Flaky zeolite immobilized Candida antarctica lipase B catalyst and method for preparing medium-chain glyceride through catalytic esterification of flaky zeolite immobilized Candida antarctica lipase B catalyst

Abstract

The invention relates to the technical field of biocatalytic synthesis of fatty glyceride, in particular to a flaky zeolite immobilized Candida antarctica lipase B catalyst and a method for preparing medium-chain glyceride through catalytic esterification of the flaky zeolite immobilized Candida antarctica lipase B catalyst. The flaky zeolite is flaky silica-alumina zeolite ZSM-5-P, and the loading capacity of the candida antarctica lipase B is 9-12% of the mass of the flaky zeolite. The candida antarctica lipase B is loaded and fixed in the flaky zeolite to prepare the solid catalyst, and the catalyst is a solid enzyme biocatalyst, is small in usage amount, high in catalytic efficiency and recyclable, is used for preparing the medium-chain glyceride, is high in efficiency, low in cost, simple in process, mild in condition, safe and environment-friendly, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of biocatalytic synthesis of fatty acid glycerides, and in particular to a flake zeolite-fixed Antarctic Candida lipase B catalyst and a method for preparing medium-chain glycerides by catalytic esterification thereof. Background Art

[0002] Medium-chain glycerides include medium-chain monoglycerides, medium-chain diglycerides and medium-chain triglycerides. As a multi-purpose dietary fat with health functions, it has attracted more and more attention. At the same time, due to its shorter carbon chain, medium-chain glycerides show metabolic characteristics of easy digestion and rapid energy supply; its structure has both hydrophilic hydroxyl groups and lipophilic carbon chains, which makes it have excellent emulsification, foaming and antibacterial properties. Based on these special physical and chemical properties and metabolic advantages, medium-chain glycerides have great application potential in infant formula milk powder, sports nutrition products, special medical formula foods, etc. in addition to traditional food, health products and pharmaceutical industries. However, natural medium-chain glycerides are relatively scarce in nature and mainly exist in vegetable oils such as calyx seed oil and camphor seed kernel oil. The catalytic esterification method can directly synthesize medium-chain glycerides from medium-chain fatty acids and glycerol, which can solve the problem of scarcity of natural medium-chain glycerides.

[0003] Catalytic esterification can generally be divided into biocatalysis and chemical catalysis. Among them, the chemical catalytic esterification reaction system has many by-products and high energy consumption, while the biocatalytic reaction system has the advantages of mild reaction conditions, low energy consumption, and little environmental pollution. In industrial production, liquid acids, liquid alkalis, and liquid enzymes are often used as catalysts for esterification reactions, but these liquid catalysts are easily soluble in the reaction system, the products are difficult to separate, and the liquid acids and liquid alkalis cannot be reused, the cost is high, and the environmental pollution is large. Therefore, the development of a new, efficient, and recyclable solid enzyme biocatalyst can achieve the goal of economical and clean production of medium-chain glycerides. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a flake zeolite-fixed Antarctic Candida lipase B catalyst and a method for preparing medium-chain glycerides by catalytic esterification thereof. The catalyst is a solid enzyme biocatalyst with a small usage amount, high catalytic efficiency, and recyclable use. It is used to prepare medium-chain glycerides with high efficiency and low cost, simple process, mild conditions, safety and environmental protection, and has broad application prospects.

[0005] To achieve the above object, the present invention provides the following technical solutions: The invention provides a flake zeolite-fixed Antarctic Candida lipase B catalyst. The flake zeolite is a flake silica-alumina zeolite ZSM-5-P. The loading amount of the Antarctic Candida lipase B (CALB) is 9-12% of the mass of the flake zeolite.

[0006] CALB is a globular protein composed of 317 amino acids and has a folded three-dimensional structure. Its active site is composed of a catalytic triad (Asp187, His224, Ser105). Asp187 locates and fixes His224 through a hydrogen bond, and His224 and Ser105 work together to form the active center of the catalytic reaction. CALB has strong catalytic activity for both water-insoluble and water-soluble substances, and has a wide range of substrate adaptability. At the same time, its high stereoselectivity enables it to accurately control the structure of the product in the esterification reaction, and the catalytic efficiency is high; in addition, the catalytic reaction of CALB usually undergoes a cycle of free enzyme → transition state 1 → acyl enzyme intermediate → transition state 2 → free enzyme, so that it can be recycled after solidification. In the esterification reaction of the present invention, it starts with the catalytic action of the serine hydroxyl group in the lipase, causing a nucleophilic attack on the carbonyl group of decanoic acid. This interaction produces an acyl-enzyme intermediate binding, followed by the glycerol molecule entering the active site of the enzyme, undergoing deprotonation facilitated by the histidine residue, which then acts as a nucleophile to bind to the acyl group leading to the formation of a second enzyme intermediate, which subsequently collapses, producing a monoglyceride and promoting the regeneration of the catalytic residue within the enzyme. The resulting monoglyceride exhibits nucleophilic properties, allowing it to participate in the subsequent reaction to produce a diester. Similarly, the diester formed also has nucleophilic properties and continues to participate in the reaction to produce a triester.

[0007] Furthermore, the preparation method of the catalyst comprises the following steps: (1) adding tetraethyl orthosilicate slowly to a tetrapropylammonium hydroxide solution, then sequentially adding isopropyl alcohol and sodium hydroxide, aluminum nitrate nonahydrate, and urea to the obtained system, stirring and aging the obtained mixture at room temperature to obtain a mixed gel, and then washing, drying, grinding, and calcining the mixed gel after hydrothermal crystallization to obtain a sheet-like zeolite ZSM-5-P; (2) mixing the flaky zeolite ZSM-5-P obtained in step (1) with an aqueous solution of ammonium nitrate, and subjecting the mixture to ion exchange in an oil bath at 80° C. for 12 h, followed by washing and drying to obtain a proton-type zeolite HZSM-5-P; (3) reacting the proton-type zeolite HZSM-5-P obtained in step (2) with (3-mercaptopropyl)trimethoxysilane and n-hexane at room temperature for 24 h, washing and drying to obtain mercapto zeolite ZSM-5-P-SH; (4) The thiolated zeolite ZSM-5-P-SH obtained in step (3) was reacted with Antarctic Candida lipase B (CALB) in a 37°C water bath for 12 h, and then washed and dried to obtain a flaky zeolite-immobilized Antarctic Candida lipase B catalyst.

[0008] The present invention forms a high-efficiency solid catalyst containing lipase on the surface by grafting thiol groups on the sheet-like silica-alumina zeolite ZSM-5-P and loading Antarctic Candida lipase B on the surface of ZSM-5-P-SH zeolite by covalent bonding. By introducing lipase on the surface of the sheet-like mesoporous silica-alumina ZSM-5-PM zeolite, it can promote the catalytic reaction of lipase with large-sized medium-chain fatty acid molecules, so that the solid catalyst has the activity of highly efficient catalysis of the esterification reaction of capric acid and glycerol, the catalytic efficiency is higher, and it can be recycled.

[0009] Furthermore, in step (1), the molar ratio of SiO2:Al2O3:TPAOH:urea:H2O:NaOH:IPA in the mixed solution is 1:0.0074:0.3:0.6:29:0.05:0.03; the temperature of the hydrothermal crystallization is 180°C, and the treatment time is 48 h.

[0010] Furthermore, in step (2), the sheet-like zeolite ZSM-5-P is mixed with an aqueous ammonium nitrate solution at a solid-liquid mass volume (g / mL) ratio of 1:10, and the concentration of ammonium nitrate in the aqueous ammonium nitrate solution is 1 mol / L.

[0011] Furthermore, in step (3), the mass ratio of the proton-type zeolite HZSM-5-P to (3-mercaptopropyl)trimethoxysilane and n-hexane is 2:1:66.

[0012] The present invention also provides a method for preparing medium-chain glycerides by catalytic esterification using the above-mentioned flaky zeolite-immobilized Antarctic Candida lipase B, comprising the following steps: S1. Adding reactants capric acid and glycerol sequentially into a round-bottom flask reaction apparatus; S2. Adding the above-mentioned flake zeolite-immobilized Antarctic Candida lipase B catalyst to the reactant of S1; S3. At a set temperature, the esterification reaction is carried out with stirring. The product after the reaction is separated by centrifugation, and the upper liquid is taken to obtain medium-chain glycerides. Specifically, the medium-chain glycerides include medium-chain monoglycerides, medium-chain diglycerides and medium-chain triglycerides, and more specifically include 1-capric acid monoglyceride, 2-capric acid monoglyceride, 1.3-capric acid diglyceride, 2.3-capric acid diglyceride, and capric acid triglyceride.

[0013] The specific reaction formula is as follows:

[0014] Furthermore, in S1, the molar ratio of capric acid to glycerol is 0.5:1-1:1, preferably 1:1.

[0015] Furthermore, in S2, the addition amount of the flake zeolite-immobilized Candida antarctica lipase B catalyst is 1.6-4.3% of the total mass of the capric acid, preferably 2.3-4.3%.

[0016] Furthermore, in S3, the temperature of the esterification reaction is 35-90°C, preferably 70°C; and the time is 2-6h, preferably 4h.

[0017] Beneficial effects of the present invention: The present invention loads and fixes Antarctic Candida lipase B into flaky zeolite to prepare a solid enzyme biocatalyst, fully utilizing the characteristics of high specific surface area and regular pore structure of flaky zeolite. Antarctic Candida lipase B is evenly distributed on the surface of the flaky zeolite, which not only has high catalytic efficiency and good universality, but also can be recycled for multiple times. The use of the flaky zeolite for preparing medium-chain glycerides has the advantages of simple process, mild reaction conditions, low cost, and environmental friendliness, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The scanning electron micrograph (a) and the perspective electron micrograph (b) of the Candida antarctica lipase B catalyst immobilized on the flaky zeolite in Example 2 of the present invention are shown; Figure 2 This is a comparative analysis diagram of different lipase loading amounts used in preparing medium-chain glycerides using flake zeolite-immobilized Antarctic Candida lipase B as a catalyst in Example 1 of the present invention; Figure 3 This is a comparative analysis diagram of different esterification reaction times for preparing medium-chain glycerides using flake zeolite-immobilized Candida antarctica lipase B as a catalyst in Example 2 of the present invention; Figure 4 This is a comparative analysis diagram of different molar ratios of capric acid to glycerol materials for preparing medium-chain glycerides using flake zeolite-immobilized Candida antarctica lipase B as a catalyst in Example 3 of the present invention; Figure 5 This is a comparative analysis diagram of different reaction temperatures for preparing medium-chain glycerides using flake zeolite-immobilized Antarctic Candida lipase B as a catalyst in Example 4 of the present invention; Figure 6 This is a comparative analysis chart of different catalyst dosages for preparing medium-chain glycerides using flaky zeolite-immobilized Candida antarctica lipase B as a catalyst in Example 5 of the present invention.

[0019] Figure 7 This is a comparative analysis chart of the number of times different catalysts are recycled in preparing medium-chain glycerides using flaky zeolite-immobilized Candida antarctica lipase B as a catalyst in Example 6 of the present invention.

[0020] The present invention is further described below in conjunction with the accompanying drawings and embodiments. DETAILED DESCRIPTION

[0021] In order to better illustrate the technical scheme of the present invention, the scheme of the present invention will be explained below in conjunction with embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If specific techniques or conditions are not indicated in the embodiments, they are carried out according to the techniques described in the literature or reference books in this area or according to the product instructions. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products.

[0022] The above-mentioned technical features of the present invention and the technical features specifically described below (such as implementation cases) can be combined with each other to form a new or preferred technical solution.

[0023] The preparation process of the medium-chain glyceride of the present invention is as follows: first, the Antarctic Candida lipase B is immobilized with the sheet zeolite ZSM-5-P, then the reactants capric acid and glycerol are added in sequence in the reaction device, and then the immobilized lipase is added to the reactants, and the esterification reaction is carried out by stirring, and the product medium-chain glyceride is obtained after the reaction. The reaction formula is shown as follows:

[0024] In the process of preparing medium-chain glycerides by reacting capric acid with glycerol using immobilized lipase as catalyst, the reaction conversion rate was determined by Agilent GC-7890B gas chromatograph using HP-5 column.

[0025] The method for analyzing the content of medium-chain glycerides is as follows: take 250µL of the reaction solution, centrifuge the upper layer, add 250µL of n-hexadecane (25mg / mL) as the internal standard; take 1µL of the sample for injection, and determine the amount of medium-chain glycerides in the reactant by gas chromatography. Among them, the conversion rate is determined by an Agilent GC-7890B gas chromatograph from Agilent Technologies, Inc., USA, equipped with an ionization flame detector (FID) and an HP-5 capillary column (30m×0.32mm×0.25μm). The specific test conditions are: high-purity nitrogen (99.99%) is used as the carrier gas, the injection port temperature is 320℃, the detector temperature is 320℃, and the column box temperature heating program is an initial temperature of 100℃, maintained for 1min, and then increased to 280℃ at a heating rate of 20℃ / min, and maintained for 13min. The conversion rate of capric acid is calculated from the measured amount of medium-chain glycerides, and the calculation method is as follows:

[0026] In the formula, and represent the molar content of octanoic acid before the reaction and the molar content of octanoic acid remaining after the reaction, respectively; The amount of medium-chain monoglycerides and remaining capric acid was calculated by gas chromatography peak area (internal standard method, n-hexadecane was used as the internal standard).

[0027]

[0028]

[0029]

[0030] In the formula, , and Represent the molar content of medium-chain monoglycerides, medium-chain diglycerides and medium-chain triglycerides, respectively.

[0031] Example 1: Effect of different loading amounts of Antarctic Candida lipase B on the preparation of medium-chain glycerides A method for preparing medium-chain glycerides by esterification catalyzed by flaky zeolite-immobilized Antarctic Candida lipase B: (1) Immobilization of Antarctic Candida lipase B using flaky zeolite ZSM-5-P, the method is as follows: Slowly add tetrapropylammonium hydroxide solution to tetrapropylammonium silicate, then add isopropanol and sodium hydroxide, aluminum nitrate nonahydrate, and urea to the obtained system in sequence, wherein the molar ratio of SiO2:Al2O3:TPAOH:urea:H2O:NaOH:IPA in the mixed solution is 1:0.0074:0.3:0.6:29:0.05:0.03. The obtained mixed solution is stirred and aged at room temperature to obtain a mixed gel, which is hydrothermally crystallized at 180°C for 48 hours, washed, dried, ground, and calcined to obtain a sheet zeolite ZSM-5-P; the sheet zeolite ZSM-5-P is then mixed with an ammonium nitrate aqueous solution (1 mol / L) at a solid-liquid mass volume (g / mL) ratio of 1:10, and ion exchange is carried out in an 80°C oil bath for 12 hours, and then washed and dried to obtain a proton zeolite HZSM-5-P; the obtained proton zeolite HZSM-5-P is obtained. ZSM-5-P was mixed with (3-mercaptopropyl)trimethoxysilane and n-hexane in a mass ratio of 2:1:66 and reacted at room temperature for 24 hours. After the reaction, the mixture was washed and dried to obtain the thiolated zeolite ZSM-5-P-SH. The thiolated zeolite ZSM-5-P-SH was then reacted with 6%-13% (based on the weight of the zeolite, w / w) Antarctic Candida lipase B in a water bath at 37°C for 12 hours, and then washed and dried to obtain a flaky zeolite-immobilized Antarctic Candida lipase B catalyst.

[0032] (2) Add the reactants capric acid and glycerol into a round-bottom flask, with the molar ratio of capric acid to glycerol being 1:1; (3) adding 2.3% of the total mass of decanoic acid to the reactants and immobilized Candida antarctica lipase B catalyst; (4) The esterification reaction was carried out with stirring at a temperature of 40°C for 4 h. The product after the reaction was separated by centrifugation and the upper layer was collected to obtain medium-chain glycerides.

[0033] The reaction solution was taken regularly for medium-chain glyceride analysis. The results were as follows: Figure 2 As shown. Figure 2 It can be seen that with the increase of lipase loading, the conversion rate of capric acid gradually increased. When the loading of Antarctic Candida lipase B was 9% (based on zeolite weight, w / w), the conversion rate of capric acid reached 56.7%, and the distribution rates of medium-chain monoglycerides, medium-chain diglycerides and medium-chain triglycerides were 67.8%, 29.6% and 2.6% respectively. After that, with the increase of Antarctic Candida lipase B loading, the conversion rate of capric acid tended to be stable. Therefore, it is better to select a loading of 9-13% of Antarctic Candida lipase B.

[0034] Example 2: Effect of different esterification reaction times on the preparation of medium-chain glycerides A method for preparing medium-chain glycerides by esterification catalyzed by flaky zeolite-immobilized Antarctic Candida lipase B: (1) Immobilization of Antarctic Candida lipase B using flaky zeolite ZSM-5-P, the method is as follows: Slowly add tetrapropylammonium hydroxide solution to tetrapropylammonium silicate, then add isopropanol and sodium hydroxide, aluminum nitrate nonahydrate, and urea to the obtained system in sequence, wherein the molar ratio of SiO2:Al2O3:TPAOH:urea:H2O:NaOH:IPA in the mixed solution is 1:0.0074:0.3:0.6:29:0.05:0.03. The obtained mixed solution is stirred and aged at room temperature to obtain a mixed gel, which is hydrothermally crystallized at 180°C for 48 hours, washed, dried, ground, and calcined to obtain a sheet zeolite ZSM-5-P; the sheet zeolite ZSM-5-P is then mixed with an ammonium nitrate aqueous solution (1 mol / L) at a solid-liquid mass volume (g / mL) ratio of 1:10, and ion exchange is carried out in an 80°C oil bath for 12 hours, and then washed and dried to obtain a proton zeolite HZSM-5-P; the obtained proton zeolite ZSM-5-P was mixed with (3-mercaptopropyl)trimethoxysilane and n-hexane in a mass ratio of 2:1:66 and reacted at room temperature for 24 hours. After the reaction, the mixture was washed and dried to obtain the thiolated zeolite ZSM-5-P-SH. The thiolated zeolite ZSM-5-P-SH was then reacted with 9% (based on the weight of the zeolite, w / w) Antarctic Candida lipase B in a water bath at 37°C for 12 hours, and then washed and dried to obtain the flaky zeolite-immobilized Antarctic Candida lipase B catalyst.

[0035] (2) Add the reactants capric acid and glycerol into a round-bottom flask, with the molar ratio of capric acid to glycerol being 1:1; (3) adding 2.3% of the total mass of decanoic acid to the reactants and immobilized Candida antarctica lipase B catalyst; (4) Stirring for esterification reaction at 40°C for 2 h to 6 h. The product after the reaction is separated by centrifugation and the upper layer is collected to obtain medium-chain glycerides.

[0036] The reaction solution was taken regularly for medium-chain glyceride analysis. The results were as follows: Figure 3 As shown. Figure 3 It can be seen that as the reaction time increases, the conversion rate of capric acid gradually increases. When the reaction time is 4 hours, the conversion rate of capric acid reaches 56.7%, and the distribution rates of the product medium-chain monoglyceride, medium-chain diglyceride and medium-chain triglyceride are 67.8%, 29.6% and 2.6%. After that, as the reaction time increases, the conversion rate of capric acid tends to be stable. Therefore, it is more ideal to select an esterification reaction time of 4 hours.

[0037] Example 3: Effect of different molar ratios of capric acid and glycerol on the preparation of medium-chain glycerides A method for preparing medium-chain glycerides by esterification catalyzed by flaky zeolite-immobilized Antarctic Candida lipase B: (1) Immobilization of Antarctic Candida lipase B using flaky zeolite ZSM-5-P, the method is as follows: TEOS was slowly added to tetrapropylammonium hydroxide solution, and then isopropyl alcohol and sodium hydroxide, aluminum nitrate nonahydrate, and urea were added to the obtained system in sequence, wherein the molar ratio of SiO2:Al2O3:TPAOH:urea:H2O:NaOH:IPA in the mixed solution was 1:0.0074:0.3:0.6:29:0.05:0.03. The obtained mixed solution was stirred and aged at room temperature to obtain a mixed gel, which was hydrothermally crystallized at 180°C for 48 hours. h later, washing, drying, grinding and calcining were performed to obtain a flake zeolite ZSM-5-P; the flake zeolite ZSM-5-P was mixed with an aqueous ammonium nitrate solution (1 mol / L) at a solid-liquid mass volume (g / mL) ratio of 1:10, and ion exchange was carried out in an oil bath at 80°C for 12 h, followed by washing and drying to obtain a proton-type zeolite HZSM-5-P; the obtained proton-type zeolite HZSM-5-P was mixed with (3-mercaptopropyl)trimethoxysilane and n-hexane at a mass ratio of 2:1:66, and reacted at room temperature for 24 h. After the reaction was completed, washing and drying were performed to obtain a thiolated zeolite ZSM-5-P-SH; the thiolated zeolite ZSM-5-P-SH was reacted with 9% (based on the weight of the zeolite, w / w) Antarctic Candida lipase B in a water bath at 37°C for 12 h, and then washed and dried to obtain a flake zeolite-immobilized Antarctic Candida lipase B catalyst.

[0038] (2) adding the reactants capric acid and glycerol into a round-bottom flask, wherein the molar ratio of capric acid to glycerol is 0.5:1-4:1; (3) adding 2.3% of the total mass of decanoic acid to the reactants and immobilized Candida antarctica lipase B catalyst; (4) The esterification reaction was carried out with stirring at a temperature of 40°C for 4 hours. The product after the reaction was separated by centrifugation and the upper layer was collected to obtain medium-chain glycerides.

[0039] The reaction solution was taken regularly for medium-chain glyceride analysis. The results were as follows: Figure 4 As shown. Figure 4 It can be seen that as the molar ratio of capric acid to glycerol increases, the conversion rate of capric acid gradually increases. When the molar ratio of capric acid to glycerol is 1:1, the conversion rate of capric acid is the highest, reaching 56.7%, and the distribution rates of the product medium-chain monoglycerides, medium-chain diglycerides and medium-chain triglycerides are 67.8%, 29.6% and 2.6%. After that, as the molar ratio of capric acid to glycerol increases, the conversion rate of capric acid gradually decreases. Therefore, it is most ideal to select a molar ratio of capric acid to glycerol of 1:1.

[0040] Example 4: Effect of different esterification reaction temperatures on the preparation of medium-chain glycerides A method for preparing medium-chain glycerides by esterification catalyzed by flaky zeolite-immobilized Antarctic Candida lipase B: (1) Immobilization of Antarctic Candida lipase B using flaky zeolite ZSM-5-P, the method is as follows: Tethyl orthosilicate was slowly added to a tetrapropylammonium hydroxide solution, and then isopropyl alcohol and sodium hydroxide, aluminum nitrate nonahydrate and urea were sequentially added to the obtained system and mixed, wherein the molar ratio of SiO2:Al2O3:TPAOH:urea:H2O:NaOH:IPA in the mixed solution was 1:0.0074:0.3:0.6:29:0.05:0.03. The obtained mixed solution was stirred and aged at room temperature to obtain a mixed gel, and the mixed gel was hydrothermally crystallized at a temperature of 180°C for 48 hours, washed, dried, ground, and calcined to obtain a flake zeolite ZSM-5-P; the flake zeolite ZSM-5-P was then mixed with an aqueous solution of ammonium nitrate (1 mol / L) at a solid-liquid mass volume (g / mL) ratio of 1:10, and ion exchange was carried out in an oil bath at 80°C for 12 hours, and then washed and dried to obtain a proton-type zeolite HZSM-5-P; the obtained proton-type zeolite HZSM-5-P was mixed with (3-mercaptopropyl)trimethoxysilane and n-hexane at a mass ratio of 2:1:66, and reacted at room temperature for 24 hours. h. After the reaction is completed, the thiolated zeolite ZSM-5-P-SH is obtained by washing and drying. The thiolated zeolite ZSM-5-P-SH is then reacted with 9% (based on the weight of zeolite, w / w) Antarctic Candida lipase B in a water bath at 37°C for 12 h, and then washed and dried to obtain a flaky zeolite-immobilized Antarctic Candida lipase B catalyst.

[0041] (2) adding the reactants capric acid and glycerol into a round-bottom flask, wherein the molar ratio of capric acid to glycerol is 0.5:1-4:1; (3) adding 2.3% of the total mass of decanoic acid to the reactants and immobilized Candida antarctica lipase B catalyst; (4) Stirring for esterification reaction at a temperature of 35°C-90°C for 4 hours. The product after the reaction is separated by centrifugation and the upper layer is collected to obtain medium-chain glycerides.

[0042] The reaction solution was taken regularly for medium-chain glyceride analysis. The results were as follows: Figure 5 As shown. Figure 5 It can be seen that as the temperature increases, the conversion rate of capric acid gradually increases. When the reaction temperature is 70°C, the conversion growth rate of capric acid is the highest, reaching 67.1%, and the distribution rates of the product medium-chain monoglyceride, medium-chain diglyceride and medium-chain triglyceride are 69.5%, 28.0% and 2.5%; then as the reaction temperature increases, the conversion growth rate of capric acid tends to be flat. Therefore, the preferred esterification reaction temperature is 70°C-90°C.

[0043] Example 5: Effect of different catalyst addition amounts on the preparation of medium-chain glycerides A method for preparing medium-chain glycerides by esterification catalyzed by flaky zeolite-immobilized Antarctic Candida lipase B: (1) Immobilization of Antarctic Candida lipase B using flaky zeolite ZSM-5-P, the method is as follows: Slowly add tetrapropylammonium hydroxide solution to tetrapropylammonium silicate, then add isopropanol and sodium hydroxide, aluminum nitrate nonahydrate, and urea to the obtained system in sequence, wherein the molar ratio of SiO2:Al2O3:TPAOH:urea:H2O:NaOH:IPA in the mixed solution is 1:0.0074:0.3:0.6:29:0.05:0.03. The obtained mixed solution is stirred and aged at room temperature to obtain a mixed gel, which is hydrothermally crystallized at 180°C for 48 hours, washed, dried, ground, and calcined to obtain a sheet zeolite ZSM-5-P; the sheet zeolite ZSM-5-P is then mixed with an ammonium nitrate aqueous solution (1 mol / L) at a solid-liquid mass volume (g / mL) ratio of 1:10, and ion exchange is carried out in an 80°C oil bath for 12 hours, and then washed and dried to obtain a proton zeolite HZSM-5-P; the obtained proton zeolite ZSM-5-P was mixed with (3-mercaptopropyl)trimethoxysilane and n-hexane in a mass ratio of 2:1:66 and reacted at room temperature for 24 hours. After the reaction, the mixture was washed and dried to obtain the thiolated zeolite ZSM-5-P-SH. The thiolated zeolite ZSM-5-P-SH was then reacted with 9% (based on the weight of the zeolite, w / w) Antarctic Candida lipase B in a water bath at 37°C for 12 hours, and then washed and dried to obtain the flaky zeolite-immobilized Antarctic Candida lipase B catalyst.

[0044] (2) adding the reactants capric acid and glycerol into a round-bottom flask, wherein the molar ratio of capric acid to glycerol is 0.5:1-4:1; (3) adding 1.6% to 4.3% of the total mass of decanoic acid to the reactants and immobilized Candida antarctica lipase B catalyst; (4) The esterification reaction was carried out with stirring at a temperature of 40°C for 4 hours. The product after the reaction was separated by centrifugation and the upper layer was collected to obtain medium-chain glycerides.

[0045] The reaction solution was taken regularly for medium-chain glyceride analysis. The results were as follows: Figure 6 As shown. Figure 6 It can be seen that when the amount of immobilized lipase catalyst is 2.3% (based on the weight of capric acid, w / w), the conversion rate of capric acid reaches 56.7%, and the distribution rates of medium-chain monoglycerides, medium-chain diglycerides and medium-chain triglycerides are 67.8%, 29.6% and 2.6% respectively. After that, as the amount of immobilized lipase increases, the conversion rate of capric acid tends to be stable. Therefore, the preferred amount of catalyst added is 2.3-4.3% (based on the weight of capric acid, w / w).

[0046] Example 6: Effect of catalyst recycling times on the preparation of medium-chain glycerides A method for preparing medium-chain glycerides by esterification catalyzed by flaky zeolite-immobilized Antarctic Candida lipase B: (1) Immobilization of Antarctic Candida lipase B using flaky zeolite ZSM-5-P, the method is as follows: Slowly add tetrapropylammonium hydroxide solution to tetrapropylammonium silicate, then add isopropanol and sodium hydroxide, aluminum nitrate nonahydrate, and urea to the obtained system in sequence, wherein the molar ratio of SiO2:Al2O3:TPAOH:urea:H2O:NaOH:IPA in the mixed solution is 1:0.0074:0.3:0.6:29:0.05:0.03. The obtained mixed solution is stirred and aged at room temperature to obtain a mixed gel, which is hydrothermally crystallized at 180°C for 48 hours, washed, dried, ground, and calcined to obtain a sheet zeolite ZSM-5-P; the sheet zeolite ZSM-5-P is then mixed with an ammonium nitrate aqueous solution (1 mol / L) at a solid-liquid mass volume (g / mL) ratio of 1:10, and ion exchange is carried out in an 80°C oil bath for 12 hours, and then washed and dried to obtain a proton zeolite HZSM-5-P; the obtained proton zeolite ZSM-5-P was mixed with (3-mercaptopropyl)trimethoxysilane and n-hexane in a mass ratio of 2:1:66 and reacted at room temperature for 24 hours. After the reaction, the mixture was washed and dried to obtain the thiolated zeolite ZSM-5-P-SH. The thiolated zeolite ZSM-5-P-SH was then reacted with 9% (based on the weight of the zeolite, w / w) Antarctic Candida lipase B in a water bath at 37°C for 12 hours, and then washed and dried to obtain the flaky zeolite-immobilized Antarctic Candida lipase B catalyst.

[0047] (2) adding the reactants capric acid and glycerol into a round-bottom flask, wherein the molar ratio of capric acid to glycerol is 0.5:1-4:1; (3) adding 2.3% of the total mass of decanoic acid to the reactants and immobilized Candida antarctica lipase B catalyst; (4) stirring to carry out esterification reaction at a temperature of 40°C for a reaction time of 4 hours, separating the product after the reaction by centrifugation, and taking the upper layer to obtain medium-chain glycerides; (5) The lower catalyst layer was washed with buffer solution and n-hexane and then vacuum dried. Steps (3) and (4) were repeated and the process was repeated five times.

[0048] After each reaction, the reaction solution was taken for medium-chain glyceride analysis. The results were as follows: Figure 7 As shown. Figure 7 It can be seen that the conversion rate of capric acid after the first reaction is 61.1%, and the conversion rate of capric acid after the fifth reaction is 50.7%, which only decreases by 10.4% after five cycles, indicating that the immobilized Antarctic Candida lipase B catalyst obtained by the present invention can be recycled for multiple times.

[0049] Comparative Example 1 The preparation method of medium-chain glycerides comprises the following steps: adding reactants capric acid and glycerol in a reaction device in a molar ratio of capric acid to glycerol of 1:1, then adding a catalyst of Antarctic Candida lipase B fixed with macroporous acrylic resin in an amount of 2.3% of the total mass of capric acid, stirring for transesterification reaction, the reaction temperature is 40°C, the reaction time is 4h, and after the reaction, the product medium-chain glycerides are obtained.

[0050] After 4 hours of reaction, the reaction solution was taken for medium-chain glyceride content analysis, and the conversion rate of capric acid was calculated to be 39.4% based on the determined medium-chain glyceride content.

[0051] Test example The flake zeolite immobilized Candida antarctica lipase B catalyst prepared in Example 2 was used as a test sample for characterization. The results are as follows: Figure 1 The scanning electron microscope (SUPRA 55, Carl Zeiss AG, Germany) was used under high vacuum and an accelerating voltage of 5 kV to obtain the scanning electron microscope image. Figure 1 (a) is shown in the figure; the transmission electron microscope (JEM-2100, JEOL Ltd.) was used at an accelerating voltage of 200 kV, a point resolution of 0.23 nm, and a line resolution of 0.14 nm. Figure 1 As shown in (b). Figure 1 (a) It can be observed that the zeolite-immobilized Candida antarctica lipase B catalyst is in the form of two-dimensional long hexagonal sheets. Figure 1 The transmission electron microscopy image in (b) further illustrates the above phenomenon.

[0052] From the above embodiments and comparative examples, it can be seen that in the reaction of preparing medium-chain glycerides from capric acid and glycerol by fixing Antarctic Candida lipase B with macroporous acrylic resin as a catalyst, the conversion rate of capric acid is 39.4%, while under the same conditions, the conversion rate of capric acid is as high as 56.7% in the reaction of preparing medium-chain glycerides from capric acid and glycerol with fixing Antarctic Candida lipase B with flake zeolite ZSM-5-P as a catalyst, and the conversion rate of capric acid is increased by 17.3%, and the catalytic efficiency is greatly improved. This is mainly because the surface area of ​​​​the flake zeolite ZSM-5 is larger than that of the macroporous acrylic resin, and the zeolite-loaded lipase is more than that of the macroporous acrylic resin, so the conversion rate is higher; the two-dimensional flake zeolite ZSM-5 is more conducive to the diffusion adsorption of reactants and the desorption of products; the macroporous acrylic acid fixation of lipase is mainly physical adsorption, which is easy to fall off and reduce activity, while the flake zeolite ZSM-5-P uses covalent bonding to fix lipase, which has strong interaction and good stability.

[0053] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A flaky zeolite-immobilized Antarctic Candida lipase B catalyst, characterized in that: The flake zeolite is flake silica-alumina zeolite ZSM-5-P, and the loading amount of the Antarctic Candida lipase B is 9-12% of the mass of the flake zeolite.

2. The flaky zeolite-immobilized Candida antarctica lipase B catalyst according to claim 1, characterized in that: The preparation method of the catalyst comprises the following steps: (1) adding tetraethyl orthosilicate slowly to a tetrapropylammonium hydroxide solution, then sequentially adding isopropyl alcohol and sodium hydroxide, aluminum nitrate nonahydrate, and urea to the obtained system, stirring and aging the obtained mixture at room temperature to obtain a mixed gel, and then washing, drying, grinding, and calcining the mixed gel after hydrothermal crystallization to obtain a sheet-like zeolite ZSM-5-P; (2) mixing the flaky zeolite ZSM-5-P obtained in step (1) with an aqueous solution of ammonium nitrate, and subjecting the mixture to ion exchange in an oil bath at 80° C. for 12 h, followed by washing and drying to obtain a proton-type zeolite HZSM-5-P; (3) reacting the proton-type zeolite HZSM-5-P obtained in step (2) with (3-mercaptopropyl)trimethoxysilane and n-hexane at room temperature for 24 h, washing and drying to obtain mercapto zeolite ZSM-5-P-SH; (4) The thiolated zeolite ZSM-5-P-SH obtained in step (3) was reacted with Antarctic Candida lipase B (CALB) in a 37°C water bath for 12 h, and then washed and dried to obtain a flaky zeolite-immobilized Antarctic Candida lipase B catalyst.

3. The flaky zeolite-immobilized Antarctic Candida lipase B catalyst according to claim 2, characterized in that: In step (1), the molar ratio of SiO2:Al2O3:TPAOH:urea:H2O:NaOH:IPA in the mixed solution is 1:0.0074:0.3:0.6:29:0.05:0.03; the temperature of the hydrothermal crystallization is 180°C, and the treatment time is 48 h.

4. The flaky zeolite-immobilized Candida antarctica lipase B catalyst according to claim 2, characterized in that: In step (2), the sheet-like zeolite ZSM-5-P is mixed with an aqueous ammonium nitrate solution at a solid-liquid mass volume ratio of 1:10, and the concentration of ammonium nitrate in the aqueous ammonium nitrate solution is 1 mol / L.

5. The flaky zeolite-immobilized Antarctic Candida lipase B catalyst according to claim 2, characterized in that: In step (3), the mass ratio of the proton-type zeolite HZSM-5-P to (3-mercaptopropyl)trimethoxysilane and n-hexane is 2:1:

66.

6. A method for preparing medium-chain glycerides by esterification catalyzed by lipase B of Candida antarctica immobilized on zeolite, characterized in that: The following steps are involved: S1. Adding reactants capric acid and glycerol sequentially into a round-bottom flask reaction apparatus; S2. Adding the flake zeolite-immobilized Antarctic Candida lipase B catalyst to the reactant of S1 as claimed in any one of claims 1 to 5; S3. At the set temperature, the esterification reaction is carried out with stirring. The product after the reaction is separated by centrifugation, and the upper layer liquid is taken to obtain medium-chain glycerides.

7. The method according to claim 6, characterized in that In S1, the molar ratio of capric acid to glycerol is 0.5:1-1:1, preferably 1:

1.

8. The method according to claim 6, characterized in that In S2, the addition amount of the flake zeolite-immobilized Candida antarctica lipase B catalyst is 1.6-4.3% of the total mass of the capric acid, preferably 2.3-4.3%.

9. The method according to claim 6, characterized in that In S3, the esterification reaction is carried out at a temperature of 35-90°C and for a time of 2-6 hours.

10. The method according to claim 9, characterized in that The temperature of the esterification reaction is 70° C. and the time is 4 hours.

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