Preparation method of medium and long-chain triglyceride rich in DHA and product thereof

Abstract

The present invention discloses a preparation method and product of medium- and long-chain triglycerides rich in DHA. By modifying the imprinted lipase with a non-ionic surfactant, an imprinted lipase with higher catalytic activity is obtained, thereby improving the reaction efficiency of the acidolysis reaction; in the present invention, a vacuum is applied to the system after the acidolysis reaction, so that the main reaction is changed from the acidolysis reaction to the esterification reaction, and by-products in the product, including monoglyceride and diglyceride, undergo an esterification reaction to be converted into triglyceride, thereby increasing the yield of triglyceride in the product. At the same time, in the subsequent molecular distillation deacidification stage, the contents of glycidyl ester and chloropropanol ester can be reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of oils and fats, and particularly relates to a preparation method and a product of medium- and long-chain triglycerides rich in DHA. Background Art

[0002] Docosahexaenoic acid (DHA, C22:6n-3) is an important component of the cell membranes of the retina and cerebral gray matter, accounting for 3-5% of the dry weight of the tissue, and is of great significance for improving the vision and intelligence level of infants. DHA is the metabolic end product of n-3 fatty acids and can be metabolically synthesized in the body through its dietary essential fatty acid precursor α-linolenic acid (C18:3n-3). However, due to the relatively low activity levels of fatty acid desaturase and elongase in infants, the endogenous synthesis of DHA cannot meet the physiological needs of their brain and visual development, etc. Therefore, it is extremely important for infants to ingest DHA from the diet. Breast milk is the best food for infants, providing all essential nutrients. When infants cannot obtain breast milk feeding, infant formula milk powder becomes a good substitute for breast milk. Infant formula milk powder is a breast milk substitute based on the existing understanding of breast milk, simulating the components and even the physical structure of breast milk. The DHA in formula milk powder mainly comes from single-cell microbial fermentation, and the added amount is based on the content in breast milk. The main fatty acids in DHA oil are DHA, eicosapentaenoic acid, and palmitic acid. As is well known, the digestive systems of infants, especially newborns and premature infants, are underdeveloped. At the same time, due to steric hindrance, the activity of lipase in the body towards long-chain polyunsaturated fatty acids is relatively low, which will affect the digestion, absorption, and metabolism (bioavailability) of DHA in infants. Therefore, developing structured lipid products with high DHA bioavailability and applying them to formula milk powder is of great significance for promoting the healthy growth and development of infants.

[0003] Medium- and long-chain triglycerides are a type of structured triglyceride that contains medium-chain fatty acids and long-chain fatty acids on the glycerol backbone at the same time. Medium-chain fatty acids are relatively more water-soluble, can be rapidly hydrolyzed in the stomach, and are absorbed into the liver through the portal vein to supply immediate energy. At the same time, the diglycerides or monoglycerides produced during gastric digestion can act as emulsifiers to increase the solubility of lipids in the small intestine, making up for the defects of the digestive system to a certain extent. Therefore, such products are widely used in clinical practice as important energy sources and supplements of functional fatty acids for patients with pancreatic lipase deficiency and bile salt deficiency. Medium- and long-chain triglycerides, as a special type of functional oil and fat, on the one hand, use medium-chain fatty acids to provide immediate energy, and on the other hand, use long-chain fatty acids to provide nutrition. Therefore, using medium- and long-chain triglycerides as a carrier to provide a DHA source for infants will help improve the digestion and utilization efficiency of DHA by infants.

[0004] At present, the preparation methods of medium- and long-chain triglycerides mainly include chemical methods and enzymatic methods. The chemical method has low cost and high reaction efficiency. However, due to the use of alkaline catalysts, it requires a relatively high reaction temperature and generates more side reactions, resulting in a decrease in the nutritional value and safety of the product. The enzymatic method has been widely used in the production of high-value functional lipids due to its mild conditions, fewer by-products, strong selectivity, and easy separation. The enzymatic production of medium- and long-chain triglycerides can be further divided into random transesterification and acidolysis reactions. Acidolysis reaction can utilize the positional selectivity of lipase to directionally replace the fatty acids on triglycerides, which is of great significance for the production of triglycerides with special structures.

[0005] However, one of the main problems faced by current enzymatic catalytic reactions is the high price of enzymes and low catalytic efficiency. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section, as well as in the abstract and title of the present application, to avoid obscuring the purpose of this section, the abstract, and the title. However, such simplifications or omissions shall not be used to limit the scope of the present invention.

[0007] In view of the above and / or problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing medium- and long-chain triglycerides rich in DHA.

[0009] To solve the above technical problems, the present invention provides the following technical solution: A method for preparing medium- and long-chain triglycerides rich in DHA, comprising:

[0010] Performing fractional crystallization on the saturated triglycerides in DHA algal oil by programmed cooling, separating the saturated fat to obtain DHA concentrated oil;

[0011] Using lipase imprinted with a non-ionic surfactant dissolved in isopropanol solution as a catalyst to catalyze the acidolysis reaction of medium-chain fatty acids and DHA concentrated oil;

[0012] After the reaction is completed, evacuate the reaction system, and esterify the by-products monoglyceride and diglyceride in the reaction into triglyceride;

[0013] Removing fatty acids by molecular distillation to obtain medium- and long-chain triglyceride products.

[0014] As a preferred embodiment of the preparation method of the present invention, wherein: the fractional crystallization process includes:

[0015] After heating the DHA algal oil to 60 - 80 °C for complete melting, the temperature is decreased to 15 - 25 °C at a rate of 10 - 20 °C / h;

[0016] The temperature is further decreased to 6 - 10 °C at a rate of 3 - 6 °C / h and maintained for 10 - 24 h;

[0017] Solid fat is separated by filtration or centrifugation to obtain DHA concentrated oil.

[0018] As a preferred embodiment of the preparation method of the present invention, wherein: for the imprinted lipase, its preparation method includes,

[0019] Dissolve the non - ionic surfactant in a mixed solution of isopropanol and water at a concentration of 20 - 100 mg / L by mass fraction, and add the immobilized lipase with a mass fraction of 10 - 30%;

[0020] Stir the mixture at 100 - 200 rpm for 30 - 60 min at 25 °C, and filter to obtain lipase;

[0021] Remove water by freeze - drying under vacuum, elute the surfactant imprinting template on the immobilized lipase with a non - polar solvent, filter the lipase and dry it under vacuum to remove the organic solvent, obtaining the imprinted lipase.

[0022] As a preferred embodiment of the preparation method of the present invention, wherein: the lipase includes lipase Novozym435, lipase Lipozyme RM IM, and lipase NS40086.

[0023] As a preferred embodiment of the preparation method of the present invention, wherein: the non - ionic surfactant includes Tween 20, Tween 40, and Tween 80.

[0024] As a preferred embodiment of the preparation method of the present invention, wherein: for the mixed solution of isopropanol and water, the content of isopropanol is greater than 70 wt%.

[0025] As a preferred embodiment of the preparation method of the present invention, wherein: the acidolysis reaction includes,

[0026] Mix medium - chain fatty acids and DHA algal oil in a molar ratio of 2 - 5:1, then add the imprinted lipase accounting for 5 - 10 wt% of the substrate weight to the mixture, fill with nitrogen, react at a temperature of 50 - 70 °C for 6 - 10 h, filter the lipase, and obtain the acidolysis reaction product.

[0027] As a preferred embodiment of the preparation method of the present invention, wherein: the esterification reaction includes,

[0028] After the acidolysis reaction is completed, the reaction system is evacuated to carry out the esterification reaction. The vacuum degree is 10 - 30 mbar, the reaction time is 6 - 10 h, the stirring rate is 500 - 800 rmp, the reaction temperature is 50 - 70 °C. After the reaction is completed, the lipase is filtered.

[0029] As a preferred embodiment of the preparation method of the present invention, wherein: the molecular distillation conditions are: the distillation temperature is 160 - 180 °C, the pressure is 2 - 5 Pa, and the condenser temperature is 25 - 35 °C.

[0030] Another object of the present invention is to overcome the deficiencies in the prior art and provide a medium and long-chain triglyceride rich in DHA prepared by a preparation method of a medium and long-chain triglyceride rich in DHA, with the glycidyl ester content less than 0.3 mg / kg and the chloropropanol ester content less than 0.3 mg / kg.

[0031] Advantages of the present invention:

[0032] (1) By programmed cooling in the present invention, the saturated triglycerides in DHA algal oil are subjected to crystallization fractionation to obtain DHA concentrated oil, thereby increasing the DHA content in the final product;

[0033] (2) In the present invention, a non-ionic surfactant-imprinted lipase dissolved in an isopropanol solution is used to obtain an imprinted lipase with higher catalytic activity, thereby improving the reaction efficiency of the acidolysis reaction; since the non-ionic surfactant covers the surface of the lipase, the hydrophobicity of the lipase surface is increased, avoiding the excessive contact with polar substances resulting in the loss of surface moisture, thereby improving the catalytic stability of the lipase; at the same time, isopropanol or an isopropanol aqueous solution with a certain concentration is used as the solvent to dissolve the surfactant and the lipase, improving the imprinting effect, thereby further enhancing the activity and stability of the enzyme;

[0034] (3) By applying a vacuum degree to the system after the acidolysis reaction in the present invention, the main reaction is changed from the acidolysis reaction to the esterification reaction, enabling the by-products in the product, including monoglyceride and diglyceride, to undergo an esterification reaction to be converted into triglyceride, thereby increasing the triglyceride yield of the product. At the same time, in the subsequent molecular distillation deacidification stage, the contents of glycidyl ester and chloropropanol ester can be reduced. Specific embodiments

[0035] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention is provided in conjunction with the examples of the specification.

[0036] In the following description, numerous specific details are set forth to provide a thorough understanding of the present invention. However, the present invention may be practiced in other ways different from those described herein. Persons skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0037] Secondly, the so-called "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation manner of the present invention. The appearances of "in one embodiment" in different places in this specification do not all refer to the same embodiment, nor are they separate or alternative embodiments that are mutually exclusive of other embodiments.

[0038] Determination of the activity of the lipase of the present invention towards medium-chain fatty acids:

[0039] Mix DHA oil (2 g) with capric acid in a molar ratio of 1:3, add molecular sieve for dehydration. Subsequently, add lipase accounting for 3 wt% of the total mass of the substrate to the mixture, and use a round-bottom flask for catalytic reaction. Set the reaction temperature at 50 °C, the stirring speed at 600 rpm. After reacting for 2 h, take out the product for analysis of the fatty acid composition;

[0040] Under these reaction conditions, the lipase activity is defined as: the content (%) of capric acid bound to the fish oil glycerol backbone per hour.

[0041] Table 1. Fatty acid composition of DHA algal oil

[0042] Fatty acid Content (%) C14:0 2.41 C16:0 33.01 C18:0 1.31 C18:1 0.62 C20:4 1.31 C20:5 1.22 C22:5 10.68 C22:6 (DHA) 48.66

[0043] Example 1

[0044] DHA algal oil contains a relatively high content of palmitic acid. First, remove the saturated triglycerides by fractionation to further increase the content of DHA in the oil.

[0045] After heating DHA algal oil to 60 °C to melt it thoroughly, cool it down by a programmed cooling method. Lower the temperature to 20 °C at a rate of 10 °C / h, and then lower the temperature to 10 °C at a rate of 3 °C / h, and hold for 10 h. Separate the solid fat by filtration or centrifugation to obtain liquid oil, and the DHA content in the liquid oil is 59.55%.

[0046] Improve the activity of lipase by the method of surfactant bioimprinting. Using the imprinted lipase as a catalyst, obtain medium- and long-chain triglycerides rich in DHA through acidolysis reaction.

[0047] The imprinting conditions were as follows: The non-ionic surfactant Tween 20 was dissolved in a mixed solution of isopropanol and water (isopropanol content was 70%) at a concentration of 20 mg / L by mass, and was fully dispersed. Then, immobilized lipase Novozym 435 with a mass fraction of 10% was added thereto. The mixture was stirred at 100 rpm for 60 min at 25°C, and the lipase was obtained by filtration. The water was removed by freeze-vacuum drying;

[0048] The surfactant imprinting template on the immobilized lipase Novozym 435 was eluted with the non-polar solvent n-hexane. Subsequently, the lipase was filtered and dried in a vacuum dryer at room temperature for 24 h to remove the organic solvent, and the imprinted lipase was obtained.

[0049] The activity of the imprinted lipase Novozym 435 was 6.32%, and the activity of the non-imprinted lipase Novozym 435 was 3.71%.

[0050] Octanoic acid and DHA algal oil were mixed at a ratio of 2:1 (w / w). Subsequently, the imprinted lipase accounting for 5 wt% of the substrate weight was added to the mixture, nitrogen was charged, and the reaction was carried out at 60°C for 6 h. The lipase was filtered to obtain the acidolysis reaction product.

[0051] The compositions of octanoic acid, DHA, and glycerides in the acidolysis reaction product are shown in the following table.

[0052] Table 2. Compositions of octanoic acid, DHA, and glycerides in the acidolysis reaction product

[0053]

[0054]

[0055] The enzyme-catalyzed acidolysis reaction is a two-step reaction. The lipase first hydrolyzes triglycerides to form monoglycerides and diglycerides, and then esterifies with fatty acids as acyl donors to complete the acidolysis.

[0056] Therefore, monoglycerides and diglycerides, as by-products of the acidolysis reaction, will reduce the yield of triglycerides in the product. After the acidolysis reaction is completed, by directly evacuating the acidolysis reaction system, the main reaction is changed from the acidolysis reaction to the esterification reaction. Using the free fatty acids, glycerides, and imprinted lipase in the acidolysis reaction system, the fatty acids react with monoglycerides and diglycerides to carry out the esterification reaction to form triglycerides, thereby improving the product yield. The vacuum degree of the esterification reaction system is 20 mbar, the reaction time is 6 h, the stirring rate is 500 rmp, and the reaction temperature is 60°C. After the reaction is completed, the lipase is filtered, and the compositions of octanoic acid, DHA, and glycerides in the esterification reaction product are analyzed as follows.

[0057] Table 3. Composition of caprylic acid, DHA and glycerides in the esterification reaction products

[0058] Fatty acid Content (%) Caprylic acid 19.37 DHA 55.42 Glyceride Content (%) Monoglyceride 0.58 Diglyceride 0.64 Triglyceride 98.78

[0059] After the esterification reaction, the monoglyceride and diglyceride in the product are converted into triglyceride. At the same time, since a large amount of fatty acids in the system are caprylic acid, the esterification reaction is mainly carried out by caprylic acid with monoglyceride and diglyceride, so the caprylic acid content of triglycerides in the system is also increased, indicating that the content of medium and long-chain triglycerides is also increased.

[0060] Molecular distillation was used to remove free fatty acids in the esterification reaction system. The molecular distillation conditions were: distillation temperature of 170°C, pressure of 5 Pa, condenser temperature of 25°C. The glycidyl ester content of the obtained product was 0.23 mg / kg, and the chloropropanol ester content was 0.19 mg / kg.

[0061] Example 2

[0062] DHA algae oil contains a relatively high content of palmitic acid. First, the saturated triglycerides are removed through fractionation to further increase the DHA content in the oil.

[0063] After the DHA algae oil is heated to 70°C and fully melted, the temperature is lowered to 25°C at 15°C / h, and then to 6°C at 6°C / h, and maintained for 16 hours. The solid fat is separated by filtration or centrifugation to obtain liquid oil. The DHA content in the liquid oil is 64.21%.

[0064] The activity of lipase was enhanced by surfactant bio-imprinting, and medium- and long-chain triglycerides rich in DHA were obtained through acid hydrolysis reaction using imprinted lipase as catalyst.

[0065] The imprinting conditions are as follows: a nonionic surfactant Tween 40 is dissolved in a mixed solution of isopropanol and water (the isopropanol content is 80%) at a concentration of 50 mg / L, and fully dispersed, and 20% of immobilized lipase Lipozyme RM IM is added thereto, and the mixture is stirred at 150 rpm for 45 min at 25°C, and the lipase is obtained by filtration, and the water is removed by freeze vacuum drying;

[0066] The surfactant imprinted template on the immobilized lipase Lipozyme RM IM was eluted with a non-polar solvent n-hexane, and then the lipase was filtered and dried in a vacuum dryer at room temperature for 24 hours to remove the organic solvent to obtain the imprinted lipase. The activity of the imprinted lipase Lipozyme RM IM was 5.35%, and the activity of the non-imprinted lipase Lipozyme RM IM was 3.24%.

[0067] Caprylic acid was mixed with DHA algal oil at a ratio of 4:1. Subsequently, lipase imprinted accounting for 8 wt% of the substrate weight was added to the mixture, nitrogen was filled in, and the reaction was carried out at 70 °C for 8 h. The lipase was filtered to obtain the acidolysis reaction product. The compositions of caprylic acid, DHA, and glycerides in the acidolysis reaction product are shown in the following table.

[0068] Table 4. Compositions of caprylic acid, DHA, and glycerides in the acidolysis reaction product

[0069] Fatty acid Content (%) Capric acid 22.38 DHA 60.53 Glyceride Content (%) Monoglyceride 3.37 Diglyceride 4.16 Triglyceride 92.47

[0070] The enzyme-catalyzed acidolysis reaction is a two-step reaction.

[0071] Lipase first hydrolyzes triglycerides to form monoglycerides and diglycerides, and then esterification is carried out with fatty acids as acyl donors to complete acidolysis.

[0072] Therefore, as by-products of the acidolysis reaction, monoglycerides and diglycerides will reduce the yield of triglycerides in the product. After the acidolysis reaction, by directly evacuating the acidolysis reaction system, the main reaction is changed from the acidolysis reaction to the esterification reaction. Using the free fatty acids, glycerides, and lipase imprinted in the acidolysis reaction system, the fatty acids react with monoglycerides and diglycerides to carry out esterification reaction to form triglycerides, thereby increasing the product yield. The vacuum degree of the esterification reaction system is 10 mbar, the reaction time is 8 h, the stirring rate is 700 rmp, and the reaction temperature is 70 °C. After the reaction, the lipase is filtered, and the compositions of caprylic acid, DHA, and glycerides in the esterification reaction product are shown as follows.

[0073] Table 5. Compositions of caprylic acid, DHA, and glycerides in the esterification reaction product

[0074] Fatty acid Content (%) Capric acid 24.11 DHA 59.76 Glyceride Content (%) Monoglyceride 0.73 Diglyceride 0.42 Triglyceride 98.85

[0075] After the esterification reaction, monoglycerides and diglycerides in the product are converted into triglycerides. At the same time, since a large amount of fatty acids in the system are caprylic acid, the esterification reaction is mainly carried out between caprylic acid and monoglycerides and diglycerides. Therefore, the caprylic acid content of triglycerides in the system is also increased, indicating that the content of medium- and long-chain triglycerides is also increased.

[0076] Molecular distillation was used to remove the free fatty acids in the esterification reaction system. The molecular distillation conditions were: the distillation temperature was 160 °C, the pressure was 3 Pa, and the condenser temperature was 30 °C. The glycidyl ester content of the obtained product was 0.18 mg / kg, and the chloropropanol ester content was 0.14 mg / kg.

[0077] Example 3

[0078] DHA algal oil contains a relatively high content of palmitic acid. First, the saturated triglycerides are removed by fractionation to further increase the DHA content in the oil.

[0079] After heating the DHA algal oil to 80 °C until it melts completely, the temperature is decreased step by step. It is cooled to 15 °C at a rate of 20 °C / h, and then further cooled to 8 °C at a rate of 5 °C / h and maintained for 24 h. The solid fat is separated by filtration or centrifugation to obtain the liquid oil, and the DHA content in the liquid oil is 60.62%.

[0080] The activity of lipase is enhanced by surfactant bioimprinting. Using the imprinted lipase as a catalyst, medium- and long-chain triglycerides rich in DHA are obtained through acidolysis reaction.

[0081] The imprinting conditions are as follows: The non-ionic surfactant Tween 80 is dissolved in a mixed solution of isopropanol and water (isopropanol content is 90%) at a concentration of 100 mg / L by mass fraction, and dispersed thoroughly. Then, the immobilized lipase NS40086 with a mass fraction of 30% is added. The mixture is stirred at 200 rpm for 30 min at 25 °C, and the lipase is obtained by filtration. The water is removed by freeze-drying under vacuum.

[0082] The surfactant imprinting template on the immobilized lipase NS40086 is eluted with the non-polar solvent n-hexane. Subsequently, the lipase is filtered and dried in a vacuum dryer at room temperature for 24 h to remove the organic solvent, obtaining the imprinted lipase. The activity of the imprinted lipase NS40086 is 5.13%, and the activity of the non-imprinted lipase NS40086 is 3.06%.

[0083] Lauric acid and DHA algal oil are mixed at a ratio of 5:1. Subsequently, 10 wt% of the imprinted lipase based on the weight of the substrate is added to the mixture, nitrogen is filled in, and the reaction is carried out at 50 °C for 10 h. The lipase is filtered to obtain the acidolysis reaction product. The compositions of lauric acid, DHA, and glycerides in the acidolysis reaction product are shown in the following table.

[0084] Table 6. Compositions of lauric acid, DHA, and glycerides in the acidolysis reaction product

[0085] Fatty acid Content (%) Lauric acid 25.45 DHA 57.15 Glyceride Content (%) Monoglyceride 3.69 Diglyceride 4.87 Triglyceride 91.44

[0086] The enzyme-catalyzed acidolysis reaction is a two-step reaction. The lipase first hydrolyzes triglycerides to form monoglycerides and diglycerides, and then esterification occurs with fatty acids as acyl donors to complete the acidolysis. Therefore, monoglycerides and diglycerides, as by-products of the acidolysis reaction, will reduce the yield of triglycerides in the product.

[0087] After the acidolysis reaction is completed, by directly evacuating the acidolysis reaction system, the main reaction is transformed from the acidolysis reaction to the esterification reaction. Using the free fatty acids, glycerides, and imprinted lipase in the acidolysis reaction system, the fatty acids react with monoglycerides and diglycerides to form triglycerides through esterification reaction, thereby improving the product yield. The vacuum degree of the esterification reaction system is 30 mbar, the reaction time is 10 h, the stirring rate is 800 rmp, and the reaction temperature is 50 °C. After the reaction is completed, the lipase is filtered, and the compositions of lauric acid, DHA, and glycerides in the esterification reaction product are shown as follows.

[0088] Table 7. Compositions of lauric acid, DHA, and glycerides in the esterification reaction product

[0089]

[0090]

[0091] After the esterification reaction, the monoglycerides and diglycerides in the product are converted into triglycerides. At the same time, since a large amount of fatty acids in the system are lauric acid, the esterification reaction is mainly carried out between lauric acid and monoglycerides and diglycerides. Therefore, the lauric acid content of triglycerides in the system is also increased, indicating that the content of medium- and long-chain triglycerides is also increased.

[0092] Molecular distillation is used to remove the free fatty acids in the esterification reaction system. The molecular distillation conditions are: the distillation temperature is 180 °C, the pressure is 2 Pa, and the condenser temperature is 35 °C. The glycidyl ester content of the obtained product is 0.28 mg / kg, and the chloropropanol ester content is 0.22 mg / kg.

[0093] Comparative Example 1

[0094] The reaction is carried out according to the conditions of Example 1 without carrying out the esterification reaction. The specific steps are as follows:

[0095] DHA algal oil contains a relatively high content of palmitic acid. First, the saturated triglycerides are removed by fractional extraction to further increase the DHA content in the oil.

[0096] After heating the DHA algal oil to 60 °C to fully melt it, the temperature is decreased through a programmed cooling method. The temperature is decreased to 20 °C at a rate of 10 °C / h, and then decreased to 10 °C at a rate of 3 °C / h and maintained for 10 h. The solid fat is obtained by filtration or centrifugal separation, and the liquid oil is obtained. The DHA content in the liquid oil is 59.55%.

[0097] The activity of lipase is improved by the method of surfactant bio-imprinting. Using the imprinted lipase as a catalyst, medium- and long-chain triglycerides rich in DHA are obtained through acidolysis reaction.

[0098] The blotting conditions were as follows: The non-ionic surfactant Tween 20 was dissolved in a mixed solution of isopropanol and water (isopropanol content was 70%) at a concentration of 20 mg / L by mass, and fully dispersed. Then, immobilized lipase Novozym 435 with a mass fraction of 10% was added thereto. The mixture was stirred at 100 rpm for 60 min at 25 °C, and the lipase was obtained by filtration. The water was removed by freeze-vacuum drying;

[0099] The surfactant imprinting template on the immobilized lipase Novozym 435 was eluted with the non-polar solvent n-hexane. Subsequently, the lipase was filtered and dried in a vacuum dryer at room temperature for 24 h to remove the organic solvent, and the imprinted lipase was obtained.

[0100] Octanoic acid and DHA algal oil were mixed at a ratio of 2:1. Subsequently, the imprinted lipase accounting for 5 wt% of the substrate weight was added to the mixture, nitrogen was charged, and the reaction was carried out at 60 °C for 6 h. The lipase was filtered to obtain the acidolysis reaction product.

[0101] Molecular distillation was used to remove the free fatty acids in the acidolysis reaction product. The molecular distillation conditions were as follows: the distillation temperature was 170 °C, the pressure was 5 Pa, and the condenser temperature was 25 °C. The content of glycidyl esters in the obtained product was 0.57 mg / kg, and the content of chloropropanol esters was 0.36 mg / kg.

[0102] The fatty acid and glyceride composition of the molecular distillation product is shown in the following table.

[0103] Table 8. Composition of octanoic acid, DHA and glycerides in the molecular distillation product

[0104] Fatty acid Content (%) Caprylic acid 17.31 DHA 56.13 Glyceride Content (%) Monoglyceride 2.12 Diglyceride 3.27 Triglyceride 94.61

[0105] Since there is a certain amount of monoglyceride and diglyceride in the system during the molecular distillation stage, the content of glycidyl esters and chloropropanol esters will be increased to a certain extent during the molecular distillation stage. At the same time, since the esterification reaction is not carried out, the content of medium- and long-chain fatty acids in the comparative product is lower than that in Example 1, indicating that the content of medium- and long-chain triglycerides is also lower than that in Example 1. At the same time, the yield of triglycerides is also lower than that in Example 1.

[0106] Comparative Example 2

[0107] The reaction was carried out under the conditions of Example 3 without prior acidolysis and then esterification, and the acidolysis reaction was carried out under full vacuum throughout the process.

[0108] DHA algal oil contains a relatively high content of palmitic acid. First, the saturated triglycerides were removed by fractionation to further increase the content of DHA in the oil.

[0109] After heating the DHA algal oil to 80 °C until it melts completely, the temperature is decreased step by step. First, it is cooled to 15 °C at a rate of 20 °C / h, and then cooled to 8 °C at a rate of 5 °C / h, and maintained for 24 h. The solid fat is separated by filtration or centrifugation to obtain liquid oil. The DHA content in the liquid oil is 60.62%.

[0110] The activity of lipase is enhanced by surfactant bioimprinting. Using the imprinted lipase as a catalyst, medium and long-chain triglycerides rich in DHA are obtained through acidolysis reaction.

[0111] The imprinting conditions are as follows: The non-ionic surfactant Tween 80 is dissolved in a mixed solution of isopropanol and water (isopropanol content is 90%) at a concentration of 100 mg / L by mass fraction, and dispersed thoroughly. Then, immobilized lipase NS40086 with a mass fraction of 30% is added. The mixture is stirred at 200 rpm for 30 min at 25 °C, and then the lipase is obtained by filtration. The water is removed by freeze-drying under vacuum.

[0112] The surfactant imprinting template on the immobilized lipase NS40086 is eluted with the non-polar solvent n-hexane. Subsequently, the lipase is filtered and dried in a vacuum dryer at room temperature for 24 h to remove the organic solvent, obtaining the imprinted lipase. The activity of the imprinted lipase NS40086 is 5.13%, and the activity of the non-imprinted lipase NS40086 is 3.06%.

[0113] Lauric acid and DHA algal oil are mixed at a ratio of 5:1. Then, 10 wt% of the imprinted lipase based on the weight of the substrate is added to the mixture. The vacuum is pumped, and the vacuum degree is 30 mbar. The reaction is carried out at 50 °C for 10 h. The lipase is filtered to obtain the acidolysis reaction product. The compositions of lauric acid, DHA, and glycerides in the acidolysis reaction product are shown in the following table.

[0114] Table 9. Compositions of lauric acid, DHA, and glycerides in the acidolysis reaction product

[0115] Fatty acid Content (%) Lauric acid 13.72 DHA 58.33 Glyceride Content (%) Monoglyceride 0.25 Diglyceride 0.32 Triglyceride 99.43

[0116] Since the enzymatic acidolysis reaction is a two-step reaction, first hydrolysis and then esterification, a certain water content is required for the hydrolysis reaction. Pumping the vacuum removes the water in the system, making the whole reaction system tend to the esterification reaction, which inhibits the acidolysis reaction to a certain extent, thus reducing the content of medium-chain fatty acids in the product.

[0117] Comparative Example 3

[0118] Referring to the conditions of Example 1, the concentration of isopropanol is reduced to 60% and 50%. The activity of lipase under different isopropanol concentrations is compared, and other conditions are the same as those in Example 1.

[0119] The imprinting conditions were as follows: The non-ionic surfactant Tween 20 was dissolved in a mixed solution of isopropanol and water (isopropanol content was 70%) at a concentration of 20 mg / L by mass, and was fully dispersed. Then, immobilized lipase Novozym 435 with a mass fraction of 10% was added thereto. The mixture was stirred at 100 rpm for 60 min at 25 °C, and then the lipase was obtained by filtration. The water was removed by freeze-vacuum drying;

[0120] The excess surfactant imprinting template on the immobilized lipase Novozym 435 was eluted with the non-polar solvent n-hexane. Subsequently, the lipase was filtered and dried in a vacuum dryer at room temperature for 24 h to remove the organic solvent, and the imprinted lipase was obtained.

[0121] Table 10 Activity of lipase under different isopropanol concentrations

[0122] Imprinting conditions Enzyme activity 60% isopropanol 4.92％ 50% isopropanol 4.56％ Example 1 6.32％ Non-imprinted 3.71％

[0123] As the concentration of isopropanol decreased, the dissolution ability of the mixed solution for the surfactant decreased, resulting in the surfactant being unable to interact well with the lipase. Finally, the imprinting effect decreased, thereby reducing the catalytic activity of the lipase.

[0124] Comparative Example 4

[0125] Referring to the conditions of Example 1, without using isopropanol as a solvent for dissolution, other solvents including methanol, ethanol, butanol, n-hexane, and octane were used for dissolution, and the activity of lipase Novozym 435 was compared. Other conditions were the same as those in Example 1.

[0126] Table 11 Activity of imprinted enzyme under different solvents

[0127] Solvent type Enzyme activity Methanol 2.55％ Ethanol 3.86％ Butanol 5.12％ n-Hexane 4.12％ Octane 3.87％ Example 1 6.32％

[0128] The isopropanol solution has good solubility for the surfactant and good interaction with the lipase. In this way, it is more conducive to the interaction between the surfactant and the lipase, enhancing the imprinting effect and improving the enzyme activity.

[0129] Comparative Example 5

[0130] Referring to Example 2, the service lives of the imprinted lipase and the non-imprinted lipase were compared. By reacting different batches according to the reaction conditions of Example 2 and repeatedly using the lipase, the enzyme activities of the two enzymes in different batches were compared, so as to compare the catalytic stability of the enzymes.

[0131] Table 12 Catalytic activities and enzyme activity loss rates of imprinted and non-imprinted lipases under different batches

[0132] Reaction batch Imprinted Lipozyme RMIM Non-imprinted Lipozyme RMIM 1 5.35％ 3.24％ 2 5.26％ 3.21％ 3 5.18％ 3.13％ 4 5.11％ 3.06％ 5 4.92％ 2.87％ 6 4.87％ 2.65％ 7 4.46％ 2.42％ 8 4.12％ 2.16％ 9 3.73％ 1.91％ 10 3.51％ 1.44％ Enzyme activity loss rate 34.39％ 55.56％

[0133] The enzyme activity of the imprinted lipase Lipozyme RM IM is higher than that of the non-imprinted lipase Lipozyme RM IM even after 10 reaction cycles. At the same time, the loss rate of the enzyme activity of the imprinted lipase is lower than that of the non-imprinted lipase, indicating that after the same number of batches, the stability of the imprinted lipase is significantly higher than that of the non-imprinted lipase.

[0134] Non-ionic surfactants interact with lipases through hydrophobic forces, that is, the polar groups of the surfactants interact with the hydrophilic groups on the surface of the lipases, while the non-polar groups of the surfactants interact with the hydrophobic groups on the surface of the lipases. Since lipases are water-soluble themselves, it indicates that the hydrophilic groups on their surface are much higher than the hydrophobic groups. Through the action of the surfactants, the surface of the lipases is transformed from hydrophilic to hydrophobic. Therefore, during the reaction process, due to the increased hydrophobicity of the lipases, their interaction with hydrophobic substances in the system is enhanced, avoiding excessive contact of the lipases with polar substances in the reaction system and preventing the polar substances from removing the water on the surface of the lipases, which may lead to their inactivation. Thus, it has a certain protective effect on the stable catalytic structure of the lipases. Since the interaction mode between non-ionic surfactants and lipases is hydrophobic interaction, this force is relatively small and will not cause a large change in the configuration of the lipases. Therefore, it will not produce an inhibitory effect on the activity of the lipases themselves. Due to the interaction between the surfactant and the active center of the lipase, the lipase opens the lid of the active center and removes the excess water of the lipase in a certain way, thus preserving the active catalytic configuration of the lipase and enhancing the catalytic efficiency of the lipase. Therefore, the activity and catalytic stability of the lipase are enhanced by changing the structure of the lipase itself and the interaction between the lipase and the system.

[0135] In view of this, in this patented technology, first, the saturated triglycerides containing palmitic acid in DHA oil are separated by fractional crystallization, and then the DHA oil is further concentrated. At the same time, a non-ionic surfactant is used to modify the imprinted lipase to obtain an imprinted lipase with higher catalytic activity. Using this enzyme as a catalyst and DHA concentrated oil as a raw material, a directed acidolysis reaction is carried out. Finally, by evacuating the system, the system is transformed from an acidolysis reaction to an esterification reaction, and the by-products in the enzyme reaction system, namely monoglycerides and diglycerides, are esterified into triglycerides, thereby increasing the yield of triglycerides in the product and reducing the generation amount of harmful substances in the post-treatment of the product.

[0136] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the present invention.

Claims

1. A method for preparing medium- and long-chain triglycerides rich in DHA, characterized in that: Including, After heating DHA algal oil to 60 - 80 °C for full melting, the temperature is decreased to 15 - 25 °C at a rate of 10 - 20 °C / h; then the temperature is decreased to 6 - 10 °C at a rate of 3 - 6 °C / h and maintained for 10 - 24 h; solid fat is separated by filtration or centrifugation to obtain DHA concentrated oil; Using lipase imprinted with non - ionic surfactant dissolved in isopropanol solution as a catalyst, medium - chain fatty acids react with DHA concentrated oil by acidolysis reaction. Among them, the preparation method of the imprinted lipase includes dissolving non - ionic surfactant in a mixed solution of isopropanol and water at a concentration of 20 - 100 mg / L by mass fraction, adding immobilized lipase with a mass fraction of 10 - 30%; stirring the mixture at 25 °C at 100 - 200 rpm for 30 - 60 min, and filtering to obtain lipase; removing moisture by freeze - drying under vacuum, eluting the excess surfactant imprinting template on the immobilized lipase with a non - polar solvent, filtering the lipase and drying it under vacuum to remove organic solvents to obtain the imprinted lipase. The content of isopropanol in the mixed solution of isopropanol and water is greater than 70 wt%, and the non - ionic surfactant is Tween 20, Tween 40 or Tween 80; After the reaction ends, the reaction system is evacuated. Monoglyceride and diglyceride in the reaction are esterified to triglyceride. Among them, the vacuum degree for the esterification reaction by evacuating the reaction system is 10 - 30 mbar, the reaction time is 6 - 10 h, the stirring rate is 500 - 800 rpm, and the reaction temperature is 50 - 70 °C; Fatty acids are removed by molecular distillation to obtain medium - and - long - chain triglyceride products.

2. The preparation method according to claim 1, characterized in that: The lipase is Lipase Novozym 435, Lipase Lipozyme RM IM or Lipase NS40086.

3. The preparation method according to claim 1 or 2, characterized in that: The acidolysis reaction includes, Mix medium - chain fatty acids and DHA algal oil at a molar ratio of 2 - 5:1, then add imprinted lipase accounting for 5 - 10 wt% of the substrate weight to the mixture, fill with nitrogen, and react at a temperature of 50 - 70 °C for 6 - 10 h, filter the lipase to obtain the acidolysis reaction product.

4. The preparation method according to claim 1, characterized in that: The molecular distillation conditions are: the distillation temperature is 160 - 180 °C, the pressure is 2 - 5 Pa, and the condenser temperature is 25 - 35 °C.

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