Preparation method for and use of medium-chain triglyceride

Abstract

The present invention relates to the technical field of light industrial grease. Specifically disclosed are a preparation method for and a use of a medium-chain triglyceride. In the present invention, firstly, specific chemical modification is performed on phospholipase A2 by using EDC or a modification solution prepared by mixing EDC with iodoacetamide to obtain modified phospholipase A2; the modified phospholipase A2 is used as a catalyst for catalyzing a hydrolysis reaction of coconut oil; specific hydrolysis is performed to enrich C8 and C10 fatty acids, and then esterification is performed on the fatty acids and glycerol to form the medium-chain triglyceride. The chemically specifically modified phospholipase A2 of the present invention exhibits improved hydrolysis specificity for C8 and C10 fatty acids in coconut oil, so that a light-phase component having a high content of C8 and C10 fatty acids is obtained after molecular distillation of a hydrolysate; and by means of subsequent esterification with glycerol, the medium-chain triglyceride having a C8 and C10 content of up to 95% can be obtained. The medium-chain triglyceride can be used in the fields of food, nutrition, medicine, etc.

Description

Preparation method and use of medium chain triglyceride TECHNICAL FIELD

[0001] The present application belongs to the technical field of light industry oil and fat, and particularly relates to a preparation method and use of medium chain triglyceride. BACKGROUND

[0002] Medium chain fatty acid (MCFA) generally refers to a fatty acid containing a chain of six to twelve carbon atoms, mainly including octanoic acid (C8) and decanoic acid (C10) and a small amount of hexanoic acid (C6) and lauric acid (C12). When esterification occurs between the three hydroxyl groups of glycerol and the medium chain fatty acid, a medium chain triglyceride (MCT) is formed. The most typical medium chain triglyceride refers to a saturated octanoic acid triglyceride, a saturated decanoic acid triglyceride and a saturated octanoic acid-decanoic acid triglyceride complex.

[0003] The digestion and decomposition speed of medium chain triglyceride is about ten times that of long chain triglyceride, because after the medium chain triglyceride is hydrolyzed by pancreatic lipase in the body, the MCFA produced is directly transported to the liver through the hepatic portal vein and is rapidly decomposed in the liver to rapidly produce energy. Therefore, based on its special metabolic characteristics, medium chain triglyceride has been widely used in the fields of food, nutrition, medicine and the like. Medium chain triglyceride is used in clinical nutrition to treat malnutrition, fat malabsorption, chylous fistula, chronic pancreatic insufficiency, intestinal fistula, bile duct obstruction, hyperlipidemia, high cholesterol, obstructive jaundice, inflammatory bowel disease, fatty liver, pancreatitis and other related diseases. In addition, due to its excellent antioxidant properties, solubility, low viscosity, low freezing point and other characteristics, and also can reduce the heat resistance of bacteria, medium chain triglyceride has a wide application in many pharmaceutical preparations. Medium chain triglyceride can also significantly promote the digestion and absorption of fat-soluble vitamin E in the human body, and can be used as a food supplement for patients with indigestion or low energy absorption. When a drug is prepared into an emulsified preparation by using medium chain triglyceride, the bioavailability of the drug is greatly improved.

[0004] The natural sources of medium chain triglyceride are few, and the method for producing medium chain triglyceride in the industry is mainly to prepare it by a chemical method. A non-enzyme catalyst is used to catalyze the esterification of medium chain fatty acid and glycerol to synthesize medium chain triglyceride. Although the chemical method for preparing medium chain triglyceride has the advantages of simple process, low energy consumption and short reaction time, it is also accompanied by a large amount of by-products and high separation difficulty, which easily causes product pollution, odor and other adverse characteristics. SUMMARY

[0005] In view of the above problems in the prior art, the present application provides a preparation method and use of medium-chain triglyceride. The method uses chemically modified phospholipase A2 to catalyze the hydrolysis of coconut oil, and specifically hydrolyze C8 and C10 fatty acids in the coconut oil, so as to enrich the raw material for synthesizing medium-chain triglyceride, realize green catalytic conversion, have mild reaction conditions, have little influence on product properties, and further reduce production cost, and can be applied to the fields of food, nutrition and medicine.

[0006] The first object of the present application is to provide a method for improving the catalytic activity of specifically chemically modified phospholipase A2, which comprises the steps of mixing a modification solution with phospholipase A2, removing the upper solvent after reaction by centrifugation, obtaining a concentrated solution by centrifugal ultrafiltration of the remaining solution, diluting the concentrated solution, and obtaining specifically chemically modified phospholipase A2 enzyme; the modification solution is prepared by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) or by mixing EDC with iodoacetamide.

[0007] Chemical modification is the formation or destruction of covalent bonds by the introduction or removal of chemical groups. In the present application, iodoacetamide reacts with the sulfhydryl group on cysteine, and EDC is used as an activating agent for carboxyl groups. By combining the modification of enzyme amino acid side chain groups, the modification of enzyme molecule main chain "cutting" and "splicing" and side chain groups, the enzyme protein is molecularly modified, the changes of some properties and functions of the enzyme are caused, the catalytic reaction characteristics are changed, and the product with specific requirements is prepared.

[0008] Preferably, the modification solution is prepared by dissolving EDC or a mixture of EDC and iodoacetamide in PBS buffer with a concentration of 0.05-0.3 mol / L and a pH of 5-7, and adding 1%-5% (volume fraction) of anhydrous ethanol as a solubilizing agent to prepare a modification solution with a concentration of 0.1-0.5 mol / L; wherein EDC and iodoacetamide are mixed at a molar ratio of 1:1-3:1.

[0009] Preferably, the mixing reaction of the modification solution and phospholipase A2 is carried out under the following conditions: the mass ratio of the modification solution to phospholipase A2 is 40-80:50, the reaction temperature is 5-20°C, the stirring rate is 200-500 rpm, and the stirring time is 30 min-60 min.

[0010] Preferably, the remaining solution is subjected to centrifugal ultrafiltration through an ultrafiltration membrane with a molecular weight cut-off of 20-50 kDa at 10-30°C and a centrifugal speed of 8000-12000 rpm for 3-10 min to obtain a concentrated solution, and then the concentrated solution is diluted with PBS having a pH of 5-7 and a concentration of 0.05-0.3 mol / L.

[0011] The second object of the present application is to provide a specific chemical modification of phospholipase A2 obtained by the above method. After specific chemical modification of phospholipase A2 by the above method, the catalytic properties are improved.

[0012] The third object of the present application is to provide the use of the above specific chemical modification of phospholipase A2 in the preparation of high-purity medium-chain triglycerides, and the preparation method of the high-purity medium-chain triglycerides is provided. The specific chemical modification of phospholipase A2 is used to catalyze the hydrolysis of coconut oil, which can enrich C8 and C10 fatty acids, and after esterification with glycerol, high-purity medium-chain triglycerides are obtained.

[0013] The above objects of the present application are achieved by the following technical solutions:

[0014] A preparation method of medium-chain triglycerides, comprising the following steps:

[0015] S1, the specific chemical modification of phospholipase A2 is used to catalyze the hydrolysis reaction of coconut oil, and then the lower aqueous phase is removed by centrifugation to obtain a mixed hydrolysis product containing free fatty acids, monoglycerides, diglycerides and triglycerides;

[0016] S2, the hydrolysis product obtained in step S1 is subjected to molecular distillation treatment to obtain heavy and light phase components, respectively;

[0017] S3, the light phase component obtained in step S2 is mixed with glycerol in a specific ratio, a certain amount of molecular sieve is added, and a lipase is used as a catalyst to catalyze the esterification reaction, and the medium-chain triglyceride is obtained;

[0018] Preferably, the amount of the specific chemical modification of phospholipase A2 added in step S1 is 5-15wt%, the temperature of the catalytic hydrolysis is 40-80℃, more preferably 60-70℃; the stirring rate is 500rpm-700rpm, and the reaction time is 60-180min, more preferably 120min.

[0019] Preferably, the centrifugation conditions in step S1 are that the centrifugal speed is 4000-8000rpm, more preferably 7000rpm, and the centrifugation time is 5-30min, more preferably 10min.

[0020] Preferably, the molecular distillation conditions in step S2 are that the distillation temperature is 150-230℃, more preferably 170℃; the vacuum degree is 100pa-1000pa, the scraping film rotation speed is 150rpm-350rpm, and the distillation time is 120-180min.

[0021] Preferably, the mixing molar ratio of the light phase component to glycerol in step S3 is 2-4:1; more preferably 3:1.

[0022] Preferably, the amount of molecular sieve added in step S3 is 0.1-1.5 wt%; more preferably 1.2 wt%. The purpose of adding the molecular sieve in the present application is to remove the water molecules produced in the esterification reaction moderately, so that the reaction proceeds in the forward direction and the occurrence of hydrolysis reaction is reduced.

[0023] Preferably, phosphoric acid can also be added in the esterification reaction in step S3, and the amount of addition is 0.01-0.05 wt%; more preferably 0.02%. The purpose of adding phosphoric acid in the present application is that the phosphoric acid added in the preparation process plays the role of moderate reversible hydrolysis of glycerides, improving the opportunity of short-chain fatty acids to combine with glycerides in the esterification process. At the same time, as a food additive, phosphoric acid also plays a role in preservative and adsorption of metal ions, reducing the inhibition of metal ions on the activity of enzymes in the reaction process, and improving the esterification efficiency of enzymes.

[0024] Preferably, the esterification conditions in step S3 are as follows: the lipase is Novozym 435, the amount of lipase added is 2-20 wt%, more preferably 10 wt%; the reaction temperature is 45-85℃, more preferably 65℃; the reaction time is 50-100 min, more preferably 80 min; and the stirring speed is 200-700 rpm, more preferably 500 rpm.

[0025] The present application uses modified phospholipase A2 to obtain fatty acids used as raw materials for the synthesis of medium-chain triglycerides. The advantages of using modified biological enzymes to catalyze the preparation of medium-chain triglycerides are high catalytic activity, high product specificity, mild reaction conditions, few by-products, no pollution and odor, and green, safe and environmentally friendly.

[0026] The fourth object of the present application is to provide a medium-chain triglyceride prepared by the above method. The medium-chain triglyceride is mainly composed of C8 and C10 fatty acids, and the main component of the triglyceride is caprylocaprin.

[0027] Another object of the present application is to provide the use of the above-mentioned medium-chain triglyceride oil. The oil can be used in the fields of food, nutrition and medicine, such as fast energy supplement, ketogenic diet meal, diluent, emulsified preparation, parenteral nutrition fat emulsion, etc.

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

[0029] The present application is based on the hydrolysis specificity of chemically modified phospholipase A2, enriches C8 and C10 fatty acids in natural coconut oil, and then esterifies with glycerol to synthesize triglyceride, to obtain high-purity medium-chain triglyceride, wherein the content of C8 and C10 fatty acids is as high as 95%, has the metabolic characteristics of medium-chain triglyceride, is rapidly metabolized and absorbed, and can be used as a raw material for fat emulsion in clinical nutrition to treat and improve malabsorption, and as a main fat component in a ketogenic diet to rapidly produce ketones. DETAILED DESCRIPTION

[0030] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0031] The test methods used in the following embodiments are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified; and for process parameters not specifically mentioned, conventional techniques can be referred to. The lipases used in the examples are purchased from Novozymes.

[0032] The present application aims to replace the traditional chemical method for preparing medium-chain triglyceride by the green and safe modification method of enzyme method, to improve the catalytic properties of phospholipase A2 after specific chemical modification, to enrich C8 and C10 fatty acids, and to obtain medium-chain triglyceride after esterification with glycerol. EMBODIMENT

[0033] A preparation method of medium-chain triglyceride, comprising the following steps:

[0034] (1) EDC (1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride) is mixed uniformly with iodine acetamide at a molar ratio of 1.6:1, then added to PBS buffer (0.2 mol / L, pH=5.5), and 2% volume fraction of anhydrous ethanol is added to assist dissolution, stirred uniformly, configured into a 0.3 mol / L modification solution, and stored at 4℃ for standby.

[0035] (2) Take 50 g of modification solution and 50 g of phospholipase A2, mix them, and then place them in a reactor. Set the reaction temperature to 20°C. When the temperature is constant, use a magnetic stirrer to stir (rotation speed 200 rpm). Perform the reaction under the condition of water circulation pump vacuum (absolute pressure = 7000 Pa). After 30 min of reaction, centrifuge the mixed sample after specific modification at 7000 rpm for 10 min at 20°C. After removing the upper solvent, use a 30 kDa ultrafiltration membrane to centrifuge the remaining aqueous solution at 10000 rpm for 5 min at 20°C to obtain a concentrated solution. Dilute the concentrated solution with 50 mL of PBS (pH = 6.7, 0.1 mol / L) to obtain the specifically chemically modified phospholipase A2.

[0036] (3) Physically mix the specifically chemically modified phospholipase A2 enzyme solution described above with coconut oil at a mass ratio of 1:10, and perform the reaction at 70°C. Stir at a rotation speed of 500 rpm under the condition of water circulation pump vacuum (absolute pressure = 7000 Pa). After 120 min of reaction, centrifuge at 7000 rpm for 10 min to separate and remove the lower aqueous phase to obtain the hydrolysis product.

[0037] (4) Perform molecular distillation treatment on the hydrolysis product described above. The distillation temperature is 170°C, the vacuum degree is 100 Pa, the scraper membrane rotation speed is 150 rpm, and the distillation time is 2 h. The heavy phase and light phase components are obtained, respectively.

[0038] (5) Physically mix the light phase component described above with glycerol at a molar ratio of 3:1. Use Novozym 435 enzyme as the reaction catalyst, and add 10 wt% of the enzyme. Add 1.2 wt‰ of molecular sieves and 0.02 wt% of phosphoric acid. The reaction temperature is 65°C, the reaction time is 80 min, the stirring rate is 500 rpm, and the esterification reaction is complete. After centrifugation at 7000 rpm to remove the excess glycerol in the lower layer, the esterification product in the upper layer is the medium-chain triglyceride.

[0039] Fatty acid composition determination: Use Shimadzu SH-2560 (100 m x 0.2 mm i.d, 0.2 μm) capillary column, N2 as carrier gas, flow rate 1.5 mL / min. Inject 1 μL of sample with a split ratio of 40:1. The injection port temperature is 250°C, and the detector temperature is set to 270°C. The temperature program is as follows: the initial temperature is 80°C, then increase to 220°C at a rate of 4°C / min and maintain for 5 min, then increase to 240°C at a rate of 4°C / min and maintain for 10 min.

[0040] Triglyceride composition determination: Shimadzu SH-65TG (30 m x 0.2 mm i.d, 0.2 μm) capillary column was used with H2 as carrier gas at a flow rate of 1.7 mL / min. 1 μL of sample was injected with a split ratio of 25:1. The injector temperature was 350 °C and the detector temperature was set at 350 °C. The temperature program was: initial temperature of 280 °C for 1.5 min, then ramped at 10 °C / min to 340 °C for 9.5 min, then ramped at 1 °C / min to 350 °C for 12 min.

[0041] The C8 and C10 fatty acid contents of the medium-chain triglyceride obtained in this example were 54.2% and 41.4%, respectively, and the contents of the triglycerides were caprylic triglyceride 18.2%, capric triglyceride 15.6%, and caprylic-capric triglyceride 61.4%, respectively. Example

[0042] A method for preparing a medium-chain triglyceride, comprising the following steps:

[0043] The difference from Example 1 is that (5) the light phase component and glycerol are physically mixed at a molar ratio of 3:1, Novozym 435 enzyme is used as a reaction catalyst, the addition amount is 10 wt%, 1.2 wt‰ of molecular sieve is added, no phosphoric acid is added, the reaction temperature is 65 °C, the stirring speed is 500 rpm, after the esterification reaction is completed, the excess glycerol in the lower layer is removed by centrifugation at 7000 rpm, and the esterification product in the upper layer is obtained, which is the medium-chain triglyceride.

[0044] The other steps and conditions are the same as in Example 1.

[0045] The C8 and C10 fatty acid contents of the medium-chain triglyceride obtained in this example were 50.8% and 42.6%, respectively, and the contents of the triglycerides were caprylic triglyceride 13.4%, capric triglyceride 11.5%, and caprylic-capric triglyceride 58.5%, respectively. Example

[0046] A method for preparing a medium-chain triglyceride, comprising the following steps:

[0047] The difference from Example 1 is that in step (3), the specifically chemically modified phospholipase A2 enzyme solution and coconut oil are physically mixed at a mass ratio of 1.5:10, the reaction is carried out at 70 °C, the reaction is carried out under the condition of water circulating pump vacuum (absolute pressure = 7000 Pa), the stirring speed is 500 rpm, the reaction is completed for 120 min, and the hydrolysis product is obtained by separating and removing the lower layer of water phase at 7000 rpm for 10 min.

[0048] Other steps and conditions are the same as in Example 1.

[0049] The C8 and C10 fatty acid contents in the medium-chain triglyceride obtained in this example are 47.8% and 37.5%, respectively, and the contents of the triglycerides are 9.8% caprylic acid triglyceride, 15.5% capric acid triglyceride, and 54.3% caprylic-capric acid triglyceride, respectively. Example

[0050] A method for preparing a medium-chain triglyceride, comprising the following steps:

[0051] The difference from Example 1 is that in step (3), the specific chemically modified phospholipase A2 enzyme solution is physically mixed with coconut oil at a mass ratio of 1:10, and the reaction is carried out at 60°C. The reaction is carried out under the condition of water circulating pump vacuum (absolute pressure = 7000 Pa), and the stirring speed is 500 rpm. The reaction is completed after 120 min, and the hydrolysis product is obtained by removing the lower water phase by centrifugation at 7000 rpm for 10 min.

[0052] Other steps and conditions are the same as in Example 1.

[0053] The C8 and C10 fatty acid contents in the medium-chain triglyceride obtained in this example are 41.7% and 36.3%, respectively, and the contents of the triglycerides are 12.5% caprylic acid triglyceride, 7.5% capric acid triglyceride, and 55.6% caprylic-capric acid triglyceride, respectively. Example

[0054] A method for preparing a medium-chain triglyceride, comprising the following steps:

[0055] The difference from Example 1 is that in step (1), EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) is mixed with iodoacetamide at a molar ratio of 2.5:1, and then added to PBS buffer (0.2 mol / L, pH=5.5). 2% volume fraction of anhydrous ethanol is added to assist dissolution, stirred uniformly, and configured into a modification solution of 0.3 mol / L, and stored at 4°C.

[0056] Other steps and conditions are the same as in Example 1.

[0057] The C8 and C10 fatty acid contents in the medium-chain triglyceride obtained in this example are 39.4% and 29.6%, respectively, and the contents of the triglycerides are 11.8% caprylic acid triglyceride, 10.6% capric acid triglyceride, and 41.8% caprylic-capric acid triglyceride, respectively. Example

[0058] A method for preparing medium-chain triglyceride, comprising the following steps:

[0059] The difference from Example 1 is that the light phase component is physically mixed with glycerol in a molar ratio of 2:1 in step (5), Novozym 435 lipase is used as a reaction catalyst, the addition amount is 2wt%, 1.2wt‰ of molecular sieve and 0.02wt% of phosphoric acid are added, the reaction temperature is 65℃, the stirring speed is 500rpm, after the esterification reaction is completed, the excess glycerol in the lower layer is removed by centrifugal separation at 7000rpm, and the esterification product in the upper layer is the medium-chain triglyceride.

[0060] The other steps and conditions are the same as those in Example 1.

[0061] According to the method described in Example 1, the C8 and C10 fatty acid contents in the medium-chain triglyceride obtained in this example are 35.6% and 25.1% respectively, and the triglyceride contents are caprylic triglyceride 9.8%, capric triglyceride 12.5% and caprylic-capric triglyceride 36.4% respectively. Example

[0062] A method for preparing medium-chain triglyceride, comprising the following steps:

[0063] The difference from Example 1 is that EDC (1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride) is added to PBS buffer (0.2 mol / L, pH=5.5) in step (1), 2% volume fraction of anhydrous ethanol is added to assist dissolution, stirring is uniform, and a modification solution of 0.3 mol / L is prepared, which is stored at 4℃ for standby.

[0064] The other steps and conditions are the same as those in Example 1.

[0065] According to the method described in Example 1, the C8 and C10 fatty acid contents in the medium-chain triglyceride obtained in this example are 33.7% and 27.4% respectively, and the triglyceride contents are caprylic triglyceride 9.5%, capric triglyceride 13.4% and caprylic-capric triglyceride 38.5% respectively.

[0066] Comparative Example 1

[0067] A method for preparing medium-chain triglyceride, comprising the following steps:

[0068] The difference from Example 1 is that the step (2) of using a modification solution to specifically modify phospholipase A2 is not performed. The other steps and conditions are the same as those in Example 1.

[0069] The C8 and C10 fatty acid contents in the medium-chain triglyceride obtained in the example are 14.5% and 14.1% respectively, and the contents of the triglycerides are 5.6% of caprylic acid triglyceride, 6.7% of capric acid triglyceride and 17.8% of caprylic-capric acid triglyceride respectively.

[0070] Comparative Example 2

[0071] A method for preparing medium-chain triglyceride comprises the following steps:

[0072] The difference between the example and the comparative example is that the step (2) is not performed, and the unmodified phospholipase A2 enzyme solution with the same concentration is physically mixed with coconut oil at a mass ratio of 1.5:10 in the step (3), and the reaction is performed at 70°C under the condition of water circulating pump vacuum (absolute pressure = 7000 Pa) and stirring speed of 500 rpm, and the reaction is completed after 120 min, and the hydrolysis product is obtained by removing the lower water phase through 7000 rpm centrifugal separation.

[0073] The other steps and conditions are the same as those in the example.

[0074] The C8 and C10 fatty acid contents in the medium-chain triglyceride obtained in the example are 14.5% and 14.1% respectively, and the contents of the triglycerides are 5.6% of caprylic acid triglyceride, 6.7% of capric acid triglyceride and 17.8% of caprylic-capric acid triglyceride respectively.

[0075] Comparative Example 3

[0076] A method for preparing medium-chain triglyceride comprises the following steps:

[0077] The difference between the example and the comparative example is that the PEG (polyethylene glycol) and iodine acetamide are mixed uniformly at a molar ratio of 1.6:1, and then added into the PBS buffer (0.2 mol / L, pH=5.5), and 2% volume fraction of anhydrous ethanol is added for dissolution, and then stirred uniformly to prepare a 0.3 mol / L modification solution, and the modification solution is stored at 4°C for standby.

[0078] The other steps and conditions are the same as those in the example.

[0079] The C8 and C10 fatty acid contents in the medium-chain triglyceride obtained in the example are 14.5% and 14.1% respectively, and the contents of the triglycerides are 5.6% of caprylic acid triglyceride, 6.7% of capric acid triglyceride and 17.8% of caprylic-capric acid triglyceride respectively.

[0080] Comparative Example 4

[0081] A method for preparing medium-chain triglyceride, comprising the following steps:

[0082] The difference from Example 1 is that the step (2) is not performed, and the hydrolysate is subjected to molecular distillation treatment in step (4), the distillation temperature is 190℃, the vacuum degree is 100 Pa, the scraper film rotation speed is 150 rpm, and the distillation time is 2 h, to obtain heavy phase and light phase components respectively.

[0083] The other steps and conditions are the same as those in Example 1.

[0084] According to the method described in Example 1, the C8 and C10 fatty acid contents in the medium-chain triglyceride obtained in this example are 16.8% and 16.2% respectively, and the triglyceride contents are caprylic triglyceride 8.5%, capric triglyceride 3.6%, and caprylic-capric triglyceride 18.6% respectively.

[0085] Comparative Example 5

[0086] A method for preparing medium-chain triglyceride, comprising the following steps:

[0087] The difference from Example 1 is that the iodine acetamide is added to PBS buffer (0.2 mol / L, pH=5.5) and 2% volume fraction of anhydrous ethanol is added to assist dissolution in step (1), and the mixture is stirred uniformly to prepare a modification solution with a concentration of 0.3 mol / L, which is stored at 4℃ for standby.

[0088] The other steps and conditions are the same as those in Example 1.

[0089] According to the method described in Example 1, the C8 and C10 fatty acid contents in the medium-chain triglyceride obtained in this example are 20.3% and 16.4% respectively, and the triglyceride contents are caprylic triglyceride 8.5%, capric triglyceride 4.3%, and caprylic-capric triglyceride 21.7% respectively.

[0090] In order to further illustrate the effect of the scheme of the present application, the intermediate products and final products prepared by the schemes of the examples and comparative examples are subjected to component analysis.

[0091] Table 1 is the fatty acid component data of the light phase components of the intermediate products after molecular distillation of the examples and comparative examples.

[0092] Table 2 is the fatty acid component data of the final products of the examples and comparative examples.

[0093] Table 3 is the triglyceride component data of the final products of the examples and comparative examples.

[0094] Table 1

[0095] Sample (%) C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 Other Example 1 48.6 37.2 6.8 2.7 0.7 0.9 1.5 1.4 0.2 Example 2 48.4 37.3 7.0 3.1 0.5 0.8 1.5 1.1 0.3 Example 3 35.9 29.8 9.5 11.8 2.3 2.6 1.3 5.7 1.1 Example 4 38.4 30.3 13.8 6.8 1.5 3.4 0.7 3.8 1.3 Example 5 36.5 28.4 17.3 6.9 3.4 1.4 2.2 3.5 0.4 Example 6 31.1 22.5 29.5 10.7 0.4 1.6 1.1 2.2 0.9 Example 7 30.7 26.5 30.9 0.3 1.8 3.7 2.1 2.8 1.2 Comparative Example 1 11.7 10.2 42.3 9.3 12.5 8.2 1.7 2.3 1.8 Comparative Example 2 9.1 13.2 40.5 11.9 7.6 7.9 5.1 2.5 2.2 Comparative Example 3 7.2 7.6 41.4 15.2 9.5 6.7 6.5 3.6 2.3 Comparative Example 4 11.8 9.7 39.5 17.2 6.4 8.5 3.7 1.7 1.5 Comparative Example 5 16.2 12.7 36.3 9.8 8.3 7.6 4.2 2.5 2.4

[0096] Table 2

[0097] Sample (%) C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 Other Coconut oil 8.7 7.8 43.2 16.2 9.4 2.6 6.7 1.6 3.8 Example 1 54.2 41.4 1.8 1.1 0.6 0.2 0.2 0.2 0.3 Example 2 50.8 42.6 2.1 1.5 0.4 0.3 0.5 0.5 1.3 Example 3 47.8 37.5 4.5 2.6 1.7 1.1 2.3 0.7 1.8 Example 4 41.7 36.3 10.7 2.3 1.4 1.3 2.9 1.5 1.9 Example 5 39.4 29.6 21.7 2.3 1.6 0.5 1.6 1.1 2.2 Example 6 35.6 25.1 25.4 3.7 2.5 0.9 2.8 1.7 2.3 Example 7 33.7 27.4 25.9 3.5 1.8 1.2 3.2 1.6 1.7 Comparative Example 1 14.5 14.1 37.8 13.6 7.9 1.8 4.7 1.5 4.1 Comparative Example 2 13.6 17.5 35.8 14.2 6.7 2.2 4.1 1.4 4.5 Comparative Example 3 12.7 12.6 39.5 12.8 8.5 2.3 5.6 1.3 4.7 Comparative Example 4 16.8 16.2 36.4 12.7 7.3 1.5 4.6 0.7 3.8 Comparative Example 5 20.3 16.4 34.7 8.5 6.5 2.2 5.8 1.8 3.8

[0098] Table 3

[0099] Triglyceride content (%) Caprylic acid triglyceride Capric acid triglyceride Caprylic acid capric acid triglyceride Example 1 8.2 15.6 61.4 Example 2 13.4 11.5 58.5 Example 3 9.8 15.5 54.3 Example 4 12.5 7.5 55.6 Example 5 11.8 10.6 41.8 Example 6 9.8 12.5 36.4 Example 7 9.5 13.4 38.5 Comparative Example 1 5.6 6.7 17.8 Comparative Example 2 7.5 4.3 15.5 Comparative Example 3 6.8 7.5 16.7 Comparative Example 4 8.5 3.6 18.6 Comparative Example 5 8.5 4.3 21.7

[0100] Conclusion: By preparing the modification liquid to specifically chemically modify phospholipase A2 to obtain modified phospholipase A2, using it as a catalyst to specifically catalyze the hydrolysis of coconut oil, and using the light phase component obtained after molecular distillation of the hydrolysis product as a raw material to synthesize medium-chain triglyceride, the results show that, compared with phospholipase A2, the modified phospholipase A2 after specific chemical modification has improved specificity for hydrolysis of C8 and C10 fatty acids in coconut oil, so that the light phase component with high content of C8 and C10 fatty acids is obtained after molecular distillation of the hydrolysis product, and through subsequent esterification with glycerol, medium-chain triglyceride with C8 and C10 content as high as 95% can be obtained, which can be used in the fields of food, nutrition and medicine. And in all the examples, in the example 1 in which the specific chemical modification of phospholipase A2 and coconut oil is mixed at a mass ratio of 1:10, the final medium-chain triglyceride obtained is the best.

[0101] The above examples of the present application are only examples for clearly illustrating the technical solutions of the present application, and are not all specific implementations of the present application. Any modifications, equivalent replacements and improvements made within the spirit and principles of the claims of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A method of increasing the catalytic activity of a specifically chemically modified phospholipase A2, characterized in that, The method comprises: mixing and reacting the modification solution with phospholipase A2, removing the upper solvent after the reaction through centrifugation, obtaining a concentrated solution through centrifugal ultrafiltration of the remaining solution, diluting the concentrated solution, and obtaining the specifically chemically modified phospholipase A2 enzyme; and the modification solution is configured by 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride or a mixture of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and iodoacetamide.

2. The method of claim 1, wherein, The modification solution is configured by mixing 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride or 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and iodoacetamide at a molar ratio of 1:1 to 3:1, then dissolving in a PBS buffer with a concentration of 0.05 to 0.3 mol / L and a pH of 5 to 7, and adding 1% to 5% by volume of anhydrous ethanol to aid dissolution, to obtain a modification solution with a concentration of 0.1 to 0.5 mol / L.

3. The method of claim 1, wherein, The mixing and reacting operation is as follows: the mass ratio of the modification solution to phospholipase A2 is 40 to 80:50, the reaction temperature is 5 to 20°C, the stirring rate is 200 to 500 rpm, and the stirring time is 30 min to 60 min.

4. The method of claim 1, wherein, The remaining solution is subjected to centrifugal ultrafiltration through an ultrafiltration membrane with a molecular weight of 20 to 50 kDa at 10 to 30°C at a centrifugal speed of 8000 to 12000 rpm for 3 to 10 min to obtain a concentrated solution, and the concentrated solution is then diluted with a PBS with a pH of 5 to 7 and a concentration of 0.05 to 0.3 mol / L.

5. A specifically chemically modified phospholipase A2 obtained by the method of any one of claims 1 to 4.

6. A method for producing medium-chain triglycerides, characterized by, The method comprises the following steps: S1. Using the specifically chemically modified phospholipase A2 enzyme of claim 5 to catalyze the hydrolysis of coconut oil, removing the lower aqueous phase through centrifugal separation to obtain a hydrolysis product; S2. Subjecting the hydrolysis product obtained in step S1 to molecular distillation to obtain heavy and light phase components, respectively; S3. Mixing the light phase component obtained in step S2 with glycerol, adding molecular sieves, and using a lipase to catalyze an esterification reaction.

7. The production method according to claim 6, characterized by, The enzyme addition amount in step S1 is 5 to 15 wt%, and the catalytic hydrolysis conditions are a temperature of 40 to 80°C, a stirring rate of 500 rpm to 700 rpm, and a reaction time of 60 to 180 min.

8. The preparation method according to claim 6, characterized in that, The molecular distillation conditions in step S2 are a distillation temperature of 150 to 230°C, a vacuum degree of 100 pa to 1000 pa, a wiper speed of 150 rpm to 350 rpm, and a distillation time of 120 to 180 min.

9. The preparation method according to claim 6, characterized in that, The molar ratio of the light phase component to glycerol in step S3 is 2 to 4:1, the addition amount of the molecular sieves is 0.1 to 1.5 wt‰, phosphoric acid can be added in the esterification reaction with an addition amount of 0.01 to 0.05 wt%, the lipase is Novozym 435, and the addition amount of the lipase is 2 to 20 wt%; the reaction temperature is 45 to 85°C, the reaction time is 50 to 100 min, and the stirring speed is 200 to 700 rpm.

10. The medium chain triglyceride produced by the method of any one of claims 6-9.