A method for preparing sesamol by enzymatic hydrolysis of sesamin phenyl glycoside using immobilized enzyme

Abstract

This invention relates to an immobilized β-galactosidase and its enzymatic hydrolysis of sesaminol glycosides to prepare sesaminol, comprising: 1) preparing magnetic Fe3O4 nanoparticles; 2) preparing magnetic mesoporous Fe3O4@SiO2; 3) preparing amino-functionalized mesoporous Fe3O4@SiO2; 4) preparing glutaraldehyde-crosslinked amino-functionalized mesoporous Fe3O4@SiO2; 5) immobilizing β-galactosidase with the glutaraldehyde-crosslinked amino-functionalized mesoporous Fe3O4@SiO2 prepared in step 4); 6) enzymatically hydrolyzing sesaminol glycosides with the immobilized β-galactosidase prepared in step 5) to prepare sesaminol. Compared with simply using free enzymes, the method of this invention has the advantages of recyclability and being green and pollution-free; compared with other β-galactosidase immobilization methods, the method of this invention has many advantages such as high enzyme loading capacity, stable operation, and reusability. At the same time, the method of this invention has the advantages of being green and efficient, and having high added value, providing a new method for the industrial preparation of sesaminol, and has extremely high market promotion value.

Description

Technical Field

[0001] The method of this invention belongs to the field of agricultural product deep processing technology, specifically relating to a method for preparing sesaminol by enzymatic hydrolysis of sesaminol glycosides using immobilized enzymes. Background Technology

[0002] Sesame is one of the world's major traditional high-quality oilseeds with a long history of cultivation. my country is the world's largest producer of sesame, earning it the title of "Sesame Kingdom." Currently, domestic sesame processing focuses on sesame oil production, with 75% of sesame seeds used for oil extraction. This process generates a large amount of sesame cake, estimated at over 500,000 tons annually in my country. Due to limitations in processing technology, domestic sesame cake is primarily used as animal feed and fertilizer, and this resource is not being fully and rationally utilized. Therefore, achieving the resource utilization of sesame cake has become an urgent industrial problem to be solved.

[0003] Studies have shown that sesame cake, in addition to being rich in protein, also contains a certain amount of sesamin glycosides. Sesamin glycosides are a type of water-soluble sesame lignan (phenolic) compound, including sesamin monoglycosides, sesamin disaccharides, and sesamin triglycosides, with a content of approximately 0.68 mg / g sesame. They exhibit weak antioxidant activity in vitro. However, research indicates that sesamin glycosides can be converted into sesaminol through chemical or biological methods. Sesaminol possesses high thermal stability, outstanding antioxidant capacity, and other beneficial physiological activities. Furthermore, due to its colorless and odorless nature, it is suitable as a food antioxidant to improve food stability and extend shelf life. It also exhibits synergistic effects with other antioxidants. Therefore, it is essential to convert sesamin glycosides into the more active sesaminol and to conduct research on its application in food systems.

[0004] Compared with traditional chemical conversion methods, enzymatic conversion methods are safer, with mild and controllable reaction conditions, effectively reducing the amount of organic solvents used. Studies have shown that β-galactosidase can effectively hydrolyze sesamin glycosides to prepare sesaminol. However, β-galactosidase is synthesized by microorganisms, has a low secretion rate, and high extraction costs, resulting in expensive commercially available β-galactosidase. Furthermore, free β-galactosidase is easily inactivated and has poor stability, which is not conducive to industrial promotion.

[0005] Compared to free enzymes, immobilized enzymes are not only reusable but also easier to separate from the reaction system. Furthermore, immobilized enzymes can be processed continuously, shortening reaction time and reducing costs, and are widely used in industry. Currently, carriers used in β-galactosidase immobilization include sodium alginate, resin, chitosan, and cellulose. However, these materials have drawbacks such as difficulty in separation. Some researchers have also used magnetic nanomaterials such as Fe3O4 as carriers. Fe3O4 magnetic nanoparticles are widely used in the field of immobilized enzymes due to their advantages such as large specific surface area, low mass transfer resistance, good biocompatibility, and superparamagnetism. However, Fe3O4 magnetic nanoparticles (MNPs) are prone to oxidation, corrosion, aggregation, or precipitation, often requiring surface coating modification. Therefore, improving their stability remains a major challenge.

[0006] With the continuous interdisciplinary development of biotechnology and materials science, new carrier modification methods and novel materials are constantly emerging, and magnetic mesoporous silica (Fe3O4@mSiO2) composite materials have come into being. Fe3O4@mSiO2 is a magnetic silicon-based composite material with magnetic Fe3O4 as the core and mesoporous SiO2 as the shell. This composite material not only prevents the oxidation and aggregation of Fe3O4, but also exhibits excellent dispersibility and rapid magnetic separation performance, and has a large specific surface area, which can improve enzyme loading. Furthermore, its surface contains abundant hydroxyl groups, making it easy to modify. Therefore, combining this composite material with β-galactosidase for the enzymatic hydrolysis of sesaminol offers advantages such as high production efficiency, controllable cost, energy saving, and ease of scale-up, making it suitable for industrial promotion and possessing high application research value. Summary of the Invention

[0007] The purpose of this invention is to overcome the defects of the prior art and provide a method for preparing sesaminol by enzymatic hydrolysis of sesaminol glycosides using immobilized enzymes. This method not only avoids the reagent residue problem existing in chemical processing methods, but also has the advantages of high production efficiency, controllable cost, green energy saving, easy scale-up, and high safety and added value of the obtained product, making it suitable for industrial promotion.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing sesaminol by enzymatic hydrolysis of sesaminol glycosides using immobilized enzymes, comprising the following steps: 1) Preparation of magnetic Fe3O4 nanoparticles; 2) Preparation of magnetic mesoporous Fe3O4@SiO2; 3) Preparation of amino-functionalized mesoporous Fe3O4@SiO2: An appropriate amount of magnetic mesoporous Fe3O4@SiO2 was mixed with ethanol, concentrated ammonia and (3-aminopropyl)triethoxysilane (APTES) to obtain a suspension. The suspension was heated at 75-85℃ and mechanically stirred for 8-12 h. Then, the amino-functionalized mesoporous Fe3O4@SiO2 was obtained by magnetic separation. After washing and drying (vacuum drying at 45-60℃ for 8-24 h), it was ready for use. 4) Preparation of glutaraldehyde-crosslinked amino-functionalized mesoporous Fe3O4@SiO2: A certain amount of amino-functionalized mesoporous Fe3O4@SiO2 was ultrasonically dispersed with an appropriate amount of glutaraldehyde solution (10-30 min), and then shaken at a constant temperature for several hours. After the reaction was completed, it was washed with water until neutral to obtain glutaraldehyde-crosslinked amino-functionalized mesoporous Fe3O4@SiO2 (Fe3O4@SiO2-NH2-glutaraldehyde), which was then kept at 4 (±1) ℃ for later use as a carrier for β-galactosidase immobilization. 5) Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase: A suitable concentration of β-galactosidase solution was mixed with a certain amount of glutaraldehyde-crosslinked amino-functionalized mesoporous Fe3O4@SiO2, and then immobilized and shaken at a constant temperature for several hours to obtain Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase. It was frozen at -20 ~ -40℃ for 2-6 hours, and then freeze-dried in a vacuum freeze dryer for 12-24 hours. It was then stored in a -20℃ freezer for later use. 6) Enzymatic preparation of sesaminol: Weigh an appropriate amount of Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase and mix it with sesaminol glycoside solution. After reacting at a constant temperature and shaking for several hours, remove and recover the Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase by magnetic adsorption. Centrifuge the remaining reaction solution and collect the precipitate. Wash it with deionized water 2-4 times to remove water-soluble impurities. Extract it with anhydrous ethanol by ultrasonic assistance 1-3 times to obtain a solution rich in sesaminol. Then add water at a volume ratio of 1:1-3, remove ethanol by rotary evaporation, and freeze-dry for 24-48 hours to obtain sesaminol with an HPLC purity of over 95%.

[0009] Further, step 2) for preparing magnetic mesoporous Fe3O4@SiO2 is as follows: After grinding the magnetic nano Fe3O4 particles, add them to a three-necked flask, add 4-6 mM NaOH aqueous solution to adjust its concentration to 0.5-1.5 mg / mL, then add a certain amount of hexadecyltrimethylammonium bromide (CTAB) to 40 mL of suspension, stir to dissolve, add an appropriate amount of 8-12% (v / v) tetraethyl orthosilicate methanol solution, stir to react for 120-180 min, then react at 110-130℃ for 22-26 h, cool to room temperature, and use a magnet to adsorb and recover the reddish-brown Fe3O4@SiO2 precipitate. After washing and drying (drying in vacuum at 40-60℃ for 8-12 h), the product is obtained.

[0010] Specifically, in step 2), the amount of cetyltrimethylammonium bromide added is 120-160 mg, and the amount of 8-12% tetraethyl orthosilicate (TEOS) methanol solution added is 500-800 μL.

[0011] Specifically, in step 3), for every 1g of magnetic mesoporous Fe3O4@SiO2, the corresponding amount of ethanol added is 120-150 mL, the amount of concentrated ammonia added is 4-6 mL, and the amount of 3-aminopropyltriethoxysilane (APTES) added is 2-4 mL.

[0012] Specifically, in step 4), the volume concentration of the glutaraldehyde solution is 2-8% (v / v), obtained by dissolving glutaraldehyde in a phosphate buffer solution at pH 7.0; the addition ratio of amino-functionalized mesoporous Fe3O4@SiO2 to glutaraldehyde solution is 10-20 mg: 0.5-2.0 mL, the shaking temperature is 25-45℃, and the shaking time is 8-24 h.

[0013] Specifically, in step 5), β-galactosidase is dissolved in citrate buffer at pH 4-6 to obtain a β-galactosidase solution with a concentration of 20-60 U / mL; the amount of glutaraldehyde cross-linked amino-functionalized mesoporous Fe3O4@SiO2 added is 10-20 mg / mL; the immobilization temperature is 25-40℃ and the immobilization time is 8-24 h.

[0014] Specifically, in step 6), sesamin glycosides are dissolved in a citrate buffer solution with a pH of 4-6 to obtain a sesamin glycoside solution. The concentration of the sesamin glycoside solution is 0.2-2.0 mg / mL. The amount of Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase added is 10-50 U / mL. The reaction temperature is 30-55℃ and the reaction time is 12-36 h.

[0015] Furthermore, in step 6), sesamin glycosides refer to one or more of sesamin trisaccharide, sesamin disaccharide, and sesamin monosaccharide.

[0016] Furthermore, in step 1), the magnetic Fe3O4 nanoparticles are prepared using an optimized hydrothermal method, specifically: 12-16 mmol FeCl3·6H2O and 12 mmol sodium acetate are dissolved in ethylene glycol, then heated at 200-210℃ for 8-12 h, cooled to room temperature, and the black Fe3O4 precipitate is recovered by magnetic adsorption. After washing and drying (drying in vacuum at 40-60℃ for 8-12 h), the Fe3O4 nanoparticles are obtained.

[0017] This invention is the first to immobilize β-galactosidase on a magnetic mesoporous silica composite material. This material not only possesses excellent dispersibility and rapid magnetic separation performance but also a large specific surface area. Even after 10 cycles of use, the enzyme activity retention rate remains above 90%. When an appropriate amount is used for the enzymatic hydrolysis of sesamin glycosides to prepare sesamin, the sesamin formation rate can reach over 96%. The application of this immobilized enzyme to prepare sesamin is simple, cost-effective, and has a high conversion rate. Furthermore, it is recyclable and reusable, making it environmentally friendly and possessing significant industrial application value.

[0018] Compared with the prior art, the method of the present invention has the following advantages and beneficial effects: 1) Compared with chemical conversion, the present invention uses an enzymatic method to prepare sesaminol, which has the advantages of mild and controllable conditions, high product safety, and easy industrialization; 2) Compared with the free enzyme method, the immobilized enzyme prepared by this invention can improve the stability of the free enzyme and achieve continuous and repeated use, which can significantly reduce the cost of raw materials, and is highly efficient, energy-saving and pollution-free. 3) β-galactosidase is immobilized using magnetic mesoporous silica material, which is easy to separate and recover, not easily oxidized and easy to store, has good dispersibility, high specific surface area and high safety; 4) The sesamin prepared by this invention has high safety and can be applied not only to subsequent research, but also to the food, pharmaceutical and other industries, and has broad application prospects. Detailed Implementation

[0019] The technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0020] Unless otherwise specified, all raw materials used in the following examples are commercially available products that can be directly purchased or prepared using conventional methods in the art. Concentrated ammonia solution, volume concentration 22-25%. Room temperature refers to 25±5℃. Example 1

[0021] A method for preparing sesaminol using immobilized enzyme hydrolysis includes the following steps: 1) Preparation of magnetic Fe3O4 nanoparticles: The optimized hydrothermal method was used for preparation, specifically as follows: FeCl3·6H2O (16 mmol) and sodium acetate (12 mmol) were first dissolved in 120 mL of ethylene glycol and stirred continuously for 30 min. The resulting homogeneous solution was then poured into a PTFE-lined autoclave (200 mL) and heated at 200 °C for 10 h. After the autoclave cooled to room temperature, the black Fe3O4 precipitate was recovered by magnetic adsorption. The precipitate was washed three times with ethanol and three times with water, and finally dried under vacuum at 50 °C for 12 h to obtain magnetic Fe3O4 nanoparticles.

[0022] 2) Preparation of magnetic mesoporous Fe3O4@SiO2: The magnetic Fe3O4 nanoparticles obtained in step 1) were ground and added to a three-necked flask. A 5 mM NaOH aqueous solution was added to adjust the concentration to 1 mg / mL. Then, 150 mg CTAB was added to 40 mL of the suspension and stirred to dissolve at 45 °C. 600 μL of a 10% (v / v) tetraethyl orthosilicate methanol solution was added dropwise and the mixture was stirred for 160 min. Finally, the mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene and reacted at 120 °C for 24 h. After cooling the obtained compound to room temperature, the reddish-brown Fe3O4@SiO2 precipitate was recovered by magnetic adsorption and washed three times with water and ethanol, respectively. Finally, it was dried in vacuum at 50 °C for 8 h to obtain magnetic mesoporous Fe3O4@SiO2.

[0023] 3) Preparation of amino-functionalized mesoporous Fe3O4@SiO2: Weigh 1 g of the magnetic mesoporous Fe3O4@SiO2 obtained in step 2) and add it to a three-necked flask. Add 120 mL of ethanol, 4 mL of concentrated ammonia, and 2 mL of APTES. The suspension is mechanically stirred and heated at 80 °C for 8 h. Then, the amino-functionalized mesoporous Fe3O4@SiO2 (Fe3O4@SiO2-NH2) is separated by magnetism. It is then washed three times with ethanol and water, respectively, and dried under vacuum at 50 °C for 12 h for later use.

[0024] 4) Glutaraldehyde cross-linked amino-functionalized mesoporous Fe3O4@SiO2: 15 mg of the amino-functionalized mesoporous Fe3O4@SiO2 obtained in step 3) was placed in a centrifuge tube, and 1.5 mL of 2% glutaraldehyde solution (obtained by dissolving glutaraldehyde in pH 7.0, 0.05M phosphate buffer) was added. The mixture was ultrasonically dispersed for 30 min, shaken at 35℃ for 8 h, and then washed with distilled water until neutral to obtain glutaraldehyde crosslinked amino-functionalized mesoporous Fe3O4@SiO2 (Fe3O4@SiO2-NH2-glutaraldehyde). The mixture was then kept at 4 (±1)℃ until it was used to immobilize β-galactosidase.

[0025] 5) Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase: 1 mL of 30 U / mL β-galactosidase solution (obtained by dissolving β-galactosidase in 0.1 M citrate buffer at pH 6.0) was added to a centrifuge tube, followed by 15 mg of glutaraldehyde-crosslinked amino-functionalized mesoporous Fe3O4@SiO2. The mixture was shaken at 30 °C for 12 h to obtain Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase. The mixture was then frozen at -20 to -40 °C for 2 h, followed by freeze-drying in a vacuum freeze dryer for 12 h, and stored at -20 °C for later use.

[0026] The enzyme activity of Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase was determined according to the enzyme activity assay method required in GB / T 33409-2016. The enzyme activity of Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase prepared under these conditions was 728.5 U / g.

[0027] 6) Enzymatic hydrolysis to prepare sesaminol: Accurately weigh 20 U of Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase obtained in step 5) into a centrifuge tube, then add 1 mL of 0.5 mg / mL sesamin trisaccharide solution (obtained by dissolving sesamin trisaccharide in pH 5.0, 0.05M citrate buffer). After constant temperature shaking at 45℃ for 12 h, remove and recover the Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase by magnetic adsorption. Centrifuge the remaining reaction solution and collect the precipitate. First, wash three times with deionized water to remove water-soluble impurities, then add anhydrous ethanol and extract three times with ultrasonic assistance for 10 min each time to obtain a solution rich in sesamin. Then add deionized water at a 1:1 volume ratio, remove ethanol by rotary evaporation, and freeze-dry for 36 h to obtain sesamin with an HPLC purity of 98.5% and a sesamin yield of 96.3%.

[0028] In the experiment, the activities of free β-galactosidase and immobilized enzyme were measured according to the method required by GB / T 33409-2016. Example 2

[0029] A method for preparing sesaminol using immobilized enzyme hydrolysis includes the following steps: 1) Preparation of magnetic Fe3O4 nanoparticles: The optimized hydrothermal method was used for preparation, specifically as follows: FeCl3·6H2O (16 mmol) and sodium acetate (12 mmol) were first dissolved in 120 mL of ethylene glycol and stirred continuously for 30 min. The resulting homogeneous solution was then poured into a PTFE-lined high-pressure autoclave (200 mL) and heated at 200 °C for 10 h. After the autoclave cooled to room temperature, the black Fe3O4 precipitate was recovered by magnetic adsorption. The precipitate was washed three times with ethanol and three times with water, and finally dried under vacuum at 50 °C for 8 h to obtain magnetic Fe3O4 nanoparticles.

[0030] 2) Preparation of magnetic mesoporous Fe3O4@SiO2: The magnetic Fe3O4 nanoparticles obtained in step 1) were ground and added to a three-necked flask. A 5 mM NaOH aqueous solution was added to adjust the concentration to 1 mg / mL. Then, 160 mg CTAB was added to 40 mL of the suspension and stirred to dissolve at 45 °C. 700 μL of a 10% (v / v) tetraethyl orthosilicate methanol solution was added dropwise and the mixture was stirred for 170 min. Finally, the mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene and reacted at 120 °C for 24 h. After cooling the obtained compound to room temperature, the reddish-brown Fe3O4@SiO2 precipitate was recovered by magnetic adsorption and washed three times with water and ethanol, respectively. Finally, it was dried in a vacuum at 50 °C for 12 h to obtain magnetic mesoporous Fe3O4@SiO2.

[0031] 3) Preparation of amino-functionalized mesoporous Fe3O4@SiO2: Weigh 500 mg of the magnetic mesoporous Fe3O4@SiO2 obtained in step 2) and add it to a three-necked flask. Add 70 mL of ethanol, 3 mL of concentrated ammonia, and 2 mL of APTES. The suspension is mechanically stirred and heated at 75 °C for 10 h. Then, the amino-functionalized mesoporous Fe3O4@SiO2 (Fe3O4@SiO2-NH2) is separated by magnetism. It is then washed three times with ethanol and water, respectively, and dried under vacuum at 50 °C for 15 h for later use.

[0032] 4) Glutaraldehyde cross-linked amino-functionalized mesoporous Fe3O4@SiO2: 10 mg of the amino-functionalized mesoporous Fe3O4@SiO2 obtained in step 3) was placed in a centrifuge tube, and 1.0 mL of 4% glutaraldehyde solution (obtained by dissolving glutaraldehyde in pH 7.0, 0.05M phosphate buffer) was added. The mixture was ultrasonically dispersed for 20 min, shaken at 30℃ for 12 h, and then washed with distilled water until neutral to obtain glutaraldehyde cross-linked amino-functionalized mesoporous Fe3O4@SiO2 (Fe3O4@SiO2-NH2-glutaraldehyde). The mixture was then kept at 4 (±1) ℃ until it was used to immobilize β-galactosidase.

[0033] 5) Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase: 2 mL of 20 U / mL β-galactosidase solution (obtained by dissolving β-galactosidase in 0.1 M citrate buffer at pH 6.0) was added to a centrifuge tube, followed by 20 mg of glutaraldehyde-crosslinked amino-functionalized mesoporous Fe3O4@SiO2. The mixture was shaken at 35 °C for 15 h to obtain Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase. The immobilized β-galactosidase was frozen at -20 to -40 °C for 2 h, then freeze-dried in a vacuum freeze dryer for 15 h and stored at -20 °C for later use.

[0034] The enzyme activity of Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase was determined according to the enzyme activity assay method required in GB / T 33409-2016. The enzyme activity of Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase prepared under these conditions was 668.9 U / g.

[0035] 6) Enzymatic hydrolysis to prepare sesaminol: Accurately weigh 25 U of Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase obtained in step 5) into a centrifuge tube, then add 1 mL of 1.0 mg / mL sesamin disodium glycoside solution (obtained by dissolving sesamin disodium glycoside in pH 5.5, 0.05M citrate buffer). After constant temperature shaking at 50℃ for 18 h, remove and recover the Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase by magnetic adsorption. Centrifuge the remaining reaction solution and collect the precipitate. First, wash three times with deionized water to remove water-soluble impurities, then add anhydrous ethanol and extract three times with ultrasonic assistance for 10 min each time to obtain a solution rich in sesamin. Then add deionized water at a 1:1 volume ratio, remove ethanol by rotary evaporation, and freeze-dry for 48 h to obtain sesamin with an HPLC purity of 98.3% and a sesamin formation rate of 97.6%. Example 3

[0036] A method for preparing sesaminol using immobilized enzyme hydrolysis includes the following steps: 1) Preparation of magnetic Fe3O4 nanoparticles: The optimized hydrothermal method was used for preparation, specifically as follows: FeCl3·6H2O (14 mmol) and sodium acetate (12 mmol) were first dissolved in 120 mL of ethylene glycol and stirred continuously for 30 min. The resulting homogeneous solution was then poured into a PTFE-lined autoclave (200 mL) and heated at 200 °C for 10 h. After the autoclave cooled to room temperature, the black Fe3O4 precipitate was recovered by magnetic adsorption. The precipitate was washed three times with ethanol and three times with water, and finally dried under vacuum at 50 °C for 10 h to obtain magnetic Fe3O4 nanoparticles.

[0037] 2) Preparation of magnetic mesoporous Fe3O4@SiO2: The magnetic Fe3O4 nanoparticles obtained in step 1) were ground and added to a three-necked flask. A 5 mM NaOH aqueous solution was added to adjust the concentration to 1 mg / mL. Then, 140 mg CTAB was added to 40 mL of the suspension and stirred to dissolve at 45 °C. 800 μL of a 10% (v / v) tetraethyl orthosilicate methanol solution was added dropwise and the mixture was stirred for 180 min. Finally, the mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene and reacted at 120 °C for 24 h. After cooling the obtained compound to room temperature, the reddish-brown Fe3O4@SiO2 precipitate was recovered by magnetic adsorption and washed three times with water and ethanol, respectively. Finally, it was dried in a vacuum at 50 °C for 10 h to obtain magnetic mesoporous Fe3O4@SiO2.

[0038] 3) Preparation of amino-functionalized mesoporous Fe3O4@SiO2: Weigh 500 mg of the magnetic mesoporous Fe3O4@SiO2 obtained in step 2) and add it to a three-necked flask. Add 60 mL of ethanol, 2 mL of concentrated ammonia, and 1 mL of APTES. The suspension is mechanically stirred and heated at 80 °C for 10 h. Then, the amino-functionalized mesoporous Fe3O4@SiO2 (Fe3O4@SiO2-NH2) is separated by magnetization. It is then washed three times with ethanol and three times with water, and dried under vacuum at 50 °C for 24 h for later use.

[0039] 4) Glutaraldehyde cross-linked amino-functionalized mesoporous Fe3O4@SiO2: 20 mg of the amino-functionalized mesoporous Fe3O4@SiO2 obtained in step 3) was placed in a centrifuge tube, and 2.0 mL of 5% glutaraldehyde solution (obtained by dissolving glutaraldehyde in pH 7.0, 0.05M phosphate buffer) was added. The mixture was ultrasonically dispersed for 20 min, shaken at 30 °C for 24 h, and then washed with distilled water until neutral to obtain glutaraldehyde crosslinked amino-functionalized mesoporous Fe3O4@SiO2 (Fe3O4@SiO2-NH2-glutaraldehyde). The mixture was then kept at 4 (±1) °C until it was used to immobilize β-galactosidase.

[0040] 5) Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase: 1 mL of 50 U / mL β-galactosidase solution (obtained by dissolving β-galactosidase in 0.1 M citrate buffer at pH 6.0) was added to a centrifuge tube, followed by 10 mg of glutaraldehyde-crosslinked amino-functionalized mesoporous Fe3O4@SiO2. The mixture was shaken at 35 °C for 18 h to obtain immobilized β-galactosidase. The immobilized β-galactosidase was then frozen at -20 to -40 °C for 2 h, followed by freeze-drying in a vacuum freeze dryer for 12 h, and stored at -20 °C for later use.

[0041] The enzyme activity of Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase was determined according to the enzyme activity assay method required in GB / T 33409-2016. The enzyme activity of Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase prepared under these conditions was 820.766 U / g.

[0042] 6) Enzymatic hydrolysis to prepare sesaminol: Accurately weigh 30 U of Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase obtained in step 5) into a centrifuge tube, then add 1.5 mL of a 1.0 mg / mL sesamin glycoside solution (sesamin glycoside is a mixture of sesamin trisaccharide, sesamin disaccharide, and sesamin monosaccharide (mass ratio 5:3:2), obtained by dissolving sesamin glycoside in pH 5.5, 0.05M citrate buffer). After constant temperature shaking at 50℃ for 24 h, remove and recover the Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase using a magnet. Centrifuge the remaining reaction solution and collect the precipitate. First, wash three times with deionized water to remove water-soluble impurities, then add anhydrous ethanol and extract three times with ultrasonic assistance for 10 min each time to obtain a solution rich in sesamin phenol. Then add deionized water at a 1:1 volume ratio, remove ethanol by rotary evaporation, and freeze-dry for 24 hours. h, that is, sesaminol with an HPLC purity of 99.2% was obtained, and the sesaminol production rate reached 98.1%.

[0043] In summary, compared with simply using free enzymes, the method of this invention has advantages such as recyclability and being green and pollution-free. Compared with other β-galactosidase immobilization methods, the method of this invention has many advantages such as high enzyme loading capacity, easy separation, stable operation, and repeated use. Furthermore, the method of this invention is green, efficient, and has high added value, providing a new method for the industrial preparation of sesaminol and possessing extremely high market promotion value.

Claims

1. A method for preparing sesaminol by enzymatic hydrolysis of sesaminol glycosides using immobilized enzymes, characterized in that, Includes the following steps: 1) Preparation of magnetic Fe3O4 nanoparticles; 2) Preparation of magnetic mesoporous Fe3O4@SiO2; 3) Preparation of amino-functionalized mesoporous Fe3O4@SiO2: Magnetic mesoporous Fe3O4@SiO2 was mixed with ethanol, concentrated ammonia and 3-aminopropyltriethoxysilane to obtain a suspension. The suspension was heated at 75-85℃ and mechanically stirred for 8-12 hours. Then it was separated by a magnet, washed and dried for later use. 4) Preparation of glutaraldehyde crosslinked amino-functionalized mesoporous Fe3O4@SiO2: Amino-functionalized mesoporous Fe3O4@SiO2 was ultrasonically dispersed with glutaraldehyde solution, and then shaken at a constant temperature. After the reaction was completed, it was washed with water until neutral to obtain glutaraldehyde crosslinked amino-functionalized mesoporous Fe3O4@SiO2. 5) Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase: After mixing the β-galactosidase solution with glutaraldehyde-crosslinked amino-functionalized mesoporous Fe3O4@SiO2, the mixture was immobilized and shaken at a constant temperature to obtain Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase. 6) Enzymatic preparation of sesaminol: Weigh Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase and mix with sesaminol glycoside solution. After constant temperature shaking reaction, remove and recover Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase by magnetic adsorption. Centrifuge the remaining reaction solution and collect the precipitate. After washing, extract with anhydrous ethanol by ultrasonic assistance, add water, remove ethanol by rotary evaporation, and freeze dry for 24-48 h to obtain sesaminol with HPLC purity of over 95%.

2. The method for preparing sesaminol by enzymatic hydrolysis of sesaminol glycosides using an immobilized enzyme as described in claim 1, characterized in that, Step 2) Preparation of magnetic mesoporous Fe3O4@SiO2: After grinding magnetic nano Fe3O4 particles, add 4-6 mM NaOH aqueous solution to adjust the concentration to 0.5-1.5 mg / mL, then add hexadecyltrimethylammonium bromide, stir to dissolve, add 8-12% tetraethyl orthosilicate methanol solution, stir to react for 120-180 min, then react at 110-130℃ for 22-26 h, cool to room temperature, and use a magnet to adsorb and recover the reddish-brown Fe3O4@SiO2 precipitate. After washing and drying, the product is obtained.

3. The method for preparing sesaminol by enzymatic hydrolysis of sesaminol glycosides using an immobilized enzyme as described in claim 2, characterized in that, In step 2), the amount of cetyltrimethylammonium bromide added is 120-160 mg, and the amount of 8-12% tetraethyl orthosilicate methanol solution added is 500-800 μL.

4. The method for preparing sesaminol by enzymatic hydrolysis of sesaminol glycosides using an immobilized enzyme as described in claim 1, characterized in that, In step 3), for every 1g of magnetic mesoporous Fe3O4@SiO2, the corresponding amount of ethanol added is 120-150mL, the amount of concentrated ammonia added is 4-6mL, and the amount of 3-aminopropyltriethoxysilane added is 2-4mL.

5. The method for preparing sesaminol by enzymatic hydrolysis of sesaminol glycosides using an immobilized enzyme as described in claim 1, characterized in that, In step 4), the volume concentration of the glutaraldehyde solution is 2-8%, obtained by dissolving glutaraldehyde in a phosphate buffer solution at pH 7.0; the addition ratio of amino-functionalized mesoporous Fe3O4@SiO2 to glutaraldehyde solution is 10-20 mg: 0.5-2.0 mL, the shaking temperature is 25-45℃, and the shaking time is 8-24 h.

6. The method for preparing sesaminol by enzymatic hydrolysis of sesaminol glycosides using an immobilized enzyme as described in claim 1, characterized in that, In step 5), β-galactosidase is dissolved in citrate buffer at pH 4-6 to obtain a β-galactosidase solution with a concentration of 20-60 U / mL; the amount of glutaraldehyde cross-linked amino-functionalized mesoporous Fe3O4@SiO2 added is 10-20 mg / mL; the immobilization temperature is 25-40℃ and the immobilization time is 8-24 h.

7. The method for preparing sesaminol by enzymatic hydrolysis of sesaminol glycosides using an immobilized enzyme as described in claim 1, characterized in that, In step 6), sesamin glycosides are dissolved in a citrate buffer solution at pH 4-6 to obtain a sesamin glycoside solution with a concentration of 0.2-2.0 mg / mL. The amount of Fe3O4@SiO2-NH2-glutaraldehyde immobilized β-galactosidase added is 10-50 U / mL. The reaction temperature is 30-55℃ and the reaction time is 12-36 h.

8. The method for preparing sesaminol by enzymatic hydrolysis of sesaminol glycosides using an immobilized enzyme as described in claim 7, characterized in that, In step 6), sesamin glycosides refer to one or more of the following: sesamin trisaccharide, sesamin disaccharide, and sesamin monosaccharide.

9. The method for preparing sesaminol by enzymatic hydrolysis of sesaminol glycosides using an immobilized enzyme as described in claim 1, characterized in that, Step 1) Preparation of magnetic nano Fe3O4 particles: 12-16 mmol FeCl3·6H2O and 12 mmol sodium acetate are dissolved in ethylene glycol, then heated at 200-210℃ for 8-12 h, cooled to room temperature, and black Fe3O4 precipitate is recovered by magnetic adsorption. After washing and drying, the precipitate is obtained.