An enzymatic preparation method for MLCT-rich oil and its product

Abstract

The invention discloses an enzymatic preparation method for MLCT-rich grease and its product, comprising the following steps: (1) in a paraffin-water emulsion template system, preparing hydrophobic hollow SiO2 mesoporous microspheres (HMSB) as a hydrophobic carrier using an organic hydrophobic silicon source and a porogen in a one-step method; then immobilizing the free enzyme on the hydrophobic carrier by an adsorption method to obtain an immobilized lipase; (2) mixing long-chain triglycerides and a medium-carbon chain raw material, adding immobilized lipase, and performing an enzymatic transesterification reaction or an acidolysis reaction under a constant temperature water bath for a certain time; after the reaction is completed, solid-liquid separation is performed, and the obtained solid product is immobilized lipase, and the obtained solution is a crude grease product rich in MLCT, and then a small amount of fatty acids are removed by molecular distillation or physical adsorption to obtain a grease product rich in MLCT. The present invention has the advantages of wide raw material adaptability, high catalytic efficiency, high MCLT content, controllable functional fatty acid content, simple operation, green and environmental protection, and is suitable for large-scale amplification production.

Description

Technical Field

[0001] The invention belongs to the field of oil modification and lipid deep processing, and particularly relates to a method for synthesizing MLCT by utilizing immobilized enzyme-catalyzed transesterification. Background Art

[0002] Medium-chain triglycerides (MLCT) contain both medium-chain and long-chain fatty acids. They are a type of structural lipid with special nutritional and physiological functions. They have the advantages of both long-chain triglycerides and medium-chain triglycerides, improve the nutritional functions of natural oils and fats, and have different metabolic pathways in the human body. They can not only quickly supply energy to the human body, but also provide the required essential fatty acids. They have positive effects on diabetes, heart disease, hyperlipidemia, hypertension and other diseases, and are an important component of functional foods. In addition, MLCT metabolites can improve the body's nitrogen balance, increase the utilization rate of nutrients, and reduce the production of inflammatory mediators. They can be used in clinical injectable fat emulsions, and natural oils can be put into pharmaceutical applications. They are an important component of parenteral nutrition. However, there are currently few types of MLCT products, so it is necessary to develop preparation methods for MLCT products with different fatty acid compositions to meet the needs of different scenarios.

[0003] Traditional MLCT production relies on chemical synthesis, but the chemical catalysts used have low selectivity and random fatty acid placement on the glycerol backbone. Furthermore, there are numerous byproducts, difficulty separating the product, high energy consumption, and environmental pollution. CN103891920 A discloses a method for preparing MLCT through chemical transesterification using sodium methoxide as a catalyst. However, separation of the product and catalyst requires cumbersome steps, including centrifugation, water washing, and vacuum drying. The resulting wastewater is also environmentally unfriendly and exhibits poor selectivity. In contrast, enzymatic catalysis for preparing MLCT can target the position of fatty acids on the glycerol backbone. The catalytic process is environmentally friendly, safe, and economical, overcoming many of the disadvantages of chemical catalysis. Because free enzymes are environmentally sensitive and easily deform and inactivate in high temperatures, organic reagents, and acidic and alkaline environments, this limits their use under various conditions. Immobilizing the enzyme not only improves its thermal tolerance but also its tolerance to organic reagents, facilitates separation of the enzyme from the product, and allows for reusable enzymes. Overall, the use of specialized immobilized enzyme catalysts offers numerous advantages, including diversified MLCT oil and fat production and simplified post-processing.

[0004] Currently, commercial immobilized lipases, such as NOV435, RMIM, and TLIM, are commonly used for catalysis. These enzymes are expensive, and free enzymes are prone to swelling when adsorbed on materials such as macroporous acrylic resins, limiting enzyme reuse and hindering the industrial production of MLCT. CN 107058413 A discloses a method for synthesizing medium- and long-chain triglycerides through enzymatic transesterification. The invention uses camphor seed kernel oil and soybean oil as raw materials, and uses immobilized lipase NOV 435 to catalyze transesterification to produce medium- and long-chain triglycerides. However, the reaction still requires more than 2 hours, and the method does not mention enzyme recycling. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an enzymatic preparation method for MLCT-rich oil in response to the deficiencies in the above-mentioned prior art, which has the advantages of high catalytic efficiency, high nutritional value of the product, simple operation, green and environmental protection, simple product separation, and suitability for large-scale production.

[0006] The technical solution adopted by the present invention to solve the above-mentioned problems is:

[0007] An enzymatic preparation method for MLCT-rich oil comprises the following steps:

[0008] (1) Preparation of immobilized lipase: In a paraffin-water emulsion template system, hydrophobic hollow SiO2 mesoporous microspheres (HMSB) were prepared as hydrophobic carriers using an organic hydrophobic silicon source and a porogen in a one-step method. The free enzyme was then immobilized on the hydrophobic carrier by adsorption to obtain immobilized lipase.

[0009] (2) Enzymatic preparation of MLCT oil: long-chain triglycerides and medium-chain raw materials are mixed, immobilized lipase is added, and an enzymatic transesterification reaction or acidolysis reaction is carried out in a constant temperature water bath for a certain period of time; after the reaction is completed, the solid and liquid are separated, and the solid product obtained is the immobilized lipase immobilized enzyme, and the obtained solution is a crude oil product rich in MLCT, and then a small amount of fatty acids are removed by molecular distillation or physical adsorption to obtain a finished oil product rich in MLCT. Wherein, the medium-chain raw material is medium-chain fatty acid triglyceride or medium-chain fatty acid; when the medium-chain raw material is medium-chain fatty acid triglyceride, step (2) undergoes an enzymatic transesterification reaction; when the medium-chain raw material is medium-chain fatty acid, step (2) undergoes an enzymatic hydrolysis reaction.

[0010] According to the above scheme, the size of the hydrophobic hollow SiO2 mesoporous microspheres (HMSB) is between 200nm and 2μm, has a cavity structure, a contact angle of more than 110°, and a pore size distribution of 3-10nm.

[0011] According to the above scheme, the specific preparation process of hydrophobic hollow SiO2 mesoporous microspheres is as follows: liquid paraffin and water are mixed in a certain volume ratio and placed in a container, surfactants and pore-forming agents are added, and ultrasonic homogenization is performed to obtain an emulsion; the emulsion is poured into a hydrophobic silicon source solution, placed in a low-temperature water bath, stirred for a certain period of time, filtered, washed with isooctane, dried, and then acid-washed to remove the pore-forming agent and surfactant, and finally filtered and dried to obtain HMSB.

[0012] Furthermore, in the specific preparation process of hydrophobic hollow SiO2 mesoporous microspheres, the volume ratio of paraffin to water is 2:1-8:1; the surfactant is one or more of oil-in-water emulsifiers such as monostearate, Span 80, Span 20, polyethylene glycol 200, polyglycerol fatty acid ester, sorbitan monolaurate, etc., and the added amount is 1.0-2.5% of the water mass; the pore-forming agent includes but is not limited to block polyether F127, triblock copolymer P123, etc., and the added amount is 1%-6% of the water mass.

[0013] Furthermore, in the specific preparation process of hydrophobic hollow SiO2 mesoporous microspheres, the hydrophobic silicon source can be one or more of methyltrichlorosilane (MTCS), ethyltrichlorosilane, butyltrichlorosilane, octyltrichlorosilane, etc., and the amount of hydrophobic silicon source added is 75%-120% of the water mass; the hydrophobic silicon source solution is prepared by dispersing the hydrophobic silicon source in isooctane, and the volume ratio of the hydrophobic silicon source to the isooctane is 3:(7-10); the temperature of the low-temperature water bath is 4-15°C; the pickling solution is a mixture of water, ethanol and hydrochloric acid in a volume ratio of 2:1:1; the pickling temperature is 60-100°C, and the time is 6-8h.

[0014] According to the above scheme, the specific process of preparing immobilized lipase by adsorption method is as follows: the free enzyme is added to phosphate buffer to prepare a certain enzyme solution concentration, and then mixed with a hydrophobic carrier at a certain solid-liquid ratio (the ratio between the mass of HMSB and the volume of the enzyme solution), incubated on a shaking table to achieve immobilization, and finally centrifuged and filtered, washed three times with PBS, and freeze-dried to obtain immobilized lipase.

[0015] Furthermore, in the specific process of preparing immobilized lipase by adsorption, the free enzyme is one or more of Candida rugosa lipase, Candida antarctica lipase, NS40086 lipase, Thermomyces lanuginosus, and Candida lipolytica lipase; the enzyme solution has a pH of 7.5-9.5 and a concentration of 10-100 mg / mL; the ratio of HMSB mass to enzyme solution volume is 1:100-3.5:100 (m / v, g / mL); the immobilization time is 30-90 minutes, and the temperature is 20-40°C; and the pH of the phosphate buffer is 7.5-9.5. During the enzyme immobilization process, the phosphate buffer has a significant impact on the amount of enzyme immobilized, so the pH of the phosphate buffer is set to a neutral to alkaline pH of 7.5-9.5. The temperature has little effect and is generally suitable at room temperature.

[0016] According to the above scheme, the carbon number of the fatty acid chain of the medium-chain fatty acid triglyceride is 6 to 12, preferably caprylic acid triglyceride rich in caprylic acid (C8) and capric acid (C10); the medium-chain fatty acid is a mixture of one or more of caprylic acid, capric acid or lauric acid in any proportion.

[0017] According to the above scheme, the long-chain triglyceride is a mixture of one or more of linseed oil, perilla oil, hemp oil, evening primrose oil, peony seed oil, sunflower oil, soybean oil, sunflower oil, conjugated linoleic acid glyceride, DHA algae oil, fish oil, garlic fruit oil, Xanthoceras sorbifolia oil, etc. in any proportion.

[0018] According to the above scheme, in step (2), the mass ratio of medium-chain triglycerides to long-chain triglycerides is 2:3-2:5 (m / m); or, the molar ratio of long-chain triglycerides to medium-chain fatty acids is 1:3-1:6.

[0019] According to the above scheme, in step (2), the amount of the immobilized enzyme added is 4%-8% of the substrate mass.

[0020] According to the above scheme, in step (2), the temperature of the constant temperature water bath is 50-90°C

[0021] According to the above scheme, in step (2), the time for the transesterification reaction is 10-60 minutes; the time for the acidolysis reaction is 1-3 hours.

[0022] According to the above scheme, in step (2), the molecular distillation has a feed rate of 10-50 mL / min, a distillation pressure of 30-50 Pa, a heating temperature of 210-260° C., and a scraper speed of 240-350 r / min.

[0023] According to the above scheme, the immobilized lipase separated in step (2) can be washed with PBS and freeze-dried before being reused; the immobilized lipase is reused 10 times, and the MLCT content in the prepared MLCT-rich oil product is still maintained at above 54%.

[0024] The MLCT-rich oil product prepared by the above method has an acid value between 0.5 and 2.0 mg KOH / g, and the mass percentage of MLCT in triglycerides is between 50% and 82%. The functional fatty acids contained in different types of MLCT include linolenic acid, nervonic acid, oleic acid, linoleic acid, DHA, EPA, ARA, etc., and the content of functional fatty acids in MLCT is above 55%. The fatty acids at the Sn1,3 positions can be medium-chain fatty acids such as caprylic acid, capric acid, and lauric acid. The total content of triglycerides is greater than 90%, and the content of diglycerides is 0.1-2.5%. All indicators meet the standard requirements for edible oils.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention uses an emulsion template method to prepare hydrophobic hollow SiO2 mesoporous microspheres as carriers. The entire process of carrier preparation and hydrophobic modification is completed in one step, without the need for organic solvents such as toluene required for post-modification. It also overcomes the cumbersome steps of existing carrier preparation requiring calcination to remove the template agent and subsequent grafting of hydrophobic groups. The preparation process is simple, the raw materials are economical and easily available, the time consumption is short, the yield is high, it is easy to scale up, and it is green, safe and pollution-free.

[0027] 2. The present invention uses the above-mentioned carrier hydrophobic hollow SiO2 mesoporous microspheres to immobilize free enzymes, and their activity and stability are higher than those of free enzymes and commercial enzymes; moreover, the mesoporous structure of the carrier can not only efficiently load the enzyme, but also facilitates mass transfer and heat transfer of the reaction substrate in the system.

[0028] 3. Based on the above, the immobilized lipase and preparation method of the present invention can be widely used to catalyze the transesterification or acidolysis reaction of long-chain triglycerides and medium-chain triglycerides or medium-chain fatty acids. Different raw materials can be selected according to demand to prepare MLCT structured esters rich in different types of fatty acids, such as algae oil and MCT can prepare DHA-rich structured esters, and linseed oil and MCT can prepare linolenic acid-rich structured esters. The MLCT content and fatty acid composition of the obtained MLCT-rich oil product are controllable, and the products are diverse, meeting the needs of different application scenarios. In addition, the crude product obtained by the enzymatic transesterification reaction or acidolysis reaction of the present invention only needs simple deacidification to prepare a finished oil product rich in MLCT, without the need for complex separation and purification steps, and the MLCT content is high.

[0029] 4. The entire process of the present invention adopts solvent-free enzyme catalysis, with mild reaction conditions, short reaction time and simple separation. Compared with traditional chemical catalysis methods, it does not produce by-products, large amounts of wastewater and secondary waste, effectively saving costs. Compared with non-aqueous enzyme catalysis methods, it does not require organic solvents, has high raw material concentration and product yield, and the immobilized enzyme can be regenerated and reused multiple times, which has great potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the SEM image of the carrier material obtained in Example 1.

[0031] Figure 2 is the contact angle of the support material obtained in Example 1 with water.

[0032] Figure 3 The pore size distribution diagram of the carrier material obtained in Example 1

[0033] Figure 4 Schematic diagram of the enzymatic transesterification reaction between medium-chain fatty acid triglycerides and long-chain fatty acid triglycerides.

[0034] Figure 5 Schematic diagram of the enzymatic acidolysis reaction of medium-chain fatty acids and long-chain fatty acid triglycerides.

[0035] Figure 6 The gas chromatograms are of the finished medium- and long-chain triglycerides synthesized by the transesterification reaction of flaxseed oil and MCT oil in Example 1 and of the physical mixture of flaxseed oil and MCT oil.

[0036] Figure 7 These are the gas chromatograms of the finished medium- and long-chain triglycerides synthesized by the transesterification reaction of algae oil and MCT oil in Example 4, and the gas chromatogram of the physical mixture of algae oil and MCT oil.

[0037] Figure 8 The content of medium- and long-chain triglycerides synthesized by 10 transesterification reactions of linseed oil and MCT oil catalyzed by the immobilized enzyme in Example 1.

[0038] Among them, MLCT stands for medium-chain triglycerides, LCT stands for long-chain triglycerides, and MCT stands for medium-chain triglycerides. DETAILED DESCRIPTION

[0039] In order to better understand the present invention, the content of the present invention is further illustrated below with reference to specific examples, but the present invention is not limited to the following examples.

[0040] MLCT products produced by enzyme-catalyzed transesterification using hollow mesoporous silica spheres were primarily detected by gas chromatography. The following conditions were used: a DB-5HT column (15 m × 0.320 mm, 0.10 μm), an inlet temperature of 380°C, an injection volume of 1 μL, and a split ratio of 50:1. High-purity helium was used as the carrier gas at a flow rate of 2 mL / min. Hydrogen flow was 32 mL / min, and air flow was 200 mL / min. The detector was an FID at 380°C. The column oven temperature program was as follows: an initial temperature of 170°C for 2 minutes, then a temperature increase of 5°C / min to 380°C for 6 minutes.

[0041] Glyceride content was determined by liquid chromatography under the following conditions: a Venusil XBP Silica 5 μm 4.6 × 250 mm column, an ELSD detector, a 30 mg / mL sample injection volume of 10 μL, a column temperature of 35°C, and an isocratic mobile phase of n-hexane / isopropanol / formic acid (15:1:0.03, v:v:v) at a flow rate of 1 mL / min.

[0042] Glyceride structure determination, chromatographic system: Shimadzu UPLC LC-30A; column: Phenomenex Kinete C18 column (100 × 2.1 mm, 2.6 μm); injection volume: 3 μL; flow rate: 0.4 mL / min; column temperature: 60°C; sample chamber temperature: 4°C. Phase A: water: methanol: acetonitrile (1:1:1 containing 5 mM ammonium acetate); phase B: isopropanol: acetonitrile (5:1 containing 5 mM ammonium acetate). Gradient elution conditions: 0.5 min, 20% phase B; 1.5 min, 40% phase B; 3 min, 60% phase B; 13 min, 95% phase B, hold for 7 min; 20% phase B for 20.1 min; 20% phase B for 25 min. Mass spectrometry system: AB Sciex TripleTOF 6600, ESI ion source, positive mode, mass range of mass spectrometry acquisition: m / z 100-1200, mass spectrometry conditions: Curtain Gas: 35.000 psi; Ion Source Gas 1: 50.000; Ion Source Gas 2: 50.00; Ion Spray Voltage: 5500.00 V; Temperature: 600°C.

[0043] Example 1

[0044] An enzymatic preparation method for MLCT-rich oil specifically comprises the following steps:

[0045] (1) Preparation of hydrophobic hollow mesoporous silica spheres: 16 mL of paraffin and 4 mL of water were placed in a beaker, 0.1 g of glyceryl monostearate was added, 0.1 g of F127 was added, ultrasonicated for 5 min, and homogenized at 5000 rpm for 5 min to prepare an emulsion. 3 mL of MTCS was dissolved in 8 mL of isooctane to obtain an MTCS solution, and the emulsion was poured into the MTCS solution and stirred at 10 ° C for 3 h. The above solution was filtered to obtain solid particles, and then isooctane was added to clean the paraffin on the surface of the material, filtered, and the obtained material was dried in an oven at 60 ° C overnight. The dried material was added to a mixed solution containing 200 mL of water, 100 mL of ethanol, and 100 mL of hydrochloric acid, stirred at 60 ° C for 6 h, and dried again to obtain a carrier-hydrophobic hollow mesoporous silica sphere with a morphology as shown. Figure 1 As shown, the size is between 200nm-2μm, and the contact angle is Figure 2 As shown, the pore size distribution is Figure 3 As shown, it is 3-10nm.

[0046] (2) Enzyme immobilization: 3 g of free enzyme Candida lipolytica lipase (CSL) was dissolved in 50 mL of phosphate buffer solution (50 mM) at pH 8.5. The carrier and enzyme solution were mixed at a solid-liquid ratio of 10 mg / mL, incubated on a shaking table at 30°C for 40 min, and centrifuged. Then, the precipitate was freeze-dried to obtain immobilized lipase.

[0047] (3) Catalytic transesterification reaction: 0.6 g of linseed oil and 0.4 g of medium-chain triglyceride (MCT, 42% caprylic acid, 58% capric acid) were placed in an 8 mL reaction bottle, 0.06 g of the above-mentioned immobilized lipase was added, and the mixture was stirred under magnetic stirring for 15 min. The supernatant was centrifuged and the supernatant was obtained, which was a crude product rich in MLCT. The crude product was molecularly distilled at a feed rate of 10 mL / min, a distillation pressure of 30 Pa, a heating temperature of 220°C, and a scraper speed of 240 r / min to obtain a refined MLCT-rich oil.

[0048] The acid value of the product was determined to be 0.6 mg KOH / g, the triglyceride content was 96.1%, the diglyceride content was 1.5%, and the proportion of MLCT in triglycerides was 77.3%. In addition, the linolenic acid content in the product was 33.18%, the total contents of caprylic acid and capric acid were 20.83% and 16.33%, respectively, and the sn-1,3 fatty acids of caprylic acid and capric acid were 23.55% and 16.44%, respectively (as shown in Table 2).

[0049] Table 1 shows the mass spectrometry data of the finished medium- and long-chain triglycerides synthesized by the transesterification reaction of linseed oil and MCT oil in Example 1. It can be seen that the diglyceride content is 1.00%, the MLCT content is 79.71%, and the content of caprylic acid, capric acid, and linolenic acid triglycerides (8:0-10:0-18:3) reaches a maximum of 14.57%.

[0050] Table 2 shows the fatty acid composition of the linseed oil and the fatty acid composition of the transesterified oil in Example 1. The caprylic acid and capric acid contents in the transesterified oil are 20.83% and 16.33%, respectively.

[0051] Table 1

[0052]

[0053] Note: The statistical data in the table are ingredients with glyceride content greater than 1%.

[0054] Table 2

[0055]

[0056] Note: The unit of data in Table 2 is %.

[0057] Example 2

[0058] An enzymatic preparation method for MLCT-rich oil specifically comprises the following steps:

[0059] (1) Preparation of carrier hydrophobic hollow mesoporous silica spheres: 16 mL of paraffin wax and 4 mL of water were placed in a beaker, 0.1 g of Span 80 and 0.1 g of F127 were added, ultrasonicated for 5 min, and homogenized at 5000 rpm for 5 min to prepare an emulsion. 3 mL of ethyltrichlorosilane was dissolved in 15 mL of isooctane to obtain an ethyltrichlorosilane solution, and the emulsion was poured into the solution and stirred at 8°C for 3 h. The above solution was filtered to obtain solid particles, and isooctane was added to clean the paraffin wax on the surface of the material. The material was filtered and dried in an oven at 60°C overnight. The dried material was added to a mixed solution containing 200 mL of water, 100 mL of ethanol, and 100 mL of hydrochloric acid, stirred at 60°C for 6 h, and dried again to obtain the carrier.

[0060] (2) Enzyme immobilization: 2 g of free enzyme Candida rugosa lipase was dissolved in 50 mL of phosphate buffer solution with a pH of 9. The carrier and enzyme solution were mixed at a solid-liquid ratio of 10 mg / mL, incubated on a shaking table at 30°C for 40 min, and centrifuged. Then, the precipitate was freeze-dried to obtain immobilized lipase.

[0061] (3) Catalytic transesterification reaction: 0.6 g of sunflower oil and 0.4 g of medium-chain triglyceride (MCT, 42% caprylic acid, 58% capric acid) were placed in an 8 mL reaction bottle, 0.06 g of the above-mentioned immobilized lipase was added, and the mixture was stirred under magnetic stirring for 15 min. The supernatant was then centrifuged and the obtained crude product was molecularly distilled at a feed rate of 20 mL / min, a distillation pressure of 40 Pa, a heating temperature of 230°C, and a scraper speed of 260 r / min to obtain an MLCT-rich oil.

[0062] The acid value of the product was determined to be 0.9 mgKOH / g, the triglyceride content was 97.3%, the diglyceride content was 0.5%, and the proportion of MLCT to triglyceride was 77.4%.

[0063] Example 3

[0064] An enzymatic preparation method for MLCT-rich oil specifically comprises the following steps:

[0065] (1) Preparation of carrier hydrophobic hollow mesoporous silica spheres: 24 mL of paraffin wax and 4 mL of water were placed in a beaker, 0.04 g of polyglycerol fatty acid ester and 0.2 g of F127 were added, and the mixture was homogenized at 8000 rpm for 5 min to prepare an emulsion. 3 mL of octyltrichlorosilane was dissolved in 20 mL of isooctane to obtain an octyltrichlorosilane solution. The emulsion was poured into the solution and stirred at 8°C for 3 h. The solid particles were filtered, isooctane was added, and the excess paraffin was washed and filtered. The obtained material was dried in an oven at 60°C overnight. The dried material was added to a mixed solution containing 200 mL of water, 100 mL of ethanol, and 100 mL of hydrochloric acid, stirred at 60°C for 6 h, and dried again to obtain the carrier.

[0066] (2) Enzyme immobilization: 3 g of free enzyme Thermomyces lanuginosus lipase was dissolved in 50 mL of phosphate buffer solution (pH 8.5). The carrier and enzyme solution were mixed at a solid-liquid ratio of 20 mg / mL. The mixture was incubated on a shaker at 30°C for 40 min and centrifuged. The precipitate was freeze-dried to obtain immobilized lipase.

[0067] (3) Catalytic transesterification reaction: 0.6 g of sunflower oil and 0.4 g of medium-chain triglyceride (MCT, 42% caprylic acid, 58% capric acid) were placed in an 8 mL reaction bottle, 0.06 g of the above-mentioned immobilized lipase was added, and the mixture was stirred under magnetic stirring for 20 min. The supernatant was centrifuged and the crude product was obtained. The crude product was molecularly distilled at a feed rate of 30 mL / min, a distillation pressure of 40 Pa, a heating temperature of 250°C, and a scraper speed of 280 r / min to obtain a refined MLCT-rich oil.

[0068] The acid value of the product was determined to be 0.7 mgKOH / g, the triglyceride content was 97.8%, the diglyceride content was 1.0%, and the proportion of MLCT to triglyceride was 80.6%.

[0069] Example 4

[0070] An enzymatic preparation method for MLCT-rich oil specifically comprises the following steps:

[0071] (1) Preparation of carrier hydrophobic hollow mesoporous silica spheres: 30 mL of paraffin wax and 4 mL of water were placed in a beaker, 0.06 g of glycerol monostearate and 0.15 g of P123 were added, ultrasonicated for 5 min, and homogenized at 10,000 rpm for 5 min to prepare an emulsion. 3 mL of MTCS was dissolved in 15 mL of isooctane to obtain an MTCS solution, and the emulsion was poured into the MTCS solution and stirred at 10°C for 3 h. The above solution was filtered to obtain solid particles, and isooctane was added to clean the surface of the material and paraffin was filtered. The obtained material was dried in an oven at 60°C overnight. The dried material was added to a mixed solution containing 200 mL of water, 100 mL of ethanol, and 100 mL of hydrochloric acid, stirred at 60°C for 6 h, and dried again to obtain the carrier.

[0072] (2) Enzyme immobilization: 3 g of free enzyme Candida albicans lipase (CSL) was dissolved in 50 mL of phosphate buffer solution at pH 7. The carrier and enzyme solution were mixed at a solid-liquid ratio of 12 mg / mL. The mixture was incubated on a shaker at 25°C for 30 min and then centrifuged. The precipitate was freeze-dried to obtain immobilized lipase.

[0073] (3) Catalytic transesterification reaction: 1.2 g of algae oil and 0.8 g of medium-chain triglyceride (MCT, 42% caprylic acid, 58% capric acid) were placed in a 20 mL reaction bottle, 0.16 g of the above-mentioned immobilized lipase was added, and the mixture was stirred magnetically for 15 min and then centrifuged to obtain the supernatant to obtain a crude product; the crude product was molecularly distilled at a feed rate of 20 mL / min, a distillation pressure of 40 Pa, a heating temperature of 260°C, and a scraper speed of 290 r / min to obtain a refined MLCT-rich oil.

[0074] The product's acid value was determined to be 2.3 mg KOH / g, with a triglyceride content of 95.7% and a diglyceride content of 0.4%. The MLCT content of triglycerides was 72.5%. Furthermore, the DHA content was 33.43%. The total caprylic and capric acid contents were 19.12% and 15.18%, respectively. The sn-1,3 fatty acid content of caprylic and capric acids was 19.05% and 12.98%, respectively (Table 4).

[0075] Table 3 shows the mass spectrometry data of the finished medium- and long-chain triglycerides synthesized by the transesterification reaction of algae oil and MCT oil in Example 4. It can be seen that the diglyceride content is 1.18%, the MLCT content is 76.99%; the content of caprylic acid, capric acid, and DHA triglycerides (8:0-10:0-22:6) reaches a maximum of 11.35%.

[0076] Table 4 shows the fatty acid composition of the algae oil and the transesterified oil in Example 4. The DHA content in the transesterified oil was 33.43%, and the caprylic acid and capric acid content were 19.12% and 15.18%, respectively.

[0077] Table 3

[0078]

[0079] Note: The statistical data in the table are ingredients with glyceride content greater than 1%.

[0080] Table 4

[0081]

[0082]

[0083] Note: The unit of data in Table 2 is %.

[0084] Example 5

[0085] An enzymatic preparation method for MLCT-rich oil specifically comprises the following steps:

[0086] (1) Preparation of carrier hydrophobic hollow mesoporous silica spheres: 16 mL paraffin and 4 mL water were placed in a beaker, 0.1 g Span 80 and 0.1 g F127 were added, ultrasonicated for 5 min, and homogenized at 5000 rpm for 5 min to prepare an emulsion. 3 mL MTCS was dissolved in 10 mL isooctane to obtain an MTCS solution, and the emulsion was poured into the MTCS solution and stirred at 8 ° C for 3 h. The above solution was filtered to obtain solid particles, and isooctane was added to clean the surface of the material. The paraffin was filtered and the obtained material was dried in an oven at 60 ° C overnight. The dried material was added to a mixed solution containing 200 mL water, 100 mL ethanol, and 100 mL hydrochloric acid, stirred at 60 ° C for 6 h, and dried again to obtain the carrier.

[0087] (2) Enzyme immobilization: 2.5 g of free NS40086 lipase was dissolved in 100 mL of phosphate buffer solution (pH 8.5). The carrier and enzyme solution were mixed at a solid-liquid ratio of 20 mg / mL. The mixture was incubated on a shaker at 25°C for 50 min and then centrifuged. The precipitate was freeze-dried to obtain immobilized lipase.

[0088] (3) Catalytic transesterification reaction: 7 g of sunflower oil and 4 g of medium-chain triglycerides (MCT, 42% caprylic acid and 58% capric acid) were placed in a 50 mL reaction bottle, 0.8 g of the above-mentioned immobilized lipase was added, and the mixture was stirred under magnetic stirring for 15 min and then centrifuged to obtain the supernatant, which was the crude product. The crude product was molecularly distilled at a feed rate of 30 mL / min, a distillation pressure of 40 Pa, a heating temperature of 260°C, and a scraper speed of 300 r / min to obtain a refined MLCT-rich oil.

[0089] The acid value of the product was determined to be 1.6 mgKOH / g, with a triglyceride content of 97.8%, a diglyceride content of 0.8%, and a MLCT content of 70.9% of the triglyceride content.

[0090] Example 6

[0091] An enzymatic preparation method for MLCT-rich oil specifically comprises the following steps:

[0092] (1) Preparation of hydrophobic hollow mesoporous silica spheres: 16 mL of paraffin wax and 4 mL of water were placed in a beaker, 0.1 g of glycerol monostearate and 0.1 g of F127 were added, ultrasonicated for 8 min, and homogenized at 9000 rpm for 5 min to prepare an emulsion. 3 mL of MTCS was dissolved in 8 mL of isooctane to obtain an MTCS solution, and the emulsion was poured into the MTCS solution and stirred at 15 ° C for 3 h. The above solution was filtered to obtain solid particles, and isooctane was added to clean the surface of the material and paraffin was filtered. The obtained material was dried in an oven at 60 ° C overnight. The dried material was added to a mixed solution containing 200 mL of water, 100 mL of ethanol, and 100 mL of hydrochloric acid, stirred at 60 ° C for 6 h, and dried again to obtain a carrier.

[0093] (2) Enzyme immobilization: 3 g of free Candida lipolytica lipase (CSL) was dissolved in 50 mL of phosphate buffer solution (pH 8.5). The carrier and enzyme solution were mixed at a solid-liquid ratio of 25 mg / mL. The mixture was incubated on a shaking table at 30°C for 30 min and then centrifuged. The precipitate was freeze-dried to obtain immobilized lipase.

[0094] (3) Catalytic transesterification reaction: 0.6 g of garlic fruit oil and 0.4 g of medium-chain triglyceride (MCT, 42% caprylic acid, 58% capric acid) were placed in an 8 mL reaction bottle, 0.1 g of the above-mentioned immobilized lipase was added, and the mixture was stirred under magnetic stirring for 15 min. The supernatant was then centrifuged to obtain a crude product. The crude product was molecularly distilled at a feed rate of 50 mL / min, a distillation pressure of 50 Pa, a heating temperature of 250°C, and a scraper speed of 300 r / min to obtain a refined MLCT-rich oil.

[0095] The product's acid value was determined to be 0.8 mgKOH / g, with a triglyceride content of 95.7%, a diglyceride content of 0.4%, and MLCT accounting for 69.6% of triglycerides.

[0096] Example 7

[0097] The process is basically the same as Example 1, except that the amount of immobilized lipase added in step (3) is 10% (wt%), and the long-chain triglyceride used is perilla oil.

[0098] The acid value of the product was determined to be 1.3 mg KOH / g, the triglyceride content was 96.9%, the diglyceride content was 1%, and the proportion of MLCT to triglyceride was 78.0%.

[0099] Table 5 shows the MLCT content of the products in Examples 1, 2, 4, 6, and 7, as well as the content of specific fatty acids in MLCT. The MLCT content is 69.6%-78%, of which the specific fatty acids are more than 55%.

[0100] Table 5

[0101]

[0102] Example 8

[0103] The method is basically the same as Example 1, except that the immobilized lipase added in step (3) is the immobilized lipase obtained by centrifugation after the first use, which is then washed with PBS and freeze-dried.

[0104] It was determined that the acid value of the product was 1.8 mgKOH / g, the triglyceride content was 96.4%, the diglyceride content was 1.3%, and the proportion of MLCT to triglyceride was 75.1%.

[0105] Figure 8 This is the MLCT content after the immobilized enzyme was recycled ten times. The MLCT content can still reach 54.2% after the immobilized enzyme was used ten times.

[0106] Example 9

[0107] The reaction was essentially the same as Example 1, except that in step (3), 1.74 g of linseed oil, 1.2 g of octanoic acid, and 0.1 g of immobilized lipase were reacted at 70°C for 3 h. The crude product was then molecularly distilled to obtain the finished product. The finished product had an acid value of 1.9 mgKOH / g and an octanoic acid insertion rate of 33.3%.

[0108] Example 10

[0109] The process was essentially the same as Example 1, except that in step (3), 1.74 g of linseed oil was reacted with 0.6 g of octanoic acid, 0.6 g of decanoic acid, and 0.1 g of immobilized lipase at 70°C for 3 h. The crude product was then molecularly distilled to obtain the finished product. The finished product had an acid value of 1.7 mgKOH / g and an octanoic acid insertion rate of 35.9%.

[0110] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this field, several improvements and changes can be made without departing from the creative concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. An enzymatic preparation method for MLCT-rich oil, characterized in that The steps include: (1) Preparation of immobilized lipase: In a paraffin-water emulsion template system, hydrophobic hollow SiO2 mesoporous microspheres were prepared as carriers using an organic hydrophobic silicon source and a porogen in a one-step method; free enzyme was then immobilized on the hydrophobic carrier by adsorption to obtain immobilized lipase; (2) Enzymatic preparation of MLCT oil: long-chain triglycerides and medium-chain raw materials are mixed, and the immobilized lipase obtained in step (1) is added, and an enzymatic transesterification reaction or acidolysis reaction is carried out in a constant temperature water bath; after the reaction is completed, the solid-liquid separation is performed, and the solid product obtained is the immobilized lipase, and the obtained solution is the crude oil product rich in MLCT, which is then deacidified to obtain a finished oil product rich in MLCT; wherein the medium-chain raw material is medium-chain fatty acid triglycerides or medium-chain fatty acids; The specific preparation process of the hydrophobic hollow SiO2 mesoporous microspheres is as follows: after mixing liquid paraffin and water, add surfactant and pore-forming agent, and ultrasonically homogenize to obtain an emulsion; pour the emulsion into a hydrophobic silicon source solution, place it in a low-temperature water bath and stir the reaction, filter, wash, dry, and then acid-wash to remove the pore-forming agent and surfactant, and finally filter and dry to obtain hydrophobic hollow SiO2 mesoporous microspheres.

2. The enzymatic preparation method of MLCT-rich oil according to claim 1, characterized in that The size of the hydrophobic hollow SiO2 mesoporous microspheres is between 200nm and 2μm, the mesopore diameter is 3-10nm, and the contact angle is above 110°.

3. The enzymatic preparation method of MLCT-rich oil according to claim 1, characterized in that The volume ratio of liquid paraffin to water is 2:1-8:1; the surfactant is one or more of glyceryl monostearate, Span 80, Span 20, polyethylene glycol 200, polyglycerol fatty acid ester, and sorbitol monolaurate, and the addition amount is 1.0%-2.5% of the water mass; the pore-forming agent is one or more of block polyether F127 and triblock copolymer P123, and the addition amount is 1%-6% of the water mass; the hydrophobic silicon source is methyltrichlorosilane , ethyltrichlorosilane, butyltrichlorosilane, octyltrichlorosilane, one or more of the hydrophobic silicon source added in an amount of 75%-120% of the mass of water; the hydrophobic silicon source solution is prepared by dispersing the hydrophobic silicon source in isooctane, and the volume ratio of the hydrophobic silicon source to the isooctane is 3: (7-10); the temperature of the low-temperature water bath is 4-15°C, and the stirring reaction time is 2-4h; the pickling solution is prepared by mixing water, ethanol and hydrochloric acid; the pickling temperature is 60-100°C, and the time is 6-8h.

4. The enzymatic preparation method of MLCT-rich oil according to claim 1, characterized in that The specific process of preparing immobilized lipase by adsorption method is as follows: free enzyme is added to phosphate buffer to prepare enzyme solution, and then the enzyme solution is mixed with a hydrophobic carrier for immobilization to obtain immobilized lipase.

5. The enzymatic preparation method of MLCT-rich oil according to claim 4, characterized in that The free enzyme is one or more of Candida rugosa lipase, Candida antarctica lipase, NS40086 lipase, Thermomyces lanuginosus, and Candida lipolytica lipase; the pH of the enzyme solution is 7.5-9.5, and the concentration is 10-100 mg / mL; the ratio of the mass of the hydrophobic carrier to the volume of the enzyme solution is (1-3.5) g:100 mL; the immobilization time is 30-90 min, and the temperature is 20-40° C.; and the pH of the phosphate buffer is 7.5-9.

5.

6. The enzymatic preparation method of MLCT-rich oil according to claim 1, characterized in that The carbon number of the fatty acid chain of the medium-chain fatty acid triglyceride is 6-12; the medium-chain fatty acid is a mixture of one or more of the fatty acids with carbon numbers of 6-12 in any proportion; the long-chain triglyceride is a mixture of one or more of linseed oil, perilla oil, hemp oil, evening primrose oil, peony seed oil, sunflower seed oil, soybean oil, conjugated linoleic acid glyceride, DHA algae oil, fish oil, garlic fruit oil, and Xanthoceras sorbifolia oil in any proportion.

7. The enzymatic preparation method of MLCT-rich oil according to claim 1, characterized in that In step (2), the mass ratio of medium-chain fatty acid triglycerides to long-chain triglycerides is 2:3-2:5, or the molar ratio of long-chain triglycerides to medium-chain fatty acids is 1:3-1:6; the amount of immobilized lipase added is 4%-8% of the substrate mass; the temperature of the constant temperature water bath is 50-90°C; the transesterification reaction time is 10-60 minutes, or the acidolysis reaction time is 1-3 hours.

8. The enzymatic preparation method of MLCT-rich oil according to claim 1, characterized in that In step (2), the deacidification is carried out by molecular distillation or physical adsorption; the feed rate of the molecular distillation is 10-50 mL / min, the distillation pressure is 30-50 Pa, the heating temperature is 210-260° C., and the scraper speed is 240-350 r / min.

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