Method for preparing cinnamon flavor ester by ultrasonic-assisted immobilized lipase and product

Abstract

The invention belongs to the technical field of processing of food lipids and flavors and fragrances, particularly discloses a method for preparing cinnamon flavor esters by ultrasonic-assisted immobilized lipase and a product, and particularly relates to a method for synthesizing methyl cinnamate, ethyl cinnamate and benzyl cinnamate by synergistic catalysis of ultrasonic waves and immobilized lipase. The invention relates to a method for preparing cinnamon flavor lipid products such as cinnamyl acetate and the like. According to the method, on the basis that the cinnamon flavor ester is prepared through catalysis of the self-made hydrophobic modified ordered mesoporous SiO2 immobilized enzyme, the ultrasonic-assisted enzymatic reaction is added, the problems that a traditional stirring reaction is large in shearing force and the enzyme is prone to inactivation are solved, and the defects that in an existing technology for preparing the cinnamon flavor ester through an enzyme method, the enzymatic activity is unstable, the catalytic efficiency is low, and the reaction time is short are overcome. And the method has the advantages of low product yield, difficulty in separation and the like, and has huge industrial application potential.

Description

Technical Field

[0001] The invention belongs to the field of immobilized enzyme development and flavor material processing, discloses a method for preparing cinnamon flavor esters, and specifically relates to a method for synthesizing cinnamon flavor esters by using ultrasound-assisted immobilized lipase catalysis and a product thereof. Background Art

[0002] Cinnamon flavor ester is a flavor ester with cinnamon aroma. It is recognized as a safe food additive by the World Health Organization and is a food additive allowed by China's "National Food Safety Standard". Taking acetic cinnamic acid as an example, it is easy to cause pollution when produced by chemical methods, and there are by-products, which limits its large-scale production and application areas. By combining cinnamyl alcohol with vinyl acetate through ester exchange, using a self-made immobilized lipase microarray as a catalyst, and enhancing the reaction process through ultrasound, the reaction time can be shortened, the production of by-products can be reduced, the pollution caused by the reaction can be reduced, and the application field can be expanded.

[0003] At present, cinnamon flavor esters are mainly prepared by chemical methods using strong acids such as concentrated sulfuric acid as catalysts, which have the disadvantages of high energy consumption, large pollution, corrosion equipment, many by-products, and difficult product separation. For example, CN 102633638A discloses a method for preparing benzyl cinnamate using concentrated sulfuric acid as a catalyst, but the method has complicated steps for separating products and catalysts, produces a large amount of acidic wastewater, and does not meet the industrialization requirements of green environmental protection and clean production. In contrast, enzymatic synthesis has attracted much attention due to its mild reaction conditions, high selectivity, and few by-products. CN 104911221 A discloses a method for enzymatic synthesis of benzyl cinnamate, but the method uses expensive commercial imported enzymes such as Lipozyme TLIM and Novozym 435, which are expensive and lead to high production costs. In addition, the enzymatic reaction time in the method is as long as 24 hours, and the reaction efficiency is low.

[0004] CN 1884459 A discloses a method for ultrasonic enhanced bio-enzyme oil degumming. The phospholipase used in this method is not immobilized, which will expose the enzyme to a strong ultrasonic environment, reduce the catalytic activity of the enzyme; and it is not easy to recover and recycle.

[0005] Therefore, it is necessary to develop more economical, practical and highly reusable immobilized enzyme catalysts to promote the industrial production of cinnamon flavor esters. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a method and product for preparing cinnamon flavor esters by using ultrasound-assisted immobilized lipase in view of the deficiencies in the above-mentioned prior art. The method is a method for synthesizing cinnamon flavor lipid products such as methyl cinnamate, ethyl cinnamate, benzyl cinnamate, and cinnamyl acetate by using ultrasound and immobilized enzyme for synergistic catalysis, and has the advantages of high catalytic efficiency, simple operation, green environmental protection, and simple product separation.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The first object of the present invention is to provide a method for preparing a hydrophobically modified ordered mesoporous SiO2 carrier. The specific preparation process of the hydrophobically modified ordered mesoporous SiO2 carrier is as follows:

[0009] (1) Preparation of ordered mesoporous SiO2 carrier: In an acidic hydrochloric acid system, add a template and a pore expander, then add tetraethyl silicate TEOS, mix thoroughly and let stand. Transfer the mixed solution to a hydrothermal kettle for aging, filter it after cooling, wash it with anhydrous ethanol, and dry it. Finally, wash the dried powder in an acidic ethanol aqueous solution, filter it, and dry it to obtain an ordered mesoporous SiO2 carrier.

[0010] (2) Hydrophobic modification of ordered mesoporous SiO2 carrier: Add the ordered mesoporous SiO2 carrier prepared in step (1) and the hydrophobic silane coupling agent into a toluene solution and disperse them by ultrasonication; place the mixture in a reactor for hydrothermal reaction, filter, wash and dry to obtain the hydrophobically modified ordered mesoporous SiO2 carrier.

[0011] Optionally, the concentration of the acidic hydrochloric acid system is 0.1-1.0M, the amount of the template added is 0.01-0.04g / mL, and the amount of the pore expander added is 0.01-0.05g / mL; the template is one or more combinations of block polyether F-127, block copolymer P123, polyethylene glycol PEG, and polyoxyethylene PEO, and the pore expander includes one or more combinations of 1,3,5-trimethylbenzene, Span 80, decane, and dodecane; and the aging temperature is 180-230°C, and the time is 24-48h.

[0012] Optionally, the hydrophobic silane coupling agent is one or more combinations of ethyltrichlorosilane, butyltrichlorosilane, octyltrichlorosilane, polymethylhydrogensiloxane, butyltriethoxysilane, octyltriethoxysilane, dodecyltriethoxysilane, and octadecyltriethoxysilane; the total addition amount of the hydrophobic silane coupling agent and tetraethyl silicate TEOS is 200-400mmol / L, wherein the molar ratio of the hydrophobic silane coupling agent to tetraethyl silicate TEOS is 1:3-1:10, the ultrasonic dispersion time is 5-15min, the hydrothermal reaction temperature is 100-150°C, the hydrothermal reaction time is 20-30h, and the drying time is 12-24h.

[0013] The hydrophobic ordered mesoporous SiO2 material prepared by the above method is hexagonal and has a size of 0.6-6 μm. The pores on the surface of the material are arranged in an orderly manner, the mesopore diameter is 10-16 nm, and the contact angle of the material after hydrophobic modification is 110-130°.

[0014] The second object of the present invention is to provide a method for preparing cinnamon flavor esters by using ultrasound-assisted immobilized lipase.

[0015] In order to achieve the above object, the present invention adopts the following technical solution:

[0016] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase, the method specifically comprising the following steps:

[0017] (1) Preparation of immobilized lipase: immobilizing the free enzyme on the hydrophobically modified ordered mesoporous SiO2 carrier prepared as described above by physical adsorption to obtain immobilized lipase;

[0018] (2) Preparation of cinnamon flavor esters by ultrasound-assisted immobilized lipase: different acyl donors are mixed with acyl acceptors, and then the immobilized lipase prepared in step (1) is added to carry out enzymatic esterification or transesterification reaction in a constant temperature water bath and under ultrasonic mixing. After the reaction is completed, the solid and liquid are separated, and the obtained solid product is the immobilized lipase, and the obtained solution is the crude product of cinnamon flavor ester.

[0019] Optionally, the immobilized lipase has a loading amount of 100-250 mg / g and a loading rate of 50-65%.

[0020] Optionally, the specific process for preparing immobilized lipase by physical adsorption is as follows:

[0021] The free enzyme is added into a phosphate buffer to prepare an enzyme solution; the enzyme solution is mixed with a hydrophobically modified mesoporous SiO2 carrier for immobilization to obtain an immobilized lipase; wherein,

[0022] The pH value of the enzyme solution is 5.0-9.0, and the concentration is 10-100 mg / mL; the immobilization time is 0.5-2 h, and the incubation temperature is 20-40° C.; the pH value of the phosphate buffer is 5.0-9.0;

[0023] The free enzyme is one of antarctic Candida lipase, porcine pancreatic lipase, Pseudomonas cepacia lipase, Candida lipase, and Candida rugosa lipase.

[0024] Optionally, in step (2), the molar ratio of the acyl donor to the acyl acceptor is 1:1-1:10; the amount of the immobilized lipase added is 1-10 g / L; the temperature of the constant temperature water bath is 30-70°C, the reaction time is 1-6 h; the ultrasonic power is 2-20 W / cm 3 The ultrasonic intermittent ratio is 9s / 3s-3s / 9s, and the ultrasonic time is 10-60min.

[0025] Furthermore, the raw material acyl donor is cinnamic acid or vinyl acetate, and the acyl acceptor is one or a combination of methanol, ethanol, butanol, benzyl alcohol, geraniol, and cinnamyl alcohol.

[0026] It should be noted that after the ultrasound-assisted immobilized lipase was repeatedly reacted for 6 times, the relative activity of the immobilized enzyme could still be maintained at more than 80%, and after being repeatedly used for 10 times, the relative activity of the immobilized enzyme could still be maintained at more than 60%.

[0027] Furthermore, the cinnamon flavor ester products prepared by the method include methyl cinnamate, ethyl cinnamate, butyl cinnamate, benzyl cinnamate, geranyl cinnamate, and cinnamyl acetate, and the product yield is 92.7-98.4%. The ultrasonic treatment can shorten the reaction time by 30-60 minutes compared with the traditional mechanical stirring reaction time.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The present invention uses a template method to prepare a hydrophobic mesoporous SiO2 material with orderly pore distribution as an immobilized enzyme carrier. The hydrophobic ordered mesoporous SiO2 material has a hexagonal structure and a material size of about 6 microns. The pores on the surface of the material are orderly and the pore size is 10-16nm. It is suitable for the fixation of lipase and the contact between enzyme molecules and substrates, and significantly improves the activity stability of biological enzymes.

[0030] 2. The present invention is simple to separate the product from the catalyst in the enzymatic esterification and transesterification reaction of cinnamon flavor esters and can realize the recycling of the catalyst. The immobilized enzyme still maintains more than 60% of its initial activity after being reused 10 times.

[0031] 3. The present invention adopts biological enzyme selective catalysis, which has the advantages of milder reaction conditions, simpler process, no by-products and wastewater generation, and immobilized enzymes can be regenerated and reused multiple times, etc., which has lower production costs and great potential for industrial application.

[0032] 4. Compared with traditional mechanical stirring, the cavitation bubbles and resonance effect generated by ultrasonic treatment can promote the full contact between substrate and enzyme and reaction mass transfer, avoiding enzyme damage and inactivation caused by mechanical stress in the stirring reaction. In addition, ultrasound assistance can change the secondary structure of lipase, improve the catalytic activity of the enzyme, thereby shortening the reaction time and improving the catalytic efficiency of enzymatic preparation of cinnamon flavor esters. Compared with the reaction time of 2h required for traditional stirring, the reaction time of ultrasound-assisted immobilized lipase can be shortened to 1h, and the catalytic efficiency is increased from 6.9mmol / g·min of traditional stirring to 13.7mmol / g·min.

[0033] 5. The immobilized enzyme disclosed in the present invention has the characteristics of high catalytic efficiency and wide adaptability. A series of different types of flavor esters, such as methyl cinnamate, ethyl cinnamate, benzyl cinnamate, cinnamyl acetate, etc., can be prepared according to different raw material acyl donors and acyl acceptors. The products are diverse and meet the needs of different application scenarios. In addition, the crude product obtained by the enzymatic esterification and transesterification reaction of the present invention only needs simple separation and purification to prepare the finished product. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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.

[0035] Figure 1 These are the SEM (a) and TEM (b) images of the support material OMS obtained in Example 1.

[0036] Figure 2 The contact angles of the carrier material OMS obtained in Example 1 with water before (a) and after (b) hydrophobic modification.

[0037] Figure 3 This is a simulation comparison of substrate mixing effects in ultrasonic-assisted and intermittent stirring.

[0038] Figure 4 This is a comparison chart of the pressure simulation of the reaction system in ultrasonic-assisted and intermittent stirring.

[0039] Figure 5 This is a comparison chart of conventional stirring and ultrasound-assisted enzymatic reaction in Example 1.

[0040] Figure 6 This is the relative activity of the immobilized enzyme catalyzing cinnamyl acetate in Example 1, which was repeated 10 times. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0042] The word "embodiment" used here as an "exemplary" does not necessarily mean that any embodiment described is superior to or better than other embodiments. Unless otherwise specified, the performance index tests in the embodiments of this application are performed using conventional test methods in the art. It should be understood that the terms described in this application are only used to describe specific implementation methods and are not used to limit the content disclosed in this application.

[0043] Unless otherwise specified, the technical and scientific terms used in this document have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0044] In order to better illustrate the content of the present application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0045] Under the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of the present application.

[0046] The invention discloses a method for preparing cinnamon flavor esters by using ultrasound-assisted immobilized lipase.

[0047] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as a limitation of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content are also considered to fall within the protection scope of the present invention.

[0048] It should be noted that the preparation of cinnamon flavor esters catalyzed by immobilized enzyme on hydrophobically modified ordered mesoporous SiO2 carrier is mainly detected by gas chromatography and liquid chromatography.

[0049] 1) Gas chromatography detection conditions are as follows:

[0050] The chromatographic column is DB-5HT (15m×0.320mm, 0.10μm), the injection port temperature is 270℃, the injection volume is 1μL, and the split ratio is 50:1. High-purity helium is used as the carrier gas with a flow rate of 2mL / min. The hydrogen flow rate is 32mL / min, and the air flow rate is 200mL / min. The detector is a FID detector with a temperature of 270℃. The column oven heating program: initial temperature 50℃ for 2min, then increase the temperature to 150℃ at 15℃ / min, and then increase the temperature to 240℃ at 10℃ / min and maintain for 1min.

[0051] 2) Liquid chromatography detection conditions are as follows:

[0052] The chromatographic column used for HPLC (Shimadzu, LC-20AD) was C18 (4.6×250mm×5μm), and the detection wavelength of the UV detector was 280nm. The mobile phase was acetonitrile:methanol=4:6, and the flow rate of the mobile phase was 0.5mL / min -1 , 20 μL was injected at a time, and the content of each component was calculated according to the peak area normalization method.

[0053] Embodiment 1:

[0054] A method and product for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase, specifically comprising the following steps:

[0055] (1) Preparation and hydrophobic modification of ordered mesoporous silica:

[0056] At an ambient temperature of 20°C, 0.05 g / mL of block polyether F-127 was dissolved in 1L of 1.0M HCl solution, and then 0.05 g / mL of 1,3,5-trimethylbenzene was added to the solution and stirred vigorously for 1 hour. Next, 20 g of tetraethyl silicate (TEOS) was added and stirred vigorously for 15 minutes. After the mixed solution was allowed to stand for 48 hours, it was transferred to an autoclave and aged at 200°C for 24 hours. After the reactor was completely cooled, the mixture was filtered, washed with anhydrous ethanol and dried. The dried powder was placed in a muffle furnace, heated to 550°C at a rate of 5°C / min, calcined for 6 hours to remove the template, and then cooled to ambient temperature at a rate of 10°C / min to obtain the final ordered mesoporous silicon (OMS).

[0057] The ordered mesoporous silica has a size of 6 μm, a pore diameter of 15 nm, and a specific surface area of ​​231.5 m 2 / g, pore volume 1.2cm 3 / g.

[0058] Next, the OMS was hydrophobically modified and a silane coupling agent (C8) was used to combine with the hydroxyl groups on the surface of the OMS to prepare OMS-C8. The specific steps are as follows:

[0059] 1.0 g of OMS sample, 100 mM C8 and 15 μL of triethylamine were added to 10 mL of toluene. In order to achieve uniform dispersion, the mixture was treated with ultrasound for 10 min. The mixture was placed in a reactor and reacted at 130 °C for 20 h. The reactant was filtered, washed with ethanol at least 3 times, and dried at 60 °C for 24 h to obtain the final OMS-C8. After hydrophobic modification, the contact angle was 135°.

[0060] (2) Preparation of special immobilized enzyme: 1 g OMS-C8 was added to 100 mL of 90 mg / mL CSL enzyme solution (dissolved in 0.1 mol / mL, pH 8.0 PBS), and the mixture was mixed in a shaker at 220 rpm at 30°C for 60 min. The precipitate after centrifugation was placed in a freeze dryer and freeze-dried to obtain the lipase microarray CSL@OMS-C8. The immobilized amount was 249.3 mg / g and the immobilized rate was 65%.

[0061] (3) Enzymatic preparation of cinnamon flavor esters: vinyl acetate: cinnamyl alcohol was added in a molar ratio of 1:5 into a 5 mL reaction bottle and placed in an ultrasonic device with an ultrasonic intensity of 14 W / cm 3 , the ultrasonic intermittent ratio was 3s / 3s, and then 2g / L CSL@OMS-C8 was added and reacted at 50℃ for 1h. Then the mixture was taken out for content determination, and the yield of cinnamyl acetate was 97.2%.

[0062] Figure 1 The SEM (a) and TEM (b) images of the carrier material OMS obtained in Example 1. From the scanning and transmission results, it can be seen that the material has a regular hexagonal structure and the surface pores are arranged in an orderly manner, which is conducive to the fixation of lipase.

[0063] Figure 2 The contact angles of the carrier material OMS obtained in Example 1 with water before (a) and after (b) hydrophobic modification. The hydrophobic angle of the material before and after modification is 15 0 Increased to 135 0 , which can better immobilize the lipase on the material and improve the catalytic activity of the lipase.

[0064] Embodiment 2:

[0065] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0066] (1) Preparation and hydrophobic modification of ordered mesoporous silica:

[0067] At an ambient temperature of 20°C, 0.01 g / mL of block polyether F-127 was dissolved in 0.5 L of 1.0 M HCl solution, and then 0.012 g / mL of 1,3,5-trimethylbenzene was added to the solution and stirred vigorously for 1 h. Next, 20 g of tetraethyl silicate (TEOS) was added and stirred vigorously for 24 h. After the mixed solution was allowed to stand for 24 h, it was transferred to a high-pressure reactor and aged at 200°C for 24 h. After the reactor was completely cooled, the mixture was filtered, washed with ethanol and dried. The dried powder was placed in a muffle furnace, heated to 550°C at a rate of 5°C / min, calcined for 6 h to remove the template, and then cooled to ambient temperature at a rate of 10°C / min to obtain the final ordered mesoporous silicon (OMS).

[0068] The ordered mesoporous silica has a size of 5 μm, a pore diameter of 15 nm, and a specific surface area of ​​203 m 2 / g, pore volume 1.1cm 3 / g.

[0069] Next, the OMS was hydrophobically modified and a silane coupling agent (C8) was used to combine with the hydroxyl groups on the surface of the OMS to prepare OMS-C8. The specific steps are as follows:

[0070] 1.0 g of OMS sample, 50 mM C8 and 15 μL of triethylamine were added to 10 mL of toluene. In order to achieve uniform dispersion, the mixture was treated with ultrasound for 10 min. The mixture was placed in a reactor and reacted at 130 °C for 20 h. The reactant was filtered, washed with ethanol at least 3 times, and dried at 60 °C for 24 h to obtain the final OMS-C8. After hydrophobic modification, the contact angle was 117°.

[0071] (2) Preparation of special immobilized enzyme: 1.5 g OMS-C8 was added to 100 mL of 90 mg / mL CSL enzyme solution (dissolved in 0.1 mol / mL, pH 8.0 PBS), and the mixture was mixed in a shaker at 220 rpm at 30°C for 60 min. The precipitate after centrifugation was placed in a freeze dryer and freeze-dried to obtain the lipase microarray CSL@OMS-C8. The immobilized amount was 249.3 mg / g and the immobilized rate was 65%.

[0072] (3) Enzymatic preparation of cinnamon flavor esters: 20 μmol of cinnamic acid and 60 μmol of methanol were added to 1 mL of organic solvent isooctane and placed in an ultrasonic device with an ultrasonic intensity of 14 W / cm 3 , the ultrasonic intermittent ratio was 3s / 3s, and then 30mgCSL@OMS-C8 was added and reacted at 70℃ for 1h. Then the mixture was taken out for content determination, and the yield of methyl cinnamate was 94.6%.

[0073] Embodiment 3:

[0074] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0075] (1) Preparation and hydrophobic modification of ordered mesoporous silica:

[0076] At an ambient temperature of 20°C, 0.01 g / mL of block polyether F-127 was dissolved in 1L of 1.0M HCl solution, and then 0.05 g / mL of 1,3,5-trimethylbenzene was added to the solution and stirred vigorously for 1 hour. Next, 20 g of tetraethyl silicate (TEOS) was added and stirred vigorously for 15 minutes. After the mixed solution was allowed to stand for 48 hours, it was transferred to a high-pressure reactor and aged at 200°C for 24 hours. After the reactor was completely cooled, the mixture was filtered, washed with ethanol and dried. The dried powder was placed in a muffle furnace, heated to 550°C at a rate of 5°C / min, calcined for 6 hours to remove the template, and then cooled to ambient temperature at a rate of 10°C / min to obtain the final ordered mesoporous silicon (OMS).

[0077] The ordered mesoporous silica has a size of 3 μm, a pore diameter of 13 nm, and a specific surface area of ​​202.8 m 2 / g, pore volume 0.9cm 3 / g.

[0078] Next, the OMS was hydrophobically modified and a silane coupling agent (C8) was used to combine with the hydroxyl groups on the surface of the OMS to prepare OMS-C8. The specific steps are as follows:

[0079] 2.0 g of OMS sample, 100 mM C8 and 15 μL of triethylamine were added to 10 mL of toluene. In order to achieve uniform dispersion, the mixture was treated with ultrasound for 5 min. The mixture was placed in a reactor and reacted at 130 °C for 20 h. The reactant was filtered, washed with ethanol at least 3 times, and dried at 60 °C for 18 h to obtain the final OMS-C8. After hydrophobic modification, the contact angle was 135°.

[0080] (2) Preparation of special immobilized enzyme: Add 1 g of OMS-C8 to 100 mL of 60 mg / mL CSL enzyme solution (dissolved in 0.1 mol / mL, pH 8.0 PBS), mix in a shaker at 220 rpm at 35°C for 30 min, and place the precipitate after centrifugation in a freeze dryer to freeze-dry to obtain the lipase microarray CSL@OMS-C8. The immobilized amount is 150 mg / g and the immobilized rate is 50%.

[0081] (3) Enzymatic preparation of cinnamic acid flavor esters: 20 μmol of cinnamic acid and 60 μmol of ethanol were added to 1 mL of organic solvent isooctane and placed in an ultrasonic device with an ultrasonic intensity of 14 W / cm 3, the ultrasonic intermittent ratio was 3s / 3s, and then 30mgCSL@OMS-C8 was added and reacted at 70℃ for 1h. Then the mixture was taken out for content determination, and the yield of ethyl cinnamate was 92.7%.

[0082] Embodiment 4:

[0083] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0084] (1) Preparation and hydrophobic modification of ordered mesoporous silica:

[0085] At an ambient temperature of 20°C, 0.01 g / mL of block polyether F-127 was dissolved in 1L of 1.0M HCl solution, and then 0.05 g / mL of 1,3,5-trimethylbenzene was added to the solution and stirred vigorously for 1 hour. Next, 20 g of tetraethyl silicate (TEOS) was added and stirred vigorously for 15 minutes. After the mixed solution was allowed to stand for 48 hours, it was transferred to a high-pressure reactor and aged at 200°C for 24 hours. After the reactor was completely cooled, the mixture was filtered, washed with ethanol and dried. The dried powder was placed in a muffle furnace, heated to 550°C at a rate of 5°C / min, calcined for 6 hours to remove the template, and then cooled to ambient temperature at a rate of 10°C / min to obtain the final ordered mesoporous silicon (OMS).

[0086] The ordered mesoporous silica has a size of 6 μm, a pore diameter of 15 nm, and a specific surface area of ​​220.8 m 2 / g, pore volume 1.2cm 3 / g.

[0087] Next, the OMS was hydrophobically modified and a silane coupling agent (C8) was used to combine with the hydroxyl groups on the surface of the OMS to prepare OMS-C8. The specific steps are as follows:

[0088] 2.0 g of OMS sample, 50 mM C8 and 15 μL of triethylamine were added to 10 mL of toluene. In order to achieve uniform dispersion, the mixture was treated with ultrasound for 5 min. The mixture was placed in a reactor and reacted at 130 °C for 24 h. The reactant was filtered, washed with ethanol at least 3 times, and dried at 60 °C for 12 h to obtain the final OMS-C8. After hydrophobic modification, the contact angle was 128°.

[0089] (2) Preparation of special immobilized enzyme: Add 1 g of OMS-C8 to 100 mL of 90 mg / mL CSL enzyme solution (dissolved in 0.05 mol / mL, pH 9.0 PBS), mix in a shaker at 220 rpm at 35°C for 40 min, and place the precipitate after centrifugation in a freeze dryer to freeze-dry to obtain the lipase microarray CSL@OMS-C8. The immobilized amount is 230 mg / g and the immobilized rate is 60%.

[0090] (3) Enzymatic preparation of cinnamic acid flavor esters: 20 μmol of cinnamic acid and 60 μmol of butanol were added to 1 mL of organic solvent isooctane and placed in an ultrasonic device with an ultrasonic intensity of 14 W / cm 3 , the ultrasonic intermittent ratio was 3s / 3s, and then 30mgCSL@OMS-C8 was added and stirred at 70℃ for 1h. Then the mixture was taken out for content determination, and the yield of butyl cinnamate was 98.3%.

[0091] Embodiment 5:

[0092] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0093] (1) Preparation and hydrophobic modification of ordered mesoporous silica:

[0094] At an ambient temperature of 20°C, 0.01 g / mL of block polyether F-127 was dissolved in 1L of 1.0M HCl solution, and then 0.05 g / mL of 1,3,5-trimethylbenzene was added to the solution and stirred vigorously for 1 hour. Next, 20 g of tetraethyl silicate (TEOS) was added and stirred vigorously for 15 minutes. After the mixed solution was allowed to stand for 48 hours, it was transferred to a high-pressure reactor and aged at 200°C for 24 hours. After the reactor was completely cooled, the mixture was filtered, washed with ethanol and dried. The dried powder was placed in a muffle furnace, heated to 550°C at a rate of 5°C / min, calcined for 6 hours to remove the template, and then cooled to ambient temperature at a rate of 10°C / min to obtain the final ordered mesoporous silicon (OMS).

[0095] The ordered mesoporous silica has a size of 6 μm, a pore diameter of 15 nm, and a specific surface area of ​​220.8 m 2 / g, pore volume 1.2cm 3 / g.

[0096] Next, the OMS was hydrophobically modified and a silane coupling agent (C8) was used to combine with the hydroxyl groups on the surface of the OMS to prepare OMS-C8. The specific steps are as follows:

[0097] 2.0 g of OMS sample, 50 mM C8 and 15 μL triethylamine were added to 10 mL toluene. In order to achieve uniform dispersion, the mixture was treated with ultrasound for 15 min; the mixture was placed in a reactor and reacted at 130 ° C for 24 h, the reactant was filtered, washed with ethanol at least 3 times, and dried at 60 ° C for 12 h to obtain the final OMS-C8, which had a contact angle of 128° after hydrophobic modification.

[0098] (2) Preparation of special immobilized enzyme: Add 1 g of OMS-C8 to 100 mL of 90 mg / mL CSL enzyme solution (dissolved in 0.05 mol / mL, pH 9.0 PBS), mix in a shaker at 220 rpm at 35°C for 40 min, and place the precipitate after centrifugation in a freeze dryer to freeze-dry to obtain the lipase microarray CSL@OMS-C8. The immobilized amount is 230 mg / g and the immobilized rate is 60%.

[0099] (3) Enzymatic preparation of cinnamic acid flavor esters: 20 μmol of cinnamic acid and 60 μmol of geraniol were added to 1 mL of organic solvent isooctane and placed in an ultrasonic device with an ultrasonic intensity of 14 W / cm 3 , the ultrasonic intermittent ratio was 3s / 3s, and then 30mg CSL@OMS-C8 was added and stirred for reaction. After 1h, the mixture was taken out for content determination, and the yield of cinnamic acid geranyl was 98.4%.

[0100] Embodiment 6:

[0101] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0102] (1) Preparation and hydrophobic modification of ordered mesoporous silica:

[0103] At an ambient temperature of 20°C, 0.01 g / mL of block polyether F-127 was dissolved in 1L of 1.0M HCl solution, and then 0.05 g / mL of 1,3,5-trimethylbenzene was added to the solution and stirred vigorously for 1 hour. Next, 20 g of tetraethyl silicate (TEOS) was added and stirred vigorously for 15 minutes. After the mixed solution was allowed to stand for 48 hours, it was transferred to a high-pressure reactor and aged at 200°C for 24 hours. After the reactor was completely cooled, the mixture was filtered, washed with ethanol and dried. The dried powder was placed in a muffle furnace, heated to 550°C at a rate of 5°C / min, calcined for 6 hours to remove the template, and then cooled to ambient temperature at a rate of 10°C / min to obtain the final ordered mesoporous silicon (OMS).

[0104] The ordered mesoporous silica has a size of 6 μm, a pore diameter of 18 nm, and a specific surface area of ​​220.8 m 2 / g, pore volume 1.2cm 3 / g.

[0105] Next, the OMS was hydrophobically modified and a silane coupling agent (C8) was used to combine with the hydroxyl groups on the surface of the OMS to prepare OMS-C8. The specific steps are as follows:

[0106] 2.0 g of OMS sample, 50 mM C8 and 15 μL triethylamine were added to 10 mL toluene. In order to achieve uniform dispersion, the mixture was treated with ultrasound for 15 min; the mixture was placed in a reactor and reacted at 130 ° C for 24 h, the reactant was filtered, washed with ethanol at least 3 times, and dried at 60 ° C for 12 h to obtain the final OMS-C8, which had a contact angle of 128° after hydrophobic modification.

[0107] (2) Preparation of special immobilized enzyme: Add 1 g of OMS-C8 to 100 mL of 90 mg / mL CSL enzyme solution (dissolved in 0.05 mol / mL, pH 9.0 PBS), mix in a shaker at 220 rpm at 35°C for 40 min, and place the precipitate after centrifugation in a freeze dryer to freeze-dry to obtain the lipase microarray CSL@OMS-C8. The immobilized amount is 230 mg / g and the immobilized rate is 60%.

[0108] (3) Enzymatic preparation of cinnamic acid flavor esters: 20 μmol of cinnamic acid and 60 μmol of benzyl alcohol were added to 1 mL of organic solvent isooctane and placed in an ultrasonic device with an ultrasonic intensity of 14 W / cm 3 , the ultrasonic intermittent ratio was 3s / 3s, and then 30mgCSL@OMS-C8 was added and stirred for reaction. After 1h, the mixture was taken out for content determination, and the yield of benzyl cinnamate was 97.6%.

[0109] Embodiment 7:

[0110] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0111] (1) Preparation and hydrophobic modification of ordered mesoporous silica. Same as Example 1.

[0112] (2) Preparation of special immobilized enzyme: same as Example 4.

[0113] (3) Enzymatic preparation of cinnamic flavor esters: The yield of cinnamyl acetate was 80.0% as in Example 1. Different buffer pH values ​​affected the lipase activity, resulting in a decrease in the yield of cinnamyl acetate.

[0114] Embodiment 8:

[0115] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0116] ((1) Preparation and hydrophobic modification of ordered mesoporous silica: Same as Example 2.

[0117] (2) Preparation of special immobilized enzyme: same as Example 4.

[0118] (3) Enzymatic preparation of cinnamon flavor esters: Same as Example 1, the yield of cinnamyl acetate was 90.5%.

[0119] Embodiment 9:

[0120] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0121] (1) Preparation and hydrophobic modification of ordered mesoporous silica: same as Example 3.

[0122] (2) Preparation of special immobilized enzyme: same as Example 5.

[0123] (3) Enzymatic preparation of cinnamon flavor esters: Same as Example 1, the yield of cinnamyl acetate was 91.9%.

[0124] It should be noted that the raw materials listed in the present invention, as well as the upper and lower limits and interval values ​​of the raw materials and enzyme amounts of the present invention, and the upper and lower limits and interval values ​​of the process parameters (such as temperature, time, and homogenization speed) can all achieve the present invention, and the embodiments are not listed one by one here.

[0125] In order to further demonstrate the beneficial effects of the present invention and to better understand the present invention, the following comparative examples are used to further illustrate the technical features disclosed in the present invention, but they should not be construed as limiting the present invention. Other improvements made by those skilled in the art based on the above invention content without inventive work are also considered to fall within the scope of protection of the present invention.

[0126] Comparative Example 1:

[0127] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0128] The method is basically the same as Example 1, except that no ultrasonic auxiliary equipment is used in step (2). The conversion rate of cinnamyl alcohol is determined to be 96.7%.

[0129] Comparative Example 2:

[0130] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0131] The same as Example 1, except that the ultrasonic intensity in step (3) is 1 W / cm 3 The conversion rate of cinnamyl alcohol was determined to be 69.6%.

[0132] Comparative Example 3:

[0133] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0134] The same as Example 1, except that the ultrasonic intensity in step (3) is 30 W / cm 3 The conversion rate of cinnamyl alcohol was determined to be 82.6%.

[0135] Comparative Example 4:

[0136] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0137] The same as Example 1, except that the ultrasonic intensity in step (3) is 500 W / cm 3 The conversion rate of cinnamyl alcohol was determined to be 89.8%.

[0138] Comparative Example 5:

[0139] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0140] The same as Example 1, except that the ultrasonic intensity in step (3) is 1000 W / cm 3 The conversion rate of cinnamyl alcohol was determined to be 84.2%.

[0141] Comparative Example 6:

[0142] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0143] The method is basically the same as Example 1, except that the intermittent ratio in step (3) is 10s / 3s, and the conversion rate of cinnamyl alcohol is 82.2%.

[0144] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0145] The method is basically the same as Example 1, except that the intermittent ratio in step (3) is 20s / 3s, and the conversion rate of cinnamyl alcohol is 84.8%.

[0146] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0147] The method is basically the same as Example 1, except that the intermittent ratio in step (3) is 3s / 10s, and the conversion rate of cinnamyl alcohol is 85.6%.

[0148] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0149] The method is basically the same as Example 1, except that the intermittent ratio in step (3) is 3s / 20s, and the conversion rate of cinnamyl alcohol is 76.5%.

[0150] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0151] The process is basically the same as Example 1, except that the reaction time in step (3) is 70 min, and the conversion rate of cinnamyl alcohol is 98.0%.

[0152] Comparative Example 11:

[0153] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0154] The process is basically the same as Example 1, except that the reaction time in step (3) is 80 min, and the conversion rate of cinnamyl alcohol is 98.5%.

[0155] Comparative Example 12:

[0156] A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase and the product thereof, specifically comprising the following steps:

[0157] The process is basically the same as Example 1, except that the reaction time in step (3) is 90 min, and the conversion rate of cinnamyl alcohol is 98.9%.

[0158] Experimental example

[0159] An enzymatic preparation method of cinnamyl acetate and its product, specifically comprising the following steps:

[0160] The method is basically the same as Example 1, except that after step (2), a repeated use experiment of lipase microarray catalyzed synthesis of cinnamyl acetate is added, that is, after the reaction is completed, the lipase is rinsed with a reaction solvent and dried to remove the raw materials, products and water attached to the enzyme, and the activated and regenerated immobilized enzyme is then put into the same reaction.

[0161] like Figure 6 As shown, after the lipase microarray was reused 10 times, although the catalytic conversion rate of cinnamyl alcohol gradually decreased, it still had a high activity.

[0162] Table 1: Evaluation of the adaptability of immobilized enzyme CSL@OMS-C8 under stirring and ultrasonic treatment conditions

[0163]

[0164]

[0165] In addition, the inventors also conducted comparative experiments on ultrasound-assisted and intermittent stirring, such as Figure 3 The figure shows the simulation comparison of substrate mixing effect in ultrasound-assisted and intermittent stirring. Figure 4 The figure shows the pressure simulation comparison of the reaction system in ultrasound-assisted and intermittent stirring. The results show that appropriate ultrasound can promote the mixing of the system and reduce the impact of mass transfer.

[0166] And, if Figure 5 The figure shows a comparison of conventional stirring and ultrasound-assisted enzymatic reactions in Example 1. The results show that the immobilized enzyme has a higher catalytic effect under ultrasound assistance, and the cinnamyl alcohol can reach the maximum conversion rate at 60 minutes.

[0167] 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 a hydrophobically modified ordered mesoporous SiO2 carrier, characterized in that: The specific preparation process of the hydrophobically modified ordered mesoporous SiO2 carrier is as follows: (1) Preparation of ordered mesoporous SiO2 carrier: In an acidic hydrochloric acid system, a template agent and a pore-enlarging agent are added, and then tetraethyl silicate (TEOS) is added, mixed thoroughly, and then allowed to stand; The mixed solution is transferred to a hydrothermal autoclave for aging, filtered after cooling, washed with anhydrous ethanol, and dried; finally, the dried powder is placed in an acidic ethanol aqueous solution for washing, filtered, and dried to obtain an ordered mesoporous SiO2 carrier; (2) Hydrophobic modification of ordered mesoporous SiO2 carrier: Add the ordered mesoporous SiO2 carrier prepared in step (1) into a toluene solution, add a hydrophobic silane coupling agent and disperse it by ultrasonication; place the mixture in a reactor for hydrothermal reaction, filter, wash and dry to obtain the hydrophobically modified ordered mesoporous SiO2 carrier.

2. The method for preparing the hydrophobically modified ordered mesoporous SiO2 carrier according to claim 1, characterized in that: The concentration of the acidic hydrochloric acid system is 0.1-1.0M, the amount of the template added is 0.01-0.04g / mL, and the amount of the pore expander added is 0.01-0.05g / mL; the template is one or more combinations of block polyether F-127, block copolymer P123, polyethylene glycol PEG, and polyoxyethylene PEO, and the pore expander includes one or more combinations of 1,3,5-trimethylbenzene, Span 80, decane, and dodecane; and the aging temperature is 180-230°C, and the time is 24-48h.

3. The method for preparing the hydrophobically modified ordered mesoporous SiO2 carrier according to claim 1, characterized in that: The hydrophobic silane coupling agent is one or more combinations of ethyltrichlorosilane, butyltrichlorosilane, octyltrichlorosilane, polymethylhydrogensiloxane, butyltriethoxysilane, octyltriethoxysilane, dodecyltriethoxysilane, and octadecyltriethoxysilane; the total addition amount of the hydrophobic silane coupling agent and tetraethyl silicate TEOS is 200-400mmol / L, wherein the molar ratio of the hydrophobic silane coupling agent to tetraethyl silicate TEOS is 1:3-1:10, the ultrasonic dispersion time is 5-15min, the hydrothermal reaction temperature is 100-150°C, the hydrothermal reaction time is 20-30h, and the drying time is 12-24h.

4. A method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase, characterized in that: The method specifically comprises the following steps: (1) Preparation of immobilized lipase: immobilizing the free enzyme on the hydrophobically modified ordered mesoporous SiO2 carrier prepared by the method of claim 1 by physical adsorption to obtain immobilized lipase; (2) Preparation of cinnamon flavor esters by ultrasound-assisted immobilized lipase: different acyl donors are mixed with acyl acceptors, and then the immobilized lipase prepared in step (1) is added to carry out enzymatic esterification or transesterification reaction in a constant temperature water bath and under ultrasonic mixing. After the reaction is completed, the solid and liquid are separated, and the obtained solid product is the immobilized lipase, and the obtained solution is the crude product of cinnamon flavor ester.

5. The method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase according to claim 4, characterized in that: The specific process for preparing immobilized lipase by physical adsorption is as follows: adding free enzyme into phosphate buffer to prepare enzyme solution, adding hydrophobically modified mesoporous SiO2 carrier, fully mixing and incubating to obtain immobilized enzyme.

6. The method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase according to claim 4 or claim 5, characterized in that: The free enzyme is one of antarctic Candida lipase, porcine pancreatic lipase, Pseudomonas cepacia lipase, Candida lipase, and Candida rugosa lipase.

7. The method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase according to claim 6, characterized in that: The pH value of the enzyme solution used for preparing the immobilized lipase is 5.0-9.0, and the concentration is 30-100 mg / mL; the immobilization time is 0.5-2 hours, the incubation temperature is 20-40° C., and the pH value of the phosphate buffer is 5.0-9.

0.

8. The method for preparing cinnamon flavor esters by ultrasound-assisted immobilized lipase according to claim 4, characterized in that: In the step (2), the molar ratio of the acyl donor to the acyl acceptor is 1:1-1:10; the amount of the immobilized lipase added is 1-10 g / L; the temperature of the constant temperature water bath is 30-70° C., the reaction time is 1-6 h; the ultrasonic power is 2-20 W / cm 3 The ultrasonic intermittent ratio is 9s / 3s-3s / 9s, and the ultrasonic time is 10-60min.

9. The method for preparing cinnamon flavor esters by using ultrasound-assisted immobilized lipase according to claim 4 or claim 8, characterized in that: The acyl donor is cinnamic acid or vinyl acetate, and the acyl acceptor is one or a combination of methanol, ethanol, butanol, benzyl alcohol, geraniol, and cinnamyl alcohol.

10. A cinnamon flavor ester prepared by the method of claim 4, characterized in that: The prepared cinnamon flavor ester products include methyl cinnamate, ethyl cinnamate, butyl cinnamate, benzyl cinnamate, geranyl cinnamate and cinnamyl acetate.

Images