Method for preparing fat-soluble acylated chlorogenic acid by enzyme method

Abstract

The invention discloses a method for preparing fat-soluble acylated chlorogenic acid through an enzyme method, and belongs to the technical field of immobilized enzyme development and fat-soluble antioxidant processing. The invention discloses a method for preparing fat-soluble acylated chlorogenic acid by an enzymic method, which comprises the following steps of: catalyzing chlorogenic acid and fatty acid vinyl ester to generate ester exchange reaction by using hydrophobic micron-sized silicon-based carrier immobilized lipase; acylated chlorogenic acid products with fat solubility and oxidation resistance, such as acetyl chlorogenic acid, butyryl chlorogenic acid, capryloyl chlorogenic acid and lauroyl chlorogenic acid, are directionally synthesized. According to the self-made immobilized lipase catalyst based on the hydrophobic micron-sized silicon-based carrier, the defects of unstable enzyme activity, difficulty in separation, low reuse efficiency and the like in the existing process for preparing fat-soluble acylated chlorogenic acid by an enzyme method are overcome; oriented, efficient and rapid synthesis of a series of acylated chlorogenic acid products with fat solubility and oxidation resistance is achieved, the preparation process is simple, safe and reliable, and the industrial application potential is huge.

Description

Technical Field

[0001] The present invention relates to the technical field of immobilized enzyme development and fat-soluble antioxidant processing, and more particularly to a method for preparing fat-soluble acylated chlorogenic acid by enzymatic method. Background Art

[0002] Chlorogenic acid (CA) is a dietary phenolic acid produced by many plants in nature, including fruits, vegetables and herbs. Studies have reported that chlorogenic acid can reduce the risk of many diseases in the human body and has multiple biological activities, such as antioxidant, antibacterial, antiviral, anti-tumor, anti-inflammatory, liver protection and hypoglycemic.

[0003] Chlorogenic acid is easily soluble in ethanol and acetone, and is also soluble in water, and its solubility increases with increasing temperature. The chlorogenic acid molecule itself has 5 hydroxyl groups and 1 carboxyl group, which results in good water solubility but poor fat solubility, and is difficult to dissolve in lipids. This property limits its application in oil-based products. Therefore, research and development of acylated chlorogenic acid products with good fat solubility has great application value and broad market prospects.

[0004] Generally speaking, the methods for preparing fat-soluble acylated chlorogenic acid mainly include chemical method and bioenzymatic method. The industrial development of chemical preparation is earlier and more mature, but a large amount of organic solvents and chemical catalysts are required in its production process, which will corrode equipment and cause environmental pollution, and there are many side reactions and complex subsequent treatment processes. Therefore, the green, efficient and mild reaction conditions bioenzymatic method for preparing fat-soluble acylated chlorogenic acid has attracted more and more attention from researchers.

[0005] Chinese invention patent CN102392057A discloses a method for synthesizing lipophilic chlorogenic acid fatty acid esters. First, ion exchange resin Amberlite IR120H and molecular sieves are added to a 0.035-0.05M methanol solution of chlorogenic acid, and the mixture is stirred at 50-55°C for 6-10 hours. After the reaction, the reaction solution is post-treated to obtain chlorogenic acid methyl ester; then octanol or dodecanol is added to the chlorogenic acid methyl ester, and Novozym 435 lipase is added. Under nitrogen protection, the mixture is stirred at 50°C for 48-76 hours. After the reaction, the reaction solution is separated and treated to obtain the chlorogenic acid fatty acid ester. The chlorogenic acid conversion rate can reach up to 92.3%.

[0006] Chinese invention patent CN112481314A discloses a method for preparing a lipophilic chlorogenic acid derivative with high inhibitory activity of amylase and glucosidase. During preparation, 100 mg of chlorogenic acid, 1 mL of vinyl laurate and 10 mL of methyl tert-butyl ether need to be added to the reaction bottle, and the amount of lipase Lipozyme RM added is 1.2 U. After reacting for 72 hours at 40 ° C and 400 rpm, the immobilized enzyme is removed by centrifugation, the solvent is removed by rotary evaporation, and the chlorogenic acid derivative product is obtained after vacuum drying. The chlorogenic acid conversion rate is as high as 76.1%. The product is identified as 4-O-laurylated chlorogenic acid by mass spectrometry and nuclear magnetic resonance.

[0007] Although the above two preparation methods can prepare lipophilic chlorogenic acid derivatives, the reaction time of the two methods is long, the conversion rate is low, and the catalyst is a foreign commercial enzyme, which is heavily dependent on imports. In addition, the process used in Chinese invention patent CN102392057A is complex and difficult to mass produce.

[0008] Therefore, providing a method for preparing fat-soluble acylated chlorogenic acid by enzymatic method is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0009] In view of this, in order to solve the defect of inefficient reaction commonly existing in the prior art, the present invention provides a method for preparing fat-soluble acylated chlorogenic acid by enzymatic method, that is, a method for catalyzing the synthesis of fat-soluble acylated chlorogenic acid by immobilizing lipase on a hydrophobic micron-sized silicon-based carrier; this method has the advantages of high reaction efficiency, simple operation, green and environmental protection, high selectivity, simple product separation and purification, and suitability for large-scale production.

[0010] In order to achieve the above object, the present invention adopts the following technical solution:

[0011] A method for preparing fat-soluble acylated chlorogenic acid by enzymatic method, the specific steps are as follows:

[0012] (1) dissolving free lipase in PBS buffer to obtain a lipase solution; adding a hydrophobic micron-sized silicon-based carrier to the lipase solution, incubating, centrifuging (immobilizing on the hydrophobic micron-sized silicon-based carrier by a multiple adsorption method), and freeze-drying to obtain immobilized lipase, with a solid loading of 100-400 mg / g and a solid loading rate of 10-60%; the hydrophobic micron-sized silicon-based carrier is a monodisperse porous silicon particle with uniform particles;

[0013] (2) adding chlorogenic acid and fatty acid vinyl ester to a reaction solvent, stirring at a constant temperature for a certain period of time, adding the immobilized lipase obtained in step (1) to carry out a transesterification reaction to obtain a crude fat-soluble acylated chlorogenic acid;

[0014] (3) The crude fat-soluble acylated chlorogenic acid obtained in step (2) is separated and purified by centrifuging at 1000-3000×g for 5-10 min or filtering using a funnel to obtain a supernatant, adding the supernatant to an organic solvent for extraction and filtering to obtain a precipitate, washing it with an organic solvent for more than three times, and finally drying to remove the residual solvent to obtain high-purity fat-soluble acylated chlorogenic acid.

[0015] Furthermore, the hydrophobic micron-sized silicon-based carrier in step (1) is a monodisperse, uniformly sized hexagonal or spherical particle; the average diagonal length of the hexagonal particles is 3-15 μm, and the thickness is 2-5 μm; the diameter of the spherical particles is 1-20 μm, and the specific surface area is 100-400 m 2 / g, the mesopore cavity size is 15-25nm, and the mesopore window size is 10-20nm.

[0016] Furthermore, the free lipase in step (1) is at least one of Candida rugosa lipase (CRL), Candida antarctica lipase (CALB), and Candida lipase (CSL).

[0017] Furthermore, the PBS buffer concentration in step (1) is 50 mM, and the pH is 5.0-9.0; the immobilization temperature of the free lipase is 20-50°C, and the time is 10-180 min; the protein content of the lipase solution is 2-10 mg / mL; and the ratio of the hydrophobic micron-sized silica-based carrier to the lipase solution is 1:20-100 (w / v, g / mL).

[0018] Furthermore, in step (2), the fatty acid vinyl ester is at least one of vinyl acetate, vinyl propionate, vinyl butyrate, vinyl caprylate, and vinyl laurate (when two or more are present, they can be mixed in any proportion); and the reaction solvent is at least one of acetonitrile, acetone, tert-butyl alcohol, isopropanol, tert-amyl alcohol, and methyl tert-butyl ether (when two or more are present, they can be mixed in any proportion).

[0019] Furthermore, in step (2), the molar ratio of chlorogenic acid to fatty acid vinyl ester is 1:1-100; the concentrations of chlorogenic acid and immobilized lipase in the reaction solvent are 5-160 mg / mL and 5-50 mg / mL, respectively; the temperature of the transesterification reaction is 30-70°C, and the time is 0.5-48h.

[0020] Furthermore, the organic solvent in step (3) is at least one of n-hexane, isooctane, methyl tert-butyl ether and petroleum ether (when two or more are used, they can be mixed in any proportion).

[0021] Furthermore, the drying method in step (3) is at least one of natural air drying at 1-40°C, vacuum freeze drying, nitrogen blowing drying, and vacuum oven drying.

[0022] Furthermore, the fat-soluble acylated chlorogenic acid is prepared by the method.

[0023] Furthermore, the fat-soluble acylated chlorogenic acid is used to improve the fat solubility and antioxidant activity of chlorogenic acid.

[0024] The preparation method of the hydrophobic micron-sized silicon-based carrier is as follows: KCl, block polyether F-127 and 1,3,5-trimethylbenzene are added to the HCl solution and stirred at room temperature for 12-24 hours. Then, tetraethyl silicate (TEOS) is added and stirred for 12-24 hours. Next, the solution is retained at 25°C for 12-24 hours, and then hydrothermally treated in an autoclave for 24 hours, the solid product is filtered out, washed with ethanol and dried to obtain a dry powder, which is placed in a muffle furnace and calcined for 4-12 hours to obtain a micron-sized silicon-based carrier (OMS). Finally, OMS and octadecyltrimethoxysilane (C 18 ), anhydrous toluene and triethylamine were reacted in a high pressure reactor for 24 hours. The solid after the reaction was collected by filtration, washed with ethanol, and dried in a forced air drying oven to obtain a hydrophobic micron-sized silica-based carrier (OMS-C 18 ).

[0025] The fat solubility of the purified acylated chlorogenic acid is increased, and its fat solubility in rapeseed oil is increased by 1.3-20 times relative to chlorogenic acid.

[0026] The purified acylated chlorogenic acid has antioxidant properties, with a DPPH free radical scavenging rate of 88.7%-93.7%, a hydroxyl free radical scavenging rate of 65.4%-85.5%, and an oxidation induction time in vegetable oil increased by 1.1-1.7 times.

[0027] After the reaction of chlorogenic acid and fatty acid vinyl ester to prepare fat-soluble acylated chlorogenic acid is completed, the immobilized lipase can be recovered and reused. After the immobilized lipase is separated by centrifugation or filtration, it can be used again in the next batch of reactions and can be reused for more than 10 times.

[0028] The fat-soluble acylated chlorogenic acid product is at least one of acetylchlorogenic acid, propionylchlorogenic acid, butyrylchlorogenic acid, octanoylchlorogenic acid, and lauroylchlorogenic acid.

[0029] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a method for preparing fat-soluble acylated chlorogenic acid by enzymatic method, which has the following beneficial effects:

[0030] (1) The present invention uses a template method to prepare a hydrophobic mesoporous SiO2 material with orderly pore distribution as an immobilized enzyme carrier, which can not only efficiently load lipase, but also significantly improve the activity and stability of lipase. The hydrolysis activity is increased by more than 1.3 times compared with the free enzyme, and after repeated use for 10 times, it still maintains more than 82.5% of the initial activity.

[0031] (2) On the basis of the above, the reaction conditions of the entire preparation process of the present invention are mild. Compared with the traditional chemical method for synthesizing fat-soluble acylated chlorogenic acid, this method does not use strong acid or strong alkaline chemical catalysts, and avoids the harsh reaction conditions of high temperature and high pressure, which is more green and safe. In addition, compared with the existing enzymatic method for synthesizing acylated chlorogenic acid, it has strong selectivity and high conversion rate, and is simple to separate and purify. Only by solvent extraction and drying, fat-soluble acylated chlorogenic acid with a purity of more than 97% can be obtained, which can meet the needs of different application scenarios.

[0032] (3) The immobilized lipase and preparation method of the present invention can be widely used to catalyze chlorogenic acid and various fatty acid vinyl esters. The products are diverse, including acylated chlorogenic acids such as acetylchlorogenic acid, butyrylchlorogenic acid, octanoylchlorogenic acid, and lauroylchlorogenic acid. The fat solubility and antioxidant activity of the prepared products are significantly improved, and the application potential in the food, medicine, cosmetics and other industries is huge. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0034] Figure 1 The accompanying figure is a schematic diagram of enzymatic synthesis of fat-soluble acylated chlorogenic acid;

[0035] Figure 2 The accompanying drawing is the H NMR spectrum of acetylchlorogenic acid in Example 1;

[0036] Figure 3 The accompanying figure is the H NMR spectrum of butyrylchlorogenic acid in Example 3;

[0037] Figure 4 The attached figure is the HPLC detection spectrum of chlorogenic acid and acylated chlorogenic acid in Examples 1-5;

[0038] Figure 5 The attached figure is the FT-IR spectrum of chlorogenic acid and acylated chlorogenic acid in Examples 1-5;

[0039] Figure 6 The accompanying drawings are diagrams showing the effects of fat-soluble chlorogenic acid prepared based on immobilized lipase on a hydrophobic micron-sized silicon-based carrier in the present invention on scavenging DPPH free radicals (a) and hydroxyl free radicals (b);

[0040] Figure 7 The attached figure shows the immobilized enzyme CSL@OMS-C in Example 6. 18The effect of repeated use of catalytic synthesis of acetylchlorogenic acid;

[0041] Figure 8 The attached figure shows the immobilized lipase CSL@OMS-C 18 Evaluation of temperature resistance (a) and solvent resistance (b) of the samples. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Candida rugosa lipase (CRL, lyophilized powder, 700 U / mg) was purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China); Candida antarctica lipase (CALB, lyophilized powder, 5000 LU / g) was purchased from Novozymes (Beijing, China); Candida sp. lipase (CSL, lyophilized powder, 20,000 U / g) was purchased from Beijing Kaitai New Century Biotechnology Co., Ltd. (Beijing, China).

[0044] Preparation of hydrophobic micron-sized silicon-based carrier: In 1L of 1M HCl solution, add 25g KCl, 10g block polyether F-127 and 12g 1,3,5-trimethylbenzene and stir at room temperature for 24h. Then, add 41.6g TEOS and stir for 24h. Next, retain the solution at 25°C for 24h, then hydrothermally treat it in an autoclave at 200°C for 24h, filter out the solid product, wash with ethanol and dry it to obtain a dry powder, which is placed in a muffle furnace and calcined at 600°C for 6h to obtain a micron-sized silicon-based carrier (OMS). Finally, 8.0g OMS and 2.7g octadecyltrimethoxysilane (C 18 ), 40 mL of anhydrous toluene and 66 μL of triethylamine were reacted in a high-pressure reactor at 130°C for 24 hours. After the reactor was completely cooled, the mixture in the reactor was taken out and filtered to collect the solid after the reaction, washed with anhydrous ethanol, and dried in a forced air drying oven for 24 hours to obtain a hydrophobic micron-sized silica-based carrier (OMS-C 18 ).

[0045] Schematic diagram of enzymatic synthesis of fat-soluble acylated chlorogenic acid is shown in Figure 1 .

[0046] Example 1

[0047] A method for preparing fat-soluble acylated chlorogenic acid by enzymatic method, comprising the following steps:

[0048] (1) Preparation of immobilized lipase: 0.1 g OMS-C was added to 10 mL of 5 mg / mL CSL enzyme solution (dissolved in 50 mM PBS, pH 9.0). 18 The mixture was incubated at 30°C in a shaker at 220 rpm for 60 min. The precipitate after centrifugation was frozen at -80°C for 6 h and then freeze-dried in a freeze dryer for 24 h to obtain the immobilized lipase CSL@OMS-C. 18 , the solid loading capacity is 200 mg / g, and the solid loading rate is 40%;

[0049] Calculation of solid loading and solid loading rate:

[0050] The BCA method was used to determine the protein content and calculate the immobilization amount and immobilization rate of lipase. The standard protein solution in the BCA kit was prepared into 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL protein standard solutions. 20 μL of target protein solution and 200 μL of BCA working solution were added to a 96-well plate, mixed evenly, and placed in an ELISA reader with a temperature controller. After incubation at 37°C for 30 minutes, the absorbance value at a wavelength of 562 nm was measured, and a protein standard curve was drawn. The calculation method of the immobilization amount and the immobilization rate is as follows:

[0051] Lipase immobilization capacity (mg / g) = ((C0-C)V) / m

[0052] Lipase immobilization rate (%) = ((C0-C) / C0) × 100%

[0053] Where: C0 is the initial protein concentration of the lipase solution (mg / mL); C is the protein concentration of the supernatant after lipase immobilization (mg / mL); V is the volume of the added enzyme solution (mL); m is the mass of the immobilized carrier (g).

[0054] (2) Enzymatic preparation of fat-soluble acylated chlorogenic acid: 100 mg of chlorogenic acid and 393 μL of vinyl acetate were added to 10 mL of tert-butyl alcohol and stirred in a magnetic stirrer at 60 °C for 60 min. Then, 200 mg of immobilized lipase CSL@OMS-C was added. 18 After stirring and reacting for 1 hour, the mixture was taken out for HPLC detection. The conversion rate of chlorogenic acid was 97.3%.

[0055] (3) Isolation and purification: The product obtained in step (2) was centrifuged at 3000×g for 10 min, the supernatant was removed, and then added to 5 volumes of n-hexane, the solid matter produced was collected by filtration, washed with n-hexane 3 times, and finally dried at room temperature using nitrogen purge to obtain the final product. The product was determined to be acetylchlorogenic acid (CA-C2) with a purity of 98.9%.

[0056] Example 2

[0057] A method for preparing fat-soluble acylated chlorogenic acid by enzymatic method, comprising the following steps:

[0058] (1) Preparation of immobilized lipase: 0.1 g OMS-C was added to 10 mL of 6 mg / mL CSL enzyme solution (dissolved in 50 mM PBS, pH 8.0). 18 The mixture was incubated at 30°C in a shaker at 220 rpm for 90 min. The precipitate after centrifugation was frozen at -80°C for 6 h and then freeze-dried in a freeze dryer for 24 h to obtain the immobilized lipase CSL@OMS-C. 18 , the solid loading capacity is 250 mg / g, and the solid loading rate is 41.6%;

[0059] (2) Enzymatic preparation of fat-soluble acylated chlorogenic acid: 100 mg of chlorogenic acid and 462 μL of vinyl propionate were added to 10 mL of tert-butyl alcohol and stirred in a magnetic stirrer at 60 °C for 60 min. Then, 200 mg of immobilized lipase CSL@OMS-C was added. 18 After continuing to stir the reaction for 1 hour, the mixture was taken out for HPLC detection. The conversion rate of chlorogenic acid was 97.8%;

[0060] (3) Isolation and purification: The product obtained in step (2) was centrifuged at 2000×g for 10 min, the supernatant was removed, and then added to 5 volumes of petroleum ether, the solid matter produced was collected by filtration, washed with petroleum ether 3 times, and finally dried naturally at room temperature to obtain the final product. The product was determined to be propionylchlorogenic acid (CA-C3) with a purity of 98.1%.

[0061] Example 3

[0062] A method for preparing fat-soluble acylated chlorogenic acid by enzymatic method, comprising the following steps:

[0063] (1) Preparation of immobilized lipase: 0.1 g OMS-C was added to 10 mL of 7 mg / mL CSL enzyme solution (dissolved in 50 mM PBS, pH 7.0). 18 The mixture was incubated at 30°C in a shaker at 220 rpm for 90 min. The precipitate after centrifugation was frozen at -80°C for 6 h and then freeze-dried in a freeze dryer for 24 h to obtain the immobilized lipase CSL@OMS-C. 18 , the solid loading capacity is 300 mg / g, and the solid loading rate is 42.8%;

[0064] (2) Enzymatic preparation of fat-soluble acylated chlorogenic acid: 100 mg of chlorogenic acid and 538 μL of vinyl butyrate were added to 10 mL of tert-butyl alcohol and stirred in a magnetic stirrer at 60 °C for 60 min. Then, 200 mg of immobilized lipase CSL@OMS-C was added. 18After stirring and reacting for 1 hour, the mixture was taken out for HPLC detection. The conversion rate of chlorogenic acid was 97.5%.

[0065] (3) Isolation and purification: The product obtained in step (2) was centrifuged at 3000×g for 10 min, the supernatant was removed, and then added to 5 volumes of methyl tert-butyl ether, the solid matter produced was filtered and collected, washed with methyl tert-butyl ether 3 times, and finally dried in a vacuum oven at 30° C. to obtain the final product. The product was determined to be butyrylchlorogenic acid (CA-C4) with a purity of 98.5%.

[0066] Structural identification of acetylchlorogenic acid (Example 1) and butyrylchlorogenic acid (Example 3): Nuclear magnetic resonance spectroscopy (H NMR) was performed using Bruker Avance III. 1 H NMR) was determined at a magnetic field of 400 MHz. Figure 2 and Figure 3 As shown, all substances are single-site substitution products.

[0067] Example 4

[0068] A method for preparing fat-soluble acylated chlorogenic acid by enzymatic method, comprising the following steps:

[0069] (1) Preparation of immobilized lipase: same as Example 1;

[0070] (2) Enzymatic preparation of fat-soluble acylated chlorogenic acid: 100 mg of chlorogenic acid and 813 μL of vinyl octanoate were added to 10 mL of tert-butyl alcohol and stirred in a magnetic stirrer at 60 °C for 60 min. Then, 200 mg of immobilized lipase CSL@OMS-C was added. 18 After stirring and reacting for 1 hour, the mixture was taken out for HPLC detection. The conversion rate of chlorogenic acid was 97.3%.

[0071] (3) Separation and purification: Same as in Example 1, the purity of octanoylchlorogenic acid (CA-C8) was 98.3%.

[0072] Example 5

[0073] A method for preparing fat-soluble acylated chlorogenic acid by enzymatic method, comprising the following steps:

[0074] (1) Preparation of immobilized lipase: same as Example 1;

[0075] (2) Enzymatic preparation of fat-soluble acylated chlorogenic acid: 100 mg of chlorogenic acid and 1095 μL of vinyl laurate were added to 10 mL of tert-butyl alcohol and stirred in a magnetic stirrer at 60 °C for 120 min to dissolve. Then, 200 mg of immobilized lipase CSL@OMS-C was added. 18After stirring and reacting for 1 hour, the mixture was taken out for HPLC detection. The conversion rate of chlorogenic acid was 97.1%.

[0076] (3) Separation and purification: Same as in Example 1, the purity of lauroyl chlorogenic acid (CA-C12) was 98.8%.

[0077] Test example

[0078] (I) High performance liquid chromatography (HPLC) detection conditions: chromatographic column: Venusil MP C18 (2) (5 μm, 4.6×250 mm), PDA detector, injection volume: 10 μL, column temperature: 40°C; mobile phase: methanol / 0.1% acetic acid aqueous solution (54:46, v:v), isocratic elution, flow rate: 0.7 mL / min, detection wavelength: 320 nm.

[0079] The HPLC detection patterns of chlorogenic acid (CA) and various products in Examples 1-5 are as follows: Figure 4 As shown, when detecting chlorogenic acid, acetylchlorogenic acid, propionylchlorogenic acid and butyrylchlorogenic acid, the volume ratio of the mobile phase methanol and 0.1% acetic acid aqueous solution is 54:46. It can be seen that the liquid chromatography peak of chlorogenic acid is located at 5.3min. When the product is detected after the reaction, the chromatographic peak of chlorogenic acid almost disappears, the peak position of acetylchlorogenic acid is located at 6.6min, the peak position of propionylchlorogenic acid is located at 9.1min, and the peak position of butyrylchlorogenic acid is 12.8min. When detecting octanoylchlorogenic acid and lauroylchlorogenic acid, the ratio is changed to methanol / 0.1% acetic acid aqueous solution (85:15, v:v), the peak position of octanoylchlorogenic acid is 9.4min, and the peak position of lauroylchlorogenic acid is 13.4min. The detection peaks are all single product peaks, and the separation effect is good.

[0080] (ii) Fourier transform infrared chromatogram (FT-RI) analysis: FT-RI (VERTEX33, Bruker) was tested using the potassium bromide tablet method in accordance with the provisions of GB / T6040. The FT-RI test results of chlorogenic acid (CA) and various products in Examples 1-5 are shown in Figure 2. Figure 5 As shown; after testing, the infrared spectrum of fat-soluble acylated chlorogenic acid was compared with that of original chlorogenic acid, 3620cm -1 The hydroxyl absorption peak of 2854cm -1 and 2926cm -1 The alkyl absorption peak of 1700cm -1 A strong ester carbonyl absorption peak appeared nearby, indicating that the lipid solubility of the new substance was enhanced and the acylation reaction between chlorogenic acid and acyl donor was successfully carried out. The reaction principle is mainly that the alcohol hydroxyl group of chlorogenic acid generates ester carbonyl, which is converted from a water-soluble group to a lipid-soluble group.

[0081] (III) Lipid solubility test: XLOGP3 online software (v3.2.2, www.sioc-ccbg.ac.cn) was used to calculate the XlogP value of each compound (the theoretical n-octanol-water partition coefficient calculated by the XLOGP3 method, representing lipophilicity). The actual solubility in rapeseed oil was determined by mixing 1g of rapeseed oil with 0.2g of chlorogenic acid, acetylchlorogenic acid, butyrylchlorogenic acid, octanoylchlorogenic acid and lauroylchlorogenic acid in a glass bottle, shaking at 37°C for 24h, and then centrifuging for 10min (2550×g) to separate insoluble chlorogenic acid or acylated chlorogenic acid. The solution was extracted with methanol and the supernatant was taken, then filtered using a 0.22μm nylon 66 disposable needle filter membrane, and the solubility concentration in rapeseed oil was determined by high performance liquid chromatography. The results are shown in Table 1.

[0082] Table 1 XlogP values ​​of chlorogenic acid and its acylated products and their solubility in rapeseed oil

[0083]

[0084] (IV) Antioxidant capacity test:

[0085] 1) In the DPPH free radical scavenging experiment, chlorogenic acid and its acylated products were first prepared into a 300 μM solution using 80% ethanol, and then 1 mL of DPPH solution (0.2 mM) and 1 mL of sample solution were fully mixed and incubated at 30°C in the dark for 30 min. The absorbance of the sample was then measured at 517 nm.

[0086]

[0087] Where A0 is the absorbance of 80% ethanol solution mixed with DPPH solution, and A1 is the absorbance of sample mixed with DPPH solution.

[0088] 2) Hydroxyl radical scavenging experiment was determined by using a hydroxyl radical test kit (Nanjing Jiancheng Bioengineering Institute, China) and following the instructions. Chlorogenic acid and acylated chlorogenic acid were dissolved in 80% ethanol to prepare a 1 mM solution as the sample solution. First, the substrate working solution (0.2 mL) and the sample solution (0.2 mL) were mixed with the hydroxyl radical solution (0.4 mL), respectively, and reacted at 37 ° C for 1 min. Next, a color developer (2 mL) was immediately added to the mixture and incubated at room temperature for 20 min. Finally, the absorbance was read at 550 nm.

[0089]

[0090] Wherein B0 and B1 are the absorbance values ​​of 80% ethanol solution and sample test respectively.

[0091] The results of DPPH free radical scavenging rate and hydroxyl free radical scavenging rate are shown in Figure 6 The antioxidant capacity of the modified acylated chlorogenic acid is comparable to or even better than that of chlorogenic acid itself. For example, the scavenging rate of hydroxyl radicals of lauroyl chlorogenic acid is 1.2 times that of chlorogenic acid.

[0092] 3) In the oxidation induction experiment, first, 60 mg of chlorogenic acid and its acylated derivatives were added to 10 mL of 80% ethanol to prepare a 6 g / L sample solution. Then, 100 μL of the sample solution was added to 3 g of vegetable oil for testing. The sample solution was replaced with 100 μL of 80% ethanol as a blank control. Finally, the sample was transferred to a test tube and heated in pure air at 110°C at a flow rate of 20 L / h, and the oxidation induction time of the oil sample was recorded. The results are shown in Table 2.

[0093] Table 2 Oxidation induction time of blank control, chlorogenic acid and its acylated products in different vegetable oils

[0094]

[0095]

[0096] Note: *Different letters represent significant differences (p<0.05).

[0097] Example 6

[0098] A method for preparing fat-soluble acylated chlorogenic acid by enzymatic method, comprising the following steps:

[0099] (1) Preparation of immobilized lipase: same as Example 1;

[0100] (2) Enzymatic preparation of fat-soluble acylated chlorogenic acid: 50 mg of chlorogenic acid and 197 μL of vinyl acetate were added to 10 mL of tert-butyl alcohol and stirred in a magnetic stirrer at 60 °C for 60 min to dissolve. Then, 100 mg of immobilized lipase CSL@OMS-C was added. 18 After continuing to stir the reaction for 2 hours, the mixture was taken out for HPLC detection. The conversion rate of chlorogenic acid was 99.5%.

[0101] The reaction solution was centrifuged at 6000 g and ambient temperature for 5 min, and the supernatant was discarded, and the precipitate was retained to separate and obtain the immobilized lipase CSL@OMS-C. 18 , refer to the operation of Example 6, and perform CSL@OMS-C 18 The conversion rate of chlorogenic acid was tested by HPLC. Figure 7As shown, it can be seen that the immobilized enzyme was reused 10 times, and the chlorogenic acid conversion rate after 2 hours of reaction was still maintained at 82.5%, so it has good reusability.

[0102] Temperature stability and solvent tolerance were evaluated using chlorogenic acid and vinyl acetate to prepare acetylchlorogenic acid (Example 6).

[0103] The temperature stability of free lipase CSL and immobilized CSL@OMS-C 18 The mixture was incubated in an oven at 120° C. for 0, 1, 2, and 3 h, and samples were taken and HPLC was used to detect the conversion rate of chlorogenic acid.

[0104] The solvent stability was evaluated by free lipase CSL and immobilized CSL@OMS-C 18 After incubation in toluene, chloroform, acetone and ethanol at 60°C for 2 h, the mixture was centrifuged and dried by nitrogen purge. The reaction was then carried out again. The conversion rate under the catalytic conditions of the unincubated lipase was taken as the initial activity and the relative activity was calculated. The reaction conditions were: tert-butyl alcohol (5 mL) as solvent, chlorogenic acid (50 mg), vinyl acetate (197 μL) and CSL@OMS-C 18 (100 mg) was reacted at 60°C with magnetic stirring at 400 rpm for 2 h. Figure 8 .

[0105] In the temperature stability evaluation, the immobilized fat can still maintain a high conversion rate under different incubation time, while the free enzyme shows a significant decrease ( Figure 8 a), indicating that immobilized lipase CSL@OMS-C 18 It has good stability and can provide good protection for lipase.

[0106] In the solvent stability test, free enzyme and immobilized lipase CSL@OMS-C in toluene 18 The relative activities of immobilized lipase and free lipase were similar, but in chloroform and acetone, the relative activity of immobilized lipase was higher than that of free lipase. In addition, in ethanol, although the relative activity of immobilized lipase was lower, it was still 18 times that of free lipase ( Figure 8 b) These indicate that immobilized lipase has higher solvent stability and can provide protection for lipase in different organic solvents.

[0107] The evaluation of temperature stability and solvent stability shows that the homemade immobilized lipase of the present invention has obvious advantages.

[0108] Example 7

[0109] A method for preparing fat-soluble acylated chlorogenic acid by enzymatic method, comprising the following steps:

[0110] (1) Preparation of immobilized lipase: same as Example 1;

[0111] (2) Enzymatic preparation of fat-soluble acylated chlorogenic acid: 100 mg of chlorogenic acid and 393 μL of vinyl acetate were added to 10 mL of acetone and stirred in a magnetic stirrer at 60 °C for 60 min. Then, 200 mg of immobilized lipase CSL@OMS-C was added. 18 After continuing to stir the reaction for 2 h, the mixture was taken out for HPLC detection. The conversion rate of chlorogenic acid was 95.5%.

[0112] (3) Separation and purification: Same as in Example 1, the purity of acetylchlorogenic acid (CA-C2) was 95.9%.

[0113] Example 8

[0114] A method for preparing fat-soluble acylated chlorogenic acid by enzymatic method, comprising the following steps:

[0115] (1) Preparation of immobilized lipase: same as Example 1;

[0116] (2) Enzymatic preparation of fat-soluble acylated chlorogenic acid: 100 mg of chlorogenic acid and 262 μL of vinyl acetate were added to 10 mL of tert-butyl alcohol and stirred in a magnetic stirrer at 70 °C for 60 min. Then, 200 mg of immobilized lipase CSL@OMS-C was added. 18 After continuing to stir the reaction for 2 hours, the mixture was taken out for HPLC detection. The conversion rate of chlorogenic acid was 95.8%.

[0117] (3) Separation and purification: Same as in Example 1, the purity of acetylchlorogenic acid (CA-C2) was 96.6%.

[0118] Example 9

[0119] A method for preparing fat-soluble acylated chlorogenic acid by enzymatic method, comprising the following steps:

[0120] (1) Preparation of immobilized lipase: same as Example 1;

[0121] (2) Enzymatic preparation of fat-soluble acylated chlorogenic acid: 100 mg of chlorogenic acid and 262 μL of vinyl acetate were added to a mixed solvent of 5 mL of tert-butyl alcohol and 5 mL of methyl tert-butyl ether, and stirred in a magnetic stirrer at 50 °C for 60 min. Subsequently, 200 mg of immobilized lipase CSL@OMS-C was added. 18 After continuing to stir the reaction for 4 hours, the mixture was taken out for HPLC detection. The conversion rate of chlorogenic acid was 93.1%.

[0122] (3) Separation and purification: Same as in Example 1, the purity of acetylchlorogenic acid (CA-C2) was 95.3%.

[0123] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing fat-soluble acylated chlorogenic acid by enzymatic method, characterized in that: The specific steps are as follows: (1) dissolving free lipase in PBS buffer to obtain a lipase solution; adding a hydrophobic micron-sized silicon-based carrier to the lipase solution, incubating, centrifuging, and freeze-drying to obtain immobilized lipase, wherein the immobilized amount is between 100 and 400 mg / g and the immobilized rate is between 10 and 60%; the hydrophobic micron-sized silicon-based carrier is a monodisperse porous silicon particle with uniform particles; (2) adding chlorogenic acid and fatty acid vinyl ester to a reaction solvent, stirring at a constant temperature for a certain period of time, adding the immobilized lipase obtained in step (1) to carry out a transesterification reaction to obtain a crude fat-soluble acylated chlorogenic acid; (3) The crude fat-soluble acylated chlorogenic acid obtained in step (2) is separated and purified by centrifuging at 1000-3000×g for 5-10 min or filtering using a funnel to obtain a supernatant, adding the supernatant to an organic solvent for extraction and filtering to obtain a precipitate, washing it with an organic solvent for more than three times, and finally drying to remove the residual solvent to obtain high-purity fat-soluble acylated chlorogenic acid.

2. The method for preparing fat-soluble acylated chlorogenic acid by enzymatic method according to claim 1, characterized in that: The hydrophobic micron-sized silicon-based carrier in step (1) is a monodisperse, uniformly sized hexagonal or spherical particle; the average diagonal length of the hexagonal particles is 3-15 μm, and the thickness is 2-5 μm; the diameter of the spherical particles is 1-20 μm, and the specific surface area is 100-400 m 2 / g, the mesopore cavity size is 15-25nm, and the mesopore window size is 10-20nm.

3. The method for preparing fat-soluble acylated chlorogenic acid by enzymatic method according to claim 1, characterized in that: The free lipase in step (1) is at least one of Candida rugosa lipase CRL, Candida antarctica lipase CALB and Candida lipase CSL.

4. The method for preparing fat-soluble acylated chlorogenic acid by enzymatic method according to claim 1, characterized in that: The PBS buffer concentration in step (1) is 50 mM, and the pH is 5.0-9.0; the immobilization temperature of the free lipase is 20-50° C., and the time is 10-180 min; the protein content of the lipase solution is 2-10 mg / mL; and the ratio of the hydrophobic micron-sized silica-based carrier to the lipase solution is 1:20-100 (w / v, g / mL).

5. The method for preparing fat-soluble acylated chlorogenic acid by enzymatic method according to claim 1, characterized in that: The fatty acid vinyl ester in step (2) is at least one of vinyl acetate, vinyl propionate, vinyl butyrate, vinyl caprylate, and vinyl laurate; and the reaction solvent is at least one of acetonitrile, acetone, tert-butyl alcohol, isopropanol, tert-amyl alcohol, and methyl tert-butyl ether.

6. The method for preparing fat-soluble acylated chlorogenic acid by enzymatic method according to claim 1, characterized in that: In step (2), the molar ratio of chlorogenic acid to fatty acid vinyl ester is 1:1-100; the concentrations of chlorogenic acid and immobilized lipase in the reaction solvent are 5-160 mg / mL and 5-50 mg / mL, respectively; the temperature of the transesterification reaction is 30-70°C, and the time is 0.5-48h.

7. The method for preparing fat-soluble acylated chlorogenic acid by enzymatic method according to claim 1, characterized in that: The organic solvent in step (3) is at least one of n-hexane, isooctane, methyl tert-butyl ether and petroleum ether.

8. The method for preparing fat-soluble acylated chlorogenic acid by enzymatic method according to claim 1, characterized in that: The drying method in step (3) is at least one of natural air drying at 1-40°C, vacuum freeze drying, nitrogen blowing drying, and vacuum oven drying.

9. The fat-soluble acylated chlorogenic acid prepared according to the method according to any one of claims 1 to 8.

10. Use of the fat-soluble acylated chlorogenic acid according to claim 9 in improving the fat solubility and antioxidant activity of chlorogenic acid.

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