultrasonic treatment, and then compound enzyme is added for compound enzymatic hydrolysis under secondary high frequency ultrasonic conditions. (4) Two-stage flash extraction and enzyme inactivation: The material after frequency conversion ultrasonic enzymatic hydrolysis is subjected to two-stage flash extraction to achieve enzyme inactivation and extraction at the same time. After centrifugation, turmeric extract is obtained. (5) Macroporous resin purification: After diluting the turmeric extract, the sample was loaded onto a macroporous resin column for adsorption, washing and elution to obtain curcumin eluent. (6) Concentration and drying: The curcumin eluent is concentrated and dried to obtain curcumin powder.

[0011] Furthermore, in step (1) of the above method for preparing curcumin, the volume ratio of petroleum ether to n-hexane is 8:2.

[0012] As described above, optimizing the pretreatment solvent ratio and using a specific ratio of mixed solvents to synergistically remove oils and fat-soluble impurities from turmeric breaks down the physical encapsulation barrier of oils on curcumin, creating favorable conditions for subsequent ethanol extraction and improving the extraction rate.

[0013] Furthermore, in step (2) of the above-mentioned method for preparing curcumin, the ethanol is an aqueous solution with a volume fraction of 70-75%, and the ratio of defatted curcumin powder to ethanol is 1:(30-40) g / mL.

[0014] Furthermore, in steps (2) and (4) of the above-mentioned method for preparing curcumin, the flash extraction speeds are 5000~6000 r / min and 7500~8500 r / min, respectively.

[0015] As described above, the above-mentioned limitations further improve the key process parameters of the primary and secondary flash extraction, ensuring that the primary flash extraction initially breaks down and dissolves the cells, while the secondary flash extraction achieves deep cell disruption and rapid mechanical enzyme inactivation at a higher rotation speed, effectively avoiding the destruction of curcumin activity caused by traditional high-temperature enzyme inactivation.

[0016] Furthermore, in step (3) of the above-mentioned method for preparing curcumin, the frequency of the first-stage low-frequency ultrasound is 20 kHz, the frequency of the second-stage high-frequency ultrasound is 80~180 kHz, and the temperature of the compound enzymatic hydrolysis is 40~45℃, and the pH value is 5.5.

[0017] Furthermore, in step (3) of the above-mentioned method for preparing curcumin, the complex enzyme includes cellulase, pectinase and β-glucanase, with a mass ratio of (5~7):(2~4):1, and the total amount of complex enzyme added is 0.1~0.2% of the material mass.

[0018] As described above, the above-mentioned optimization of the complex enzyme formulation and ratio targeting the main components of turmeric cell walls (cellulose, pectin, and glucan) makes the enzyme system of the present invention more targeted, capable of more efficient and specific degradation of cell walls, and reducing ineffective enzymatic hydrolysis side reactions.

[0019] Furthermore, in step (5) of the above-mentioned method for preparing curcumin, before loading the macroporous resin for adsorption, the curcumin extract is diluted with pure water until the volume content of ethanol is ≤16%.

[0020] As described above, the key pretreatment steps before purification are defined. Reducing the ethanol concentration weakens the solubility of curcumin in the solvent, making it easier for the macroporous resin to adsorb, significantly improving the resin's adsorption capacity and purification efficiency. This is one of the key steps in obtaining high-purity products.

[0021] Furthermore, in step (5) of the above-mentioned method for preparing curcumin, the macroporous resin is a D301 type weakly basic anion exchange resin; the eluent is an 80-95% ethanol solution.

[0022] Furthermore, the total time for frequency-conversion ultrasonic enzymatic hydrolysis in step (3) of the above-mentioned method for preparing curcumin is 20-30 minutes.

[0023] Another technical solution of the present invention is to provide curcumin prepared by the above-mentioned method.

[0024] The beneficial effects of this invention are as follows: The method for preparing curcumin according to this invention has the following advantages: (1) By combining two-stage flash extraction with variable frequency ultrasonic enzymatic hydrolysis, the effective extraction time (including cell wall disruption and enzymatic hydrolysis) is shortened from 3-4 hours in the existing technology to 20-30 minutes. Example data shows that the curcumin extraction yield reaches 5.42-5.45%; (2) An optimized complex enzyme system (cellulase: pectinase: β-glucanase = 6:3:1) specifically degrades the cell wall of turmeric, reducing the dissolution of irrelevant impurities. Combined with the highly efficient selective adsorption of D301 macroporous resin at a specific ethanol concentration (≤16%), the purity of the obtained curcumin powder can reach more than 90%, and there is no need to go through complex crystallization and recrystallization steps.

[0025] (3) The two-stage flash extraction method with instantaneous mechanical enzyme inactivation effectively avoids the thermal degradation loss of curcumin caused by traditional high-temperature enzyme inactivation. The temperature is always controlled below 45℃ throughout the extraction process, maximizing the preservation of the biological activity of curcumin.

[0026] (4) The three-stage cell disruption strategy of “first-stage flash extraction (macroscopic cell disruption) + variable frequency ultrasonic enzymatic hydrolysis (microscopic cell disruption and biodegradation) + second-stage flash extraction (final cell disruption and enzyme inactivation)” has achieved a deep integration of physical, acoustic and biological methods and solved the technical bottleneck of incomplete cell disruption by a single method.

[0027] (5) The process time is short and the energy consumption is low; the amount of enzyme used is small and the action time is short; the ethanol-water system is adopted, which is safe and low in toxicity; the use of strong acids, strong alkalis or surfactants is avoided, the product is purer, and the post-processing is simple. Attached Figure Description

[0028] Figure 1 The effects of different types of resin on the adsorption and desorption rates of curcumin in specific embodiments of the present invention. Detailed Implementation

[0029] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments and accompanying drawings.

[0030] The key concept of this invention lies in: (1) A specific ratio of petroleum ether / n-hexane mixed solvent can effectively dissolve and remove volatile oils and lipids in turmeric, breaking the physical encapsulation of curcumin crystals by the oils, allowing the subsequent ethanol solvent to directly and fully contact curcumin. After this pretreatment, the efficiency of the first-stage flash extraction is greatly improved.

[0031] (2) First-stage flash extraction, as a "pretreatment," initially breaks down the material at the macroscopic level through high-speed shearing, increasing the specific surface area of ​​the particles and disrupting the integrity of some cell walls. This creates extremely favorable conditions for subsequent "variable-frequency ultrasonic enzymatic hydrolysis": ① a larger reaction contact surface; ② a shorter diffusion path for enzymes and solvents; ③ the damaged cell walls are more easily attacked by ultrasonic cavitation and enzymes. "Variable-frequency ultrasonic enzymatic hydrolysis" is the "fine processing": low-frequency ultrasound (20 kHz) acts like a "hammer," generating powerful cavitation bubble collapse and shock waves, promoting overall mixing and penetration of the medium; high-frequency ultrasound (100 kHz) acts like a "fine needle," generating dense microcavitation, finely "perforating" and "tearing" the cell walls, greatly enhancing the contact frequency and reaction efficiency between enzymes and cell wall components (cellulose, pectin, etc.). This "mechanical coarse breaking followed by ultrasonic-enzyme synergistic fine breaking" mode achieves full-scale cell wall disruption from macroscopic to microscopic.

[0032] (3) After enzymatic hydrolysis, the enzyme reaction needs to be terminated in time to prevent product degradation or interference with subsequent processes. This invention creatively utilizes high-speed two-stage flash extraction to achieve "flash enzyme inactivation". Under high-speed shearing of over 7500 r / min, the strong mechanical force and instantaneous temperature rise can rapidly and uniformly denature and inactivate the enzyme protein. Compared with the traditional 85℃ water bath inactivation for 20 minutes, this method is extremely short (<90 seconds) and the temperature is controllable (limited overall temperature rise), maximizing the protection of heat-sensitive curcumin. At the same time, the two-stage flash extraction performs a final "sweeping" crushing and extraction of the cell residues that have undergone ultrasonic enzymatic hydrolysis and have a highly relaxed structure at the moment of enzyme inactivation, efficiently transferring the curcumin released by the enzyme into the solvent.

[0033] (4) The preparation method of this application forms a continuous and efficient system of "degreasing and permeation enhancement - high-speed coarse crushing - ultrasonic enzymatic fine crushing - high-speed enzyme inactivation and final extraction". Compared with traditional static enzymatic hydrolysis (3 hours) or continuous flow ultrasonic extraction (>60 minutes), this invention compresses the core cell wall breaking-enzymatic hydrolysis-enzyme inactivation-extraction process to within 30 minutes, which improves efficiency by several times and significantly improves product yield and purity.

[0034] Example 1 A method for preparing curcumin, comprising the following steps: (1) Pulverize the turmeric, pass it through a 60-mesh sieve, and wash it with petroleum ether and n-hexane (8:2, v / v) at 45°C for 30 min. After centrifugation, vacuum dry it to obtain turmeric powder. (2) Add 70% ethanol to turmeric powder at a material-to-liquid ratio of 1:30 g / mL, and perform flash extraction at 5000 r / min for 90 s; then perform primary ultrasonic treatment for 10 min at an ultrasonic frequency of 20 kHz, adjust the temperature to 40 ℃, and the pH value to 5.5. Add 0.1% of cellulase, pectinase and β-glucanase (mass ratio 6:3:1) to the solution, adjust the pH value to 5.5, and adjust the ultrasonic frequency to 80 kHz. Perform secondary ultrasonic enzymatic hydrolysis for 20 min; then transfer to a flash extraction vessel, perform secondary flash extraction at 7500 r / min for 60 s to inactivate enzymes, and centrifuge to obtain turmeric extract; (3) Dilute the extract with pure water to ≤16% ethanol content, load it onto a D301 macroporous resin chromatography column for adsorption, wash with pure water, and then elute with 95% ethanol. Collect the eluent, concentrate under reduced pressure to recover ethanol, and obtain curcumin extract. (4) The curcumin extract was freeze-dried under vacuum to obtain curcumin powder.

[0035] Example 2 A method for preparing curcumin, comprising the following steps: (1) Pulverize the turmeric, pass it through a 60-mesh sieve, and wash it with petroleum ether and n-hexane (8:2, v / v) at 45°C for 30 min. After centrifugation, vacuum dry it to obtain turmeric powder. (2) Add 70% ethanol to turmeric powder at a material-to-liquid ratio of 1:30 g / mL, and perform flash extraction at 5000 r / min for 90 s; then perform primary ultrasonic treatment for 10 min at an ultrasonic frequency of 20 kHz, adjust the temperature to 45℃ and the pH value to 5.5, add 0.1% of cellulase, pectinase and β-glucanase (mass ratio 6:3:1), adjust the ultrasonic frequency to 180 kHz, and perform secondary ultrasonic enzymatic hydrolysis for 20 min; then transfer to a flash extraction vessel, perform secondary flash extraction at 7500 r / min for 60 s to inactivate enzymes, and centrifuge to obtain turmeric extract; (3) Dilute the extract with pure water to ≤16% ethanol content, load it onto a D301 macroporous resin chromatography column for adsorption, wash with pure water, and then elute with 95% ethanol. Collect the eluent, concentrate under reduced pressure to recover ethanol, and obtain curcumin extract. (4) The curcumin extract was freeze-dried under vacuum to obtain curcumin powder.

[0036] Example 3 A method for preparing curcumin, comprising the following steps: (1) Pulverize the turmeric, pass it through a 60-mesh sieve, and wash it with petroleum ether and n-hexane (8:2, v / v) at 45°C for 30 min. After centrifugation, vacuum dry it to obtain turmeric powder. (2) Add 70% ethanol to turmeric powder at a material-to-liquid ratio of 1:40 g / mL, and perform flash extraction at 5000 r / min for 90 s; then perform primary ultrasonic treatment for 10 min at an ultrasonic frequency of 20 kHz, adjust the temperature to 45℃, and the pH value to 5.5. Add 0.2% of cellulase, pectinase and β-glucanase (mass ratio 6:3:1) to the solution, adjust the ultrasonic frequency to 100 kHz, and perform secondary ultrasonic enzymatic hydrolysis for 20 min; then transfer to a flash extraction vessel, perform secondary flash extraction at 7500 r / min for 60 s to inactivate enzymes, and centrifuge to obtain turmeric extract; (3) Dilute the extract with pure water to ≤16% ethanol content, load it onto a D301 macroporous resin chromatography column for adsorption, wash with pure water, and then elute with 95% ethanol. Collect the eluent, concentrate under reduced pressure to recover ethanol, and obtain curcumin extract. (4) The curcumin extract was freeze-dried under vacuum to obtain curcumin powder.

[0037] Example 4 A method for preparing curcumin, comprising the following steps: (1) Pulverize the turmeric, pass it through a 60-mesh sieve, and wash it with petroleum ether and n-hexane (8:2, v / v) at 45°C for 30 min to remove the supernatant. After centrifugation, vacuum dry it to obtain turmeric powder. (2) Add 75% ethanol to turmeric powder at a material-to-liquid ratio of 1:40 g / mL, and perform flash extraction at 5000 r / min for 90 s; then perform primary ultrasonic treatment for 10 min at an ultrasonic frequency of 20 kHz, adjust the temperature to 40℃, and the pH value to 5.5. Add 0.2% of cellulase, pectinase and β-glucanase (mass ratio 6:3:1) to the solution, adjust the ultrasonic frequency to 100 kHz, and perform secondary ultrasonic enzymatic hydrolysis for 20 min; then transfer to a flash extraction vessel, perform secondary flash extraction at 7500 r / min for 60 s to inactivate enzymes, and centrifuge to obtain turmeric extract; (3) Dilute the extract with pure water to ≤16% ethanol content, load it onto a D301 macroporous resin chromatography column for adsorption, wash with pure water, and then elute with 95% ethanol. Collect the eluent, concentrate under reduced pressure to recover ethanol, and obtain curcumin extract. (4) The curcumin extract was freeze-dried under vacuum to obtain curcumin powder.

[0038] Comparative Experiment 1: (Selection of Raw Material Pretreatment) Referring to Example 1, step (1) is carried out according to the following process: ① Crush the turmeric, pass it through a 60-mesh sieve, and then proceed directly to step (2).

[0039] ② Crush the turmeric, pass it through a 60-mesh sieve, rinse it with petroleum ether at 45°C for 30 min, centrifuge it, and then vacuum dry it to obtain turmeric powder. Proceed with step (2).

[0040] ③ Crush the turmeric, pass it through a 60-mesh sieve, wash it with hexane at 45°C for 30 min, centrifuge it, and then vacuum dry it to obtain turmeric powder. Proceed with step (2).

[0041] ④ Crush the turmeric, pass it through a 60-mesh sieve, and wash it with petroleum ether and n-hexane (8:2, v / v) at 45°C for 30 minutes. After centrifugation, vacuum dry it to obtain turmeric powder. Proceed to step (2).

[0042] Method for determining curcumin: The method was slightly modified from that of Zhao Qianqian et al. (Zhao Qianqian, Xiao Zhiyong, Zhu Cuiping, Huang Meixia, Liu Qinghua, Wang Yinghao. Optimization of the ultrasonic-internal boiling method for curcumin extraction using Box-Behnken experimental design [J]. Journal of Liaoning University of Traditional Chinese Medicine, 2021, 23(12):36-40).

[0043] Chromatographic conditions: Eclipse XDB-C18 column (150×4.6 mm, 5 μm); mobile phase: acetonitrile-4% glacial acetic acid (48:52, V / V); flow rate: 0.8 mL·min -1 Column temperature: 30℃, detection wavelength: 430 nm; injection volume: 10 μL.

[0044] The yield of turmeric extract is calculated according to formula (1), the curcumin content in turmeric extract is calculated according to formula (2), the curcumin extraction yield is calculated according to formula (3), and the purity of curcumin powder is calculated according to formula (4).

[0045] (1) (2) (3) Table 1. Effects of different pretreatment methods on curcumin extraction efficiency of turmeric raw materials Turmeric raw materials (especially rhizomes) contain approximately 5-10% volatile oils (essential oils) and a large amount of fat-soluble impurities. These oils can encapsulate or dissolve curcumin crystals, forming a physical barrier that hinders the effective contact and penetration of solvents (such as ethanol and acetone) with curcumin, leading to a decrease in extraction rate. As shown in Table 1, both petroleum ether and n-hexane are beneficial to increasing the yield and curcumin content of turmeric extract, and have a certain synergistic effect.

[0046] Comparative Experiment 2: (Selection of Enzymatic Hydrolysis Process) Referring to Example 2, step (2) is carried out according to the following processes: ① Referring to "A method for extracting curcumin from ginger" (Invention Application Publication No. CN 108147955A, hereinafter referred to as Prior Art Document 1), the specific steps are as follows: pretreatment with 10 times the amount of NaOH solution, flash extraction for 3 min, then adding a complex enzyme consisting of 0.3% cellulase, 0.1% turmeric amylase and 1.0% turmeric papain by weight of the cell wall breaking solution and adjusting the pH to 3, enzymatic hydrolysis for 3 h, high temperature enzyme inactivation treatment to obtain the enzymatic hydrolysate, then adding sodium salicylate alcohol solution to the enzymatic hydrolysate, homogenizing at 6000 r / min for 6 min, then ultrasonic extraction twice, and centrifugation to obtain the turmeric extract.

[0047] ① Referring to "A method for extracting curcumin from ginger" (Invention Application Publication No. CN 108147955A, hereinafter referred to as Prior Art Document 1), the specific steps are as follows: pretreatment with 10 times the amount of NaOH solution, flash extraction for 3 min, then adding a complex enzyme consisting of 0.3% cellulase, 0.1% turmeric amylase and 1.0% turmeric papain by weight of the cell wall breaking solution and adjusting the pH to 3, enzymatic hydrolysis for 3 h, high temperature enzyme inactivation treatment to obtain the enzymatic hydrolysate, then adding sodium salicylate alcohol solution to the enzymatic hydrolysate, homogenizing at 6000 r / min for 6 min, then ultrasonic extraction twice, and centrifugation to obtain the turmeric extract.

[0048] ② Referring to Comparative Document 1, the static enzymatic hydrolysis step was placed between the two ultrasonic extraction steps. Specifically, the following steps were performed: 10 times the amount of NaOH solution was added for pretreatment, followed by flash extraction for 3 min. Then, the enzymatic hydrolysate was added to sodium salicylate alcohol solution and homogenized at 6000 r / min for 6 min. After the first ultrasonic extraction, a complex enzyme consisting of 0.3% cellulase, 0.1% turmeric amylase, and 1.0% turmeric papain was added to the cell wall disruption solution and the pH was adjusted to 3. After enzymatic hydrolysis for 3 h, the enzyme was inactivated by high temperature to obtain the enzymatic hydrolysate. Then, the second ultrasonic extraction was performed, and the turmeric extract was obtained by centrifugation.

[0049] ③ Referring to Comparative Document 1, the enzymatic hydrolysis step was placed between the two ultrasonic extraction steps, and the compound enzyme formula of the present invention was used. Specifically, the enzymatic hydrolysate was pretreated with 10 times the amount of NaOH solution, flash extracted for 3 min, then the enzymatic hydrolysate was added to sodium salicylate alcohol solution, homogenized at 6000 r / min for 6 min, and then subjected to the first ultrasonic extraction. Then, 0.1% of cellulase, pectinase and β-glucanase (mass ratio 6:3:1) were added to the cell wall breaking solution, and the pH was adjusted to 5.5. The temperature was adjusted to 45℃, and the enzymatic hydrolysis was carried out for 20 min. The enzyme was inactivated by high temperature to obtain the enzymatic hydrolysate, and then subjected to the second ultrasonic extraction. The extract was centrifuged to obtain the turmeric extract.

[0050] ④ Referring to "A method for extracting curcumin from turmeric using a combination of biological enzymatic method and continuous flow ultrasound-assisted extraction" (Invention Application Publication No. CN 113387785 A, hereinafter referred to as Prior Art Document 2), the specific steps are as follows: Take 100 g of fine turmeric powder, add 300 mL of pure water and stir evenly. Heat in a water bath at 60℃ for 60 min to obtain turmeric slurry. Add a compound enzyme to the turmeric slurry, based on 100 g of fine turmeric powder, wherein the added amounts of cellulase are 0.8%, hemicellulase is 0.8%, pectinase is 0.5%, and amylase is 0.7%. Adjust the pH of the enzymatic hydrolysis system to 4.2, and hydrolyze at 45℃ for 3 h. Then, treat the enzymatic hydrolysis mixture at 85℃ for 20 minutes. After inactivating the enzyme and cooling, the enzymatically hydrolyzed turmeric powder was obtained by filtration. The enzymatically hydrolyzed turmeric powder was placed in a continuous flow ultrasonic extraction device. The extraction solvent (prepared with Tween-80, ethanol and water in a volume ratio of 2:70:28) was continuously pumped into the ultrasonic generator at a flow rate of 0.5 mL / min. The amount of extraction solvent used was 15 times the mass of the enzymatically hydrolyzed turmeric powder. The ultrasonic extraction time was 60 min. The extracted liquid was pumped out every 5 min using a peristaltic pump. The extracted liquid was recycled twice. The extract was centrifuged to obtain turmeric extract.

[0051] ⑤ Referring to Comparative Document 2, the compound enzyme formula of this invention is used as follows: 100 g of turmeric powder is added to 300 mL of pure water and stirred evenly. The mixture is then placed in a water bath at 60°C for 60 min to obtain turmeric slurry. 0.1% (by weight) of cellulase, pectinase, and β-glucanase (by weight ratio 6:3:1) are added to the turmeric slurry. The pH is adjusted to 5.5, and the mixture is enzymatically hydrolyzed at 45°C for 3 h. The hydrolyzed mixture is then treated at 85°C for 20 min to inactivate the enzymes. After cooling, the mixture is filtered to obtain hydrolyzed turmeric powder. The hydrolyzed turmeric powder is placed in a continuous flow ultrasonic extraction device. The extraction solvent (prepared with Tween-80, ethanol, and water in a volume ratio of 2:70:28) is continuously pumped into the ultrasonic generator at a flow rate of 0.5 mL / min. The amount of extraction solvent used is 15 times the mass of the hydrolyzed turmeric powder. The ultrasonic extraction time is 60 min. After extraction, the liquid is extracted using a peristaltic pump every 5 minutes. The extract was pumped out at min, and the extract was recycled twice. The extract was then centrifuged to obtain turmeric extract.

[0052] Table 2. Effects of different enzymatic hydrolysis processes on curcumin extraction efficiency. As shown in Table 2, the present invention selects cellulase, pectinase, and β-glucanase, targeting the main components of the cell wall, and the ratio has been optimized (6:3:1) to better match the cell wall composition of turmeric plants, resulting in stronger targeting. Furthermore, the present invention is superior to ①-⑤ in terms of solvent selection, process continuity, avoidance of strong acid / alkali / high temperature damage, and rapid enzyme inactivation. Therefore, it can release curcumin from cells and dissolve it in the solvent more efficiently and completely, thereby increasing the yield and content of turmeric extract.

[0053] Comparative Experiment 3: (Selection of Extraction Mode) Referring to Example 3, step (2) is implemented in combination according to the following modes: ① Static enzymatic hydrolysis: Add 70% ethanol to turmeric powder at a material-to-liquid ratio of 1:40 g / mL, and perform flash extraction at 5000 r / min for 90 s; then perform primary ultrasonic treatment for 10 min at an ultrasonic frequency of 20 kHz, adjust the temperature to 45℃ and the pH value to 5.5, add 0.2% of cellulase, pectinase and β-glucanase (mass ratio 6:3:1) for enzymatic hydrolysis for 20 min; then adjust the ultrasonic frequency to 100 kHz, perform secondary ultrasonic treatment for 20 min, and then transfer to a flash extraction vessel. Perform secondary flash extraction at 7500 r / min for 60 s to inactivate enzymes, and centrifuge to obtain turmeric extract.

[0054] ② Non-frequency ultrasonic enzymatic hydrolysis: Add 70% ethanol to turmeric powder at a material-to-liquid ratio of 1:40 g / mL, and perform flash extraction at 5000 r / min for 90 s; then perform primary ultrasonic treatment for 10 min at an ultrasonic frequency of 20 kHz, adjust the temperature to 45℃ and the pH to 5.5, add 0.2% of cellulase, pectinase and β-glucanase (mass ratio 6:3:1), adjust the ultrasonic frequency to 20 kHz, and perform secondary ultrasonic enzymatic hydrolysis for 20 min. Then transfer to a flash extraction vessel, perform secondary flash extraction at 7500 r / min for 60 s to inactivate the enzymes, and centrifuge to obtain the turmeric extract.

[0055] ③ “Enzymatic hydrolysis + primary flash extraction + primary ultrasound + secondary ultrasound + secondary flash extraction” mode: Add 70% ethanol to turmeric powder at a material-to-liquid ratio of 1:40 g / mL, adjust the temperature to 45℃ and the pH to 5.5, add 0.2% (by mass) of cellulase, pectinase and β-glucanase (mass ratio 6:3:1) for enzymatic hydrolysis for 20 min; primary flash extraction at 5000 r / min for 90 s; primary ultrasound treatment for 10 min at an ultrasound frequency of 20 kHz; modulate the ultrasound frequency to 100 kHz, perform secondary ultrasound enzymatic hydrolysis for 20 min, then transfer to a flash extraction vessel, perform secondary flash extraction at 7500 r / min for 60 s to inactivate enzymes, and centrifuge to obtain turmeric extract.

[0056] ④ High-temperature enzymatic hydrolysis instead of two-stage flash extraction: Add 70% ethanol to turmeric powder at a material-to-liquid ratio of 1:40 g / mL, and perform flash extraction at 5000 r / min for 90 s; then perform primary ultrasonic treatment for 10 min at an ultrasonic frequency of 20 kHz, adjust the temperature to 45℃, and the pH value to 5.5. Add 0.2% (by weight) of cellulase, pectinase, and β-glucanase (by weight ratio of 6:3:1), adjust the ultrasonic frequency to 100 kHz, and perform secondary ultrasonic enzymatic hydrolysis for 20 min. After high-temperature enzyme inactivation treatment, obtain the enzymatic hydrolysate, and centrifuge to obtain the turmeric extract.

[0057] Table 3. Effects of different extraction modes on curcumin extraction efficiency As shown in Table 3, the extraction mode of "two-stage flash extraction + variable frequency ultrasonic enzymatic hydrolysis" is beneficial to improving the yield of turmeric extract and the curcumin content. At the same time, the two-stage 60s flash extraction treatment with high shear rate has an enzyme-inactivating effect, which can replace the traditional high-temperature enzyme inactivation treatment, thereby preventing the decomposition and destruction of curcumin by high-temperature enzyme inactivation and maintaining the biological activity of curcumin.

[0058] Comparative Experiment 4: (Selection of Macroporous Resin in Curcumin Purification Process) Referring to Example 4, the turmeric extract obtained after centrifugation in step (2) was processed according to the following procedures: ① Dilute the extract with pure water to an ethanol content ≤16%. The extract was then loaded onto Diaion HP-10, SPD100, D-101, AB-8 and D301 macroporous resin chromatography columns for adsorption. After washing with pure water, the extract was eluted with 95% ethanol. The eluent was collected after elution, and the concentration of curcumin in the eluent was measured. The adsorption capacity and desorption rate of the resin were then calculated.

[0059] ② The extract was not diluted with pure water to an ethanol content of ≤16%. The extract was directly loaded onto a D301 macroporous resin chromatography column for adsorption, followed by washing with pure water and then elution with 95% ethanol. After elution, the eluent was collected, and the concentration of curcumin in the eluent after desorption was measured. The adsorption capacity and desorption rate of the resin were then calculated.

[0060] The adsorption capacity and desorption rate of macroporous resins are calculated using the following formulas: In the formula: Q – Adsorption capacity (mg / g); R—Adsorption rate (%) D—Desorption rate (%) C0—Initial concentration of the sample solution before adsorption (mg / mL); V0 — Volume of sample solution during adsorption (mL); C1—Concentration of the sample solution after adsorption (mg / mL); V1 — Volume of the eluent (mL); C2 — Concentration of the eluent after desorption from macroporous resin (mg / mL); W – Mass of macroporous resin (g).

[0061] Polarity is an important consideration when selecting a suitable resin. Non-polar resins with strong hydrophobic pores and no functional groups are suitable for adsorbing non-polar substances. For example... Figure 1 As shown, when the ethanol content of the extract is reduced to below 16%, the D301 resin exhibits the best performance, with the highest adsorption capacity (70.15 mg / g) and is extremely easy to elute (87.12%). However, if the ethanol content of the extract is not reduced and the sample is directly loaded, the adsorption capacity of curcumin decreases by 11.9%.

[0062] Furthermore, referring to Example 4, the eluents obtained from processes ① and ② were collected and concentrated under reduced pressure to recover ethanol, yielding curcumin extract. The curcumin extract was then freeze-dried under vacuum to obtain curcumin powder. The purity of the curcumin powder was then tested. Further referring to Comparative Document 1, the curcumin powder was crystallized once using a mixed solvent of ethanol and water, and then freeze-dried to obtain curcumin primary crystals. The purity of the curcumin powder was calculated according to formula (4).

[0063] (4) Table 4. Effects of different treatment methods and resin types on curcumin purity Note: 1. Purity of curcumin powder; 2. Purity of curcumin powder after primary crystallization. Table 4 shows that the D301 resin exhibits significantly better separation and purification effects for curcumin than D-101, AB-8, HP-10, and SPD100. The curcumin powder prepared using the D301 resin has the highest quality, achieving a purity comparable to the primary crystals of curcumin prepared in Comparative Document 1. Furthermore, it is further demonstrated that reducing the ethanol content of the extract to below 16% is beneficial for the adsorption of curcumin by the D301 resin, resulting in a 16.75% increase in curcumin powder quality.

[0064] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention's specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing curcumin, characterized in that, Includes the following steps: (1) Turmeric pretreatment: After pulverizing turmeric, it is rinsed with a mixed solvent of petroleum ether and n-hexane, centrifuged and dried to obtain defatted turmeric powder; (2) First-stage flash extraction: The defatted turmeric powder is placed in a flash extractor and ethanol is added for high-speed shear extraction; (3) Variable frequency ultrasonic enzymatic hydrolysis: The material after primary flash extraction is subjected to primary low frequency ultrasonic treatment, and then compound enzyme is added for compound enzymatic hydrolysis under secondary high frequency ultrasonic conditions. (4) Two-stage flash extraction and enzyme inactivation: The material after frequency conversion ultrasonic enzymatic hydrolysis is subjected to two-stage flash extraction to achieve enzyme inactivation and extraction at the same time. After centrifugation, turmeric extract is obtained. (5) Macroporous resin purification: After diluting the turmeric extract, the sample was loaded onto a macroporous resin column for adsorption, washing and elution to obtain curcumin eluent. (6) Concentration and drying: The curcumin eluent is concentrated and dried to obtain curcumin powder.

2. The method for preparing curcumin according to claim 1, characterized in that, In step (1), the volume ratio of petroleum ether to n-hexane is 8:

2.

3. The method for preparing curcumin according to claim 1, characterized in that, In step (2), the ethanol is an aqueous solution with a volume fraction of 70-75%, and the ratio of defatted turmeric powder to ethanol is 1:(30-40)g / mL.

4. The method for preparing curcumin according to claim 1, characterized in that, The flash extraction speeds in steps (2) and (4) are 5000~6000 r / min and 7500~8500 r / min, respectively.

5. The method for preparing curcumin according to claim 1, characterized in that, In step (3), the frequency of the first-level low-frequency ultrasound is 20 kHz, the frequency of the second-level high-frequency ultrasound is 80~180 kHz, the temperature of the compound enzymatic hydrolysis is 40~45℃, and the pH value is 5.

5.

6. The method for preparing curcumin according to claim 1, characterized in that, In step (3), the complex enzyme includes cellulase, pectinase and β-glucanase, with a mass ratio of (5~7):(2~4):1, and the total amount of complex enzyme added is 0.1~0.2% of the material mass.

7. The method for preparing curcumin according to claim 1, characterized in that, In step (5), before loading the macroporous resin for adsorption, the turmeric extract is diluted with pure water until the volume content of ethanol is ≤16%.

8. The method for preparing curcumin according to claim 1, characterized in that, In step (5), the macroporous resin is a D301 type weakly basic anion exchange resin; the eluent is an 80-95% ethanol solution.

9. The method for preparing curcumin according to claim 1, characterized in that, In step (3), the total time for frequency conversion ultrasonic enzymatic hydrolysis is 20-30 minutes.

10. Curcumin prepared by the method of any one of claims 1 to 9.

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