A preparation method of medium- and long-chain triglycerides enriched in n-3 polyunsaturated fatty acids and its products

Abstract

The invention discloses a preparation method of medium- and long-carbon-chain triglycerides enriched in n-3 polyunsaturated fatty acids and a product thereof. The invention adopts double imprinting of fatty acids and nonionic surfactants to improve the activity of lipase to medium-chain fatty acids and reduce the selectivity of lipase to EPA and DHA, thereby improving the content of medium-chain fatty acids in acidolysis reaction products and further enriching EPA and DHA. The reaction system of acidolysis reaction is directly utilized, a certain vacuum degree is applied, and fatty acids, glycerides and lipase existing in the reaction system are utilized to carry out esterification reaction, so that byproducts monoglycerides and diglycerides are further esterified into triglycerides, thereby improving the yield of the final product, and reducing the content of harmful substances such as glycidyl esters and chloropropanol esters in the final deacidification stage.

Description

Technical Field

[0001] The invention belongs to the technical field of oils and fats, and specifically relates to a preparation method of medium- and long-carbon-chain triglycerides enriched in n-3 polyunsaturated fatty acids and a product thereof. Background Art

[0002] n-3 polyunsaturated fatty acids have many benefits for human health, including promoting the development of infants' retina, brain and nervous system; reducing the risk of cardiovascular disease; reducing inflammation; reducing the levels of low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) in the serum of diabetic patients; inhibiting the growth and proliferation of cancer cells, thereby promoting cell apoptosis. Deep-sea fish oil is rich in n-3 polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), etc., and is the main raw material for supplementing n-3 polyunsaturated fatty acids. However, n-3 polyunsaturated fatty acids have more double bonds, and the lipase activity in the human body is not high. At the same time, for some special populations, such as infants and the elderly, due to the relatively low activity of digestive enzymes in the body, the digestion and utilization rate of deep-sea fish oil containing n-3 polyunsaturated fatty acids is low. Therefore, in order to improve the digestion and utilization rate of n-3 polyunsaturated fatty acids for these special populations, it is necessary to develop a functional lipid product with a higher digestion and utilization rate.

[0003] Medium-chain triglycerides are a type of structural lipid product that has been widely studied and used in clinical practice. Medium-chain fatty acids are highly water-soluble and can be rapidly hydrolyzed during gastric digestion and absorbed into the liver through the portal vein to provide immediate energy. The diglycerides (DAG) or monoglycerides (MAG) produced by digestion can act as emulsifiers in the small intestine to increase the specific surface area of ​​lipids, assist pancreatic lipase in lipid digestion, and to a certain extent make up for the defects caused by an imperfect digestive system. Therefore, medium-chain triglycerides are good carriers of n-3 polyunsaturated fatty acids, and can, to a certain extent, improve the digestibility and utilization of n-3 polyunsaturated fatty acids in special populations with defective digestive enzyme activity.

[0004] At present, the synthesis methods of medium- and long-chain triglycerides mainly include chemical method and enzymatic method. Enzymatic synthesis is widely used due to mild conditions, few by-products, and convenient post-processing. The main problems faced by enzymatic synthesis are the high price of enzymes and relatively low reaction activity, which leads to reduced reaction efficiency, prolonged catalytic time, and increased production costs. At the same time, since the enzyme-catalyzed reaction is a two-step reaction, triglycerides are hydrolyzed into diglycerides or monoglycerides, and then esterified to triglycerides. After the reaction is completed, diglycerides and monoglycerides will be produced as by-products, thereby reducing the yield of triglycerides in the product, and in the later deacidification stage, due to the presence of by-products, more glycidyl esters and chloropropanol esters will be produced. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] 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 enriched in n-3 polyunsaturated fatty acids.

[0008] In order to solve the above technical problems, the present invention provides the following technical solution: a method for preparing medium- and long-chain triglycerides enriched in n-3 polyunsaturated fatty acids, comprising:

[0009] The double-imprinted lipase was prepared by reacting fatty acids and non-ionic surfactants, which increased the activity of the lipase towards medium-chain fatty acids and reduced the selectivity of the lipase towards EPA and DHA.

[0010] Using imprinted lipase as a catalyst, medium-chain fatty acids and fish oil rich in n-3 polyunsaturated fatty acids are subjected to acid hydrolysis to obtain medium- and long-chain triglycerides rich in n-3 polyunsaturated fatty acids;

[0011] The vacuum degree of the medium- and long-chain triglyceride reaction system rich in n-3 polyunsaturated fatty acids is increased, the reaction is converted into an esterification reaction, the fatty acids in the acidolysis reaction are used to carry out an esterification reaction with glycerides, and the triglyceride product is obtained after the fatty acids are removed by molecular distillation.

[0012] As a preferred embodiment of the preparation method of the present invention, the preparation method of the double imprinted lipase comprises:

[0013] A nonionic surfactant and a fatty acid are added to a mixed solution of isopropanol and water to prepare a blotting solution;

[0014] Adding immobilized lipase to the imprinting solution, stirring at 100-200 rpm for 30-60 min at 25° C., filtering the lipase, and removing water and isopropanol by vacuum freeze drying;

[0015] The imprinted template on the immobilized lipase was eluted with a non-polar solvent and dried in a vacuum dryer at room temperature to obtain the double-imprinted lipase.

[0016] As a preferred embodiment of the preparation method of the present invention, the imprinting solution comprises a fatty acid concentration of 60 to 150 mg / L and a surfactant concentration of 50 to 100 mg / L; and the mass concentration of isopropanol in the mixed solution of isopropanol and water is greater than 70%.

[0017] As a preferred embodiment of the preparation method of the present invention, wherein: immobilized lipase is added to the imprinting solution, wherein the content of the immobilized enzyme is 10-30wt%.

[0018] As a preferred embodiment of the preparation method of the present invention, the fatty acid comprises C8:0, C10:0 and C12:0; the nonionic surfactant is a Tween series surfactant, including Tween 40, Tween 80 and Tween 100;

[0019] The lipase is a commercial lipase, including but not limited to Lipozyme RM IM, NS40086, and Novozym435.

[0020] As a preferred embodiment of the preparation method of the present invention, the conditions for the double imprinted lipase-catalyzed acid hydrolysis reaction are:

[0021] The molar ratio of medium-chain fatty acids to fish oil is 2-5:1, the temperature is 40-70° C., the amount of enzyme added is 6-15wt% of the weight of the fish oil, the stirring speed is 600-800rpm, and the reaction time is 6-10h.

[0022] As a preferred embodiment of the preparation method of the present invention, in the esterification reaction, the vacuum degree of the reaction system is 10 to 30 mbar, the reaction time is 5 to 10 hours, and the stirring rate is 600 to 800 rpm.

[0023] As a preferred embodiment of the preparation method of the present invention, the molecular distillation conditions are: distillation temperature is 160°C, pressure is 3Pa, and condenser temperature is 30°C.

[0024] 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 n-3 polyunsaturated fatty acids, wherein the medium- and long-chain triglyceride product has a triglyceride content of more than 98%, a glycidyl ester content of less than 0.3 mg / kg, and a chloropropanol ester content of less than 0.2 mg / kg.

[0025] Beneficial effects of the present invention:

[0026] (1) The present invention uses double imprinting of fatty acids and nonionic surfactants to enhance the activity and catalytic stability of lipase for medium-chain fatty acids and reduce the selectivity of lipase for EPA and DHA, thereby increasing the content of medium-chain fatty acids in the acidolysis reaction product and further enriching EPA and DHA;

[0027] (2) The present invention directly utilizes the reaction system of the acidolysis reaction, applies a certain vacuum degree, and utilizes the existing fatty acids, glycerides and lipase in the reaction system to carry out an esterification reaction, so that the by-products monoglyceride and diglyceride are further esterified into triglycerides, thereby improving the yield of the final product and reducing the content of harmful substances such as glycidyl esters and chloropropanol esters in the final deacidification stage. DETAILED DESCRIPTION

[0028] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0029] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0030] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0031] Determination of the activity of lipase on medium-chain fatty acids in the present invention:

[0032] Fish oil (2 g) and capric acid were mixed at a molar ratio of 1:3, and then lipase at 3 wt% of the total mass of the substrate was added to the mixture. A round-bottom flask was used for catalytic reaction. The reaction temperature was set at 50 °C and the stirring speed was 600 rpm. After 2 h of reaction, the product was taken out for analysis of fatty acid composition.

[0033] Under these reaction conditions, lipase activity was defined as the amount (%) of capric acid incorporated into the glycerol backbone of fish oil per hour.

[0034] The selectivity of lipase for EPA or DHA in the present invention is:

[0035] The reaction was carried out according to the method of lipase activity determination. After 2 hours of reaction, the content of EPA and DHA in the product was analyzed and compared with EPA and DHA in fish oil. The selectivity was defined as:

[0036] Selectivity of lipase for EPA or DHA (%) = (content of EPA or DHA in fish oil - content of EPA or DHA in reactant) / content of EPA or DHA in fish oil × 100%

[0037] The higher the selectivity, the higher the activity of the lipase towards EPA or DHA, and the more EPA or DHA is ultimately replaced, and vice versa.

[0038] Table 1. Fatty acid composition of fish oil

[0039] fatty acid Content (mol%) C14 8.87 C16 19.71 C16:1 9.1 C17 1.98 C17:1 1.12 C18 9.66 C18:1 3.35 C18:3 3.72 C20:5(EPA) 23.91 C24:1 2.76 C22:6 (DHA) 15.82

[0040] Example 1

[0041] Optimization of fatty acid types:

[0042] Fatty acids C8:0, C10:0, C12:0, C14:0, C16:0, C18:0 and C18:1 (Beijing Bailingwei Technology Co., Ltd., purity> 95%) were mixed with a mixed solution of non-ionic surfactant Tween 40, isopropanol and water (isopropanol content was 80%), the mixed solution had good solubility for fatty acids and surfactants, and could achieve a better imprinting effect, the concentration of fatty acid was 60 mg / L, the concentration of Tween 40 was 50 mg / L, and they were fully dispersed to obtain a mixed solution 1;

[0043] Then, immobilized lipase Lipozyme RM IM is added to the mixed solution 1, and the immobilized enzyme accounts for 10 wt % of the mixed solution 1;

[0044] The mixture was stirred at 200 rpm for 30 min at 25°C, the lipase was filtered, and the water and isopropanol were removed by vacuum freeze drying;

[0045] The fatty acid and surfactant imprinting templates on the immobilized lipase were eluted with a non-polar solvent n-hexane, and the lipase was placed in a vacuum dryer at room temperature for 24 hours to remove the organic solvent, thereby obtaining a double-imprinted lipase;

[0046] The catalytic activity and selectivity of imprinted lipase were compared with those of non-imprinted lipase.

[0047] Determination of lipase activity:

[0048] Fish oil (2 g) and capric acid (C10:0) were mixed at a molar ratio of 1:3. Subsequently, lipase at 3 wt% of the total mass of the substrate was added to the mixture. A round-bottom flask was used for catalytic reaction. The reaction temperature was set at 50 °C and the stirring speed was 600 rpm. After 2 h of reaction, the product was taken out for analysis of fatty acid composition.

[0049] Under these reaction conditions, lipase activity was defined as the amount (%) of capric acid incorporated into the glycerol backbone of fish oil per hour.

[0050] Table 2. Catalytic activity and selectivity of imprinted and non-imprinted lipases after 2 h of reaction

[0051]

[0052] After imprinting, the activities of medium-chain fatty acids in the enzymes were improved to varying degrees, among which the activities of C8:0, C10:0, and C12:0 were higher than those of C14:0, C16:0, C18:0, and C18:1.

[0053] At the same time, after imprinting, the activity of the enzyme towards EPA and DHA was significantly reduced, indicating that after fatty acid imprinting, the inertness of lipase towards n-3 polyunsaturated fatty acids increased.

[0054] At the same time, it can be clearly seen that the lipase after C8:0, C10:0, and C12:0 imprinting has lower activity and smaller selectivity for EPA and DHA. The reason may be that their fatty acid chains are shorter, and after imprinting, the lipase's inertness to long-chain polyunsaturated fatty acids increases.

[0055] Therefore, C8:0, C10:0, and C12:0 can be selected for imprinting, which can, on the one hand, enhance the activity of lipase on medium-chain fatty acids, and on the other hand, further reduce the activity of lipase on functional fatty acids such as EPA and DHA, thereby increasing the content of EPA and DHA in the product.

[0056] Example 2

[0057] The fatty acid C8:0 and the nonionic surfactant Tween 40 are mixed with a mixed solution of isopropanol and water (the isopropanol content is 80%). The mixed solution has good solubility for the fatty acid and the surfactant and can achieve a relatively good imprinting effect. The concentration of the fatty acid is 60 mg / L and the concentration of Tween 40 is 50 mg / L. The mixture is fully dispersed to obtain a mixed solution 1.

[0058] Then, immobilized lipase Lipozyme RM IM is added to the mixed solution 1, and the immobilized enzyme accounts for 10 wt % of the mixed solution 1;

[0059] The mixture was stirred at 200 rpm for 30 min at 25°C, the lipase was filtered, and the water and isopropanol were removed by vacuum freeze drying. The excess fatty acids and surfactant imprinting template on the immobilized lipase were eluted with a non-polar solvent n-hexane. The lipase was placed in a vacuum dryer and dried at room temperature for 24 h to obtain a double imprinted lipase.

[0060] The activity of imprinted lipase Lipozyme RM IM towards medium-chain fatty acids was 5.52%, the selectivity towards EPA was 4.73%, and the selectivity towards DHA was 8.22%;

[0061] The activity of non-imprinted lipase Lipozyme RM IM was 3.47%, the selectivity for EPA was 25.68%, and the selectivity for DHA was 20.35%.

[0062] Therefore, the activity of the obtained imprinted lipase is higher than that of the non-imprinted lipase, while the selectivity for EPA and DHA is much lower than that of the non-imprinted lipase, indicating that after fatty acid imprinting, the inertness of lipase to EPA and DHA is enhanced, which will be beneficial to the enrichment of EPA and DHA in the product.

[0063] Capric acid (C10:0) and fish oil were mixed in a molar ratio of 3:1 and added to a batch reactor, nitrogen was introduced, the temperature was raised to 60°C, and 10 wt% of imprinted lipase based on the weight of the fish oil was added to the reactor for acid hydrolysis reaction, the stirring rate was 800 rpm, and the reaction time was 6 h to obtain product 1;

[0064] The triglycerides in product 1 were subjected to fatty acid analysis, and the glyceride composition in reaction product 1 was also analyzed.

[0065] Table 3. Major fatty acid composition of acid hydrolysis products

[0066] fatty acid content(%) C10:0 28.72 C20:5(EPA) 18.43 C22:6 (DHA) 14.23

[0067] Table 4. Glyceride composition in the acid hydrolysis product

[0068] fatty acid content(%) Monoglyceride 3.26 Diacylceride 4.15 Triglycerides 92.59

[0069] The enzyme-catalyzed acid hydrolysis reaction is a two-step reaction:

[0070] First, triglycerides need to be hydrolyzed to generate diglycerides or monoglycerides, and then diglycerides or monoglycerides are used as substrates for esterification reaction. Therefore, diglycerides or monoglycerides will always exist as reaction by-products, reducing the yield and purity of the product. Since there are a large number of free fatty acids in the acid hydrolysis reaction system, the content of monoglycerides and diglycerides in the system can be greatly reduced by performing an esterification reaction under high vacuum after the acid hydrolysis reaction is completed.

[0071] After the acidolysis reaction, the fatty acids and glycerides in the original reaction system were used as raw materials and the original imprinted lipase was used as a catalyst. The vacuum degree of the system was directly reduced to carry out the esterification reaction. The vacuum degree of the system was 10 mbar, the reaction time was 5 h, and the stirring rate was 800 rpm. After the reaction, the lipase was filtered and the glyceride composition in the esterification reaction product was analyzed.

[0072] Table 5. Glyceride composition in esterification reaction products

[0073] fatty acid content(%) Monoglyceride 0.26 Diacylceride 0.64 Triglycerides 99.10

[0074] After the esterification reaction, the triglyceride content in the product is increased to 99.10%, and the yield of the obtained product is 99.10%. Molecular distillation is used to remove free fatty acids in the esterification reaction product. The molecular distillation conditions are a distillation temperature of 160°C, a pressure of 3Pa, and a condenser temperature of 30°C. The content of glycidyl ester in the obtained product is 0.16 mg / kg, and that of chloropropanol ester is 0.11 mg / kg.

[0075] Example 3

[0076] The fatty acid C10:0 and the nonionic surfactant Tween 80 are mixed with a mixed solution of isopropanol and water (the isopropanol content is 70%). The mixed solution has good solubility for the fatty acid and the surfactant and can achieve a relatively good imprinting effect. The concentration of the fatty acid is 100 mg / L and the concentration of Tween 80 is 80 mg / L. The mixture is fully dispersed to obtain a mixed solution 1.

[0077] Then, immobilized lipase NS40086 was added to the mixed solution 1, and the immobilized enzyme accounted for 20 wt % of the mixed solution 1;

[0078] The mixture was stirred at 100 rpm for 60 min at 25°C, the lipase was filtered, and the water and isopropanol were removed by vacuum freeze drying. The fatty acid and surfactant imprinting template on the immobilized lipase were eluted with a non-polar solvent octane. The lipase was placed in a vacuum dryer and dried at room temperature for 24 h to obtain a double imprinted lipase.

[0079] The activity of imprinted lipase NS40086 for medium-chain fatty acids was 5.88%, the selectivity for EPA was 9.77%, and the selectivity for DHA was 8.12%, while the activity of non-imprinted lipase NS40086 was 3.13%, the selectivity for EPA was 23.56%, and the selectivity for DHA was 17.87%. Therefore, the activity of the obtained imprinted lipase was higher than that of the non-imprinted lipase, while the selectivity for EPA and DHA was much lower than that of the non-imprinted lipase, indicating that after fatty acid imprinting, the inertness of lipase to EPA and DHA was enhanced, which would be beneficial to the enrichment of EPA and DHA in the product.

[0080] Capric acid (C8:0) and fish oil were mixed in a molar ratio of 2:1 and added to an intermittent reactor, nitrogen was introduced, the temperature was raised to 40°C, and 15 wt% of imprinted lipase based on the weight of the fish oil was added to the reactor for acid hydrolysis reaction. The stirring rate was 700 rpm and the reaction time was 8 h to obtain product 1. The triglycerides in product 1 were subjected to fatty acid analysis, and the glyceride composition in the reaction product 1 was analyzed.

[0081] Table 6. Major fatty acid composition of acid hydrolysis products

[0082] fatty acid content(%) C8:0 21.54 C20:5(EPA) 17.21 C22:6 (DHA) 13.46

[0083] Table 7. Glyceride composition in the acid hydrolysis product

[0084] fatty acid content(%) Monoglyceride 2.77 Diacylceride 3.58 Triglycerides 93.65

[0085] The enzyme-catalyzed acid hydrolysis reaction is a two-step reaction. First, triglycerides need to be hydrolyzed to generate diglycerides or monoglycerides, and then diglycerides or monoglycerides are used as substrates for esterification. Therefore, diglycerides or monoglycerides will always exist as reaction by-products, reducing the yield and purity of the product.

[0086] Since there are a large amount of free fatty acids in the acidolysis reaction system, the content of monoglyceride and diglyceride in the system can be greatly reduced by performing an esterification reaction under high vacuum after the acidolysis reaction.

[0087] After the acid hydrolysis reaction, the fatty acids and glycerides in the original reaction system were used as raw materials and the original imprinted lipase was used as a catalyst. The vacuum degree of the system was directly reduced to carry out the esterification reaction. The vacuum degree of the system was 20 mbar and the reaction time was 8 h. After the reaction, the lipase was filtered and the glyceride composition in the esterification reaction product was analyzed.

[0088] Table 8. Glyceride composition in esterification products

[0089] fatty acid content(%) Monoglyceride 0.35 Diacylceride 0.74 Triglycerides 98.91

[0090] After the esterification reaction, the triglyceride content in the product is increased to 98.91%, and the yield of the obtained product is 98.91%. Molecular distillation is used to remove free fatty acids in the esterification reaction product. The molecular distillation conditions are a distillation temperature of 160°C, a pressure of 3Pa, and a condenser temperature of 30°C. The content of glycidyl ester in the obtained product is 0.21 mg / kg, and that of chloropropanol ester is 0.17 mg / kg.

[0091] Example 4

[0092] The fatty acid C12:0 and the nonionic surfactant Tween 100 are mixed with a mixed solution of isopropanol and water (the isopropanol content is 90%). The mixed solution has good solubility for the fatty acid and the surfactant and can achieve a relatively good imprinting effect. The concentration of the fatty acid is 150 mg / L, and the concentration of Tween 100 is 100 mg / L. They are fully dispersed to obtain a mixed solution 1.

[0093] Immobilized lipase Novozym 435 was then added to the mixed solution 1, and the immobilized enzyme accounted for 30 wt % of the mixed solution 1. The mixture was stirred at 150 rpm for 45 min at 25° C., the lipase was filtered, and the water and isopropanol were removed by vacuum freeze drying. The fatty acid and surfactant imprinting template on the immobilized lipase were eluted with a non-polar solvent octane, and the lipase was placed in a vacuum dryer and dried at room temperature for 24 h to obtain a double imprinted lipase.

[0094] The activity of imprinted lipase Novozym 435 for medium-chain fatty acids was 6.57%, the selectivity for EPA was 10.15%, and the selectivity for DHA was 14.13%, while the activity of non-imprinted lipase Novozym 435 was 3.87%, the selectivity for EPA was 28.15%, and the selectivity for DHA was 23.94%. Therefore, the activity of the obtained imprinted lipase was higher than that of the non-imprinted lipase, while the selectivity for EPA and DHA was much lower than that of the non-imprinted lipase, indicating that after fatty acid imprinting, the inertness of lipase to EPA and DHA was enhanced, which would be beneficial to the enrichment of EPA and DHA in the product.

[0095] Capric acid (C12:0) and fish oil were mixed in a molar ratio of 5:1 and added to a batch reactor. Nitrogen was introduced, the temperature was raised to 70°C, and 6 wt% of the imprinted lipase based on the weight of the fish oil was added to the reactor for acid hydrolysis reaction. The stirring rate was 600 rpm and the reaction time was 10 h to obtain product 1. The triglycerides in product 1 were subjected to fatty acid analysis, and the glyceride composition in the reaction product 1 was analyzed.

[0096] Table 9. Major fatty acid composition of acid hydrolysis products

[0097] fatty acid content(%) C10:0 35.2 C20:5(EPA) 16.55 C22:6 (DHA) 12.37

[0098] Table 10. Glyceride composition in the acid hydrolysis reaction products

[0099] fatty acid content(%) Monoglyceride 3.83 Diacylceride 4.75 Triglycerides 91.42

[0100] The enzyme-catalyzed acidolysis reaction is a two-step reaction. First, triglycerides need to be hydrolyzed to generate diglycerides or monoglycerides, and then diglycerides or monoglycerides are used as substrates for esterification. Therefore, diglycerides or monoglycerides will always exist as reaction by-products, reducing the yield and purity of the product. Since there are a large number of free fatty acids in the acidolysis reaction system, the content of monoglycerides and diglycerides in the system can be greatly reduced by esterification reaction under high vacuum after the acidolysis reaction.

[0101] After the acid hydrolysis reaction, the fatty acids and glycerides in the original reaction system were used as raw materials and the original imprinted lipase was used as a catalyst. The vacuum degree of the system was directly reduced to carry out the esterification reaction. The vacuum degree of the system was 30 mbar and the reaction time was 10 h. After the reaction, the lipase was filtered and the glyceride composition in the esterification reaction product was analyzed.

[0102] Table 11. Glyceride composition in esterification products

[0103] fatty acid content(%) Monoglyceride 0.57 Diacylceride 0.73 Triglycerides 98.68

[0104] After the esterification reaction, the triglyceride content in the product is increased to 98.68%, and the yield of the obtained product is 98.68%. Molecular distillation is used to remove free fatty acids in the esterification reaction product. The molecular distillation conditions are a distillation temperature of 160°C, a pressure of 3Pa, and a condenser temperature of 30°C. The content of glycidyl ester in the obtained product is 0.28 mg / kg, and that of chloropropanol ester is 0.14 mg / kg.

[0105] Comparative Example 1

[0106] Referring to the conditions of Example 2, fatty acid C8:0 and nonionic surfactant Tween 40 were mixed with a mixed solution of isopropanol and water (isopropanol content was 60%). The mixed solution had good solubility for fatty acids and surfactants and could achieve a relatively good imprinting effect. The concentration of fatty acid was 60 mg / L and the concentration of Tween 40 was 50 mg / L. They were fully dispersed to obtain a mixed solution 1.

[0107] Then, immobilized lipase Lipozyme RM IM is added to the mixed solution 1, and the immobilized enzyme accounts for 10 wt % of the mixed solution 1;

[0108] The mixture was stirred at 200 rpm for 30 min at 25°C, the lipase was filtered, and the water and isopropanol were removed by vacuum freeze drying. The fatty acid and surfactant imprinting template on the immobilized lipase were eluted with a non-polar solvent n-hexane. The lipase was placed in a vacuum dryer and dried at room temperature for 24 h to remove the organic solvent to obtain the imprinted lipase.

[0109] The activity of the imprinted lipase towards medium-chain fatty acids was 4.11%, the selectivity towards EPA was 12.47%, and the selectivity towards DHA was 16.68%.

[0110] Compared with Example 2, the activity of the lipase in Comparative Example 1 on medium-chain fatty acids decreased, and the selectivity on EPA and DHA increased.

[0111] The content of isopropanol in the mixed solution system is crucial for the solvent of fatty acids. If the isopropanol content is too low, fatty acids and surfactants cannot be fully dissolved, thus affecting the effect.

[0112] Comparative Example 2

[0113] Referring to the conditions of Example 3, no further vacuum esterification reaction was performed after the acidolysis reaction:

[0114] The fatty acid C10:0 and the nonionic surfactant Tween 80 are mixed with a mixed solution of isopropanol and water (the isopropanol content is 70%). The mixed solution has good solubility for the fatty acid and the surfactant and can achieve a relatively good imprinting effect. The concentration of the fatty acid is 100 mg / L and the concentration of Tween 80 is 80 mg / L. The mixture is fully dispersed to obtain a mixed solution 1.

[0115] Then, immobilized lipase NS40086 was added to the mixed solution 1, and the immobilized enzyme accounted for 20 wt % of the mixed solution 1;

[0116] The mixture was stirred at 100 rpm for 60 min at 25°C, the lipase was filtered, and the water and isopropanol were removed by vacuum freeze drying. The fatty acid and surfactant imprinting template on the immobilized lipase were eluted with a non-polar solvent octane. The lipase was placed in a vacuum dryer and dried at room temperature for 24 h to remove the organic solvent to obtain the imprinted lipase.

[0117] Capric acid (C8:0) and fish oil were mixed in a molar ratio of 2:1 and added to a batch reactor, nitrogen was introduced, the temperature was raised to 40°C, and 15 wt% of imprinted lipase based on the weight of the fish oil was added to the reactor for acid hydrolysis reaction, the stirring rate was 700 rpm, and the reaction time was 8 h to obtain product 1;

[0118] After filtering the lipase, the glyceride composition of the intermediate product was 2.77% monoglyceride, 3.58% diglyceride, and 93.65% triglyceride;

[0119] Free fatty acids in the reaction product were removed by molecular distillation. The molecular distillation conditions were as follows: a distillation temperature of 160° C., a pressure of 3 Pa, and a condenser temperature of 30° C. The triglyceride yield of the obtained product was the triglyceride content in the acidolysis reaction, i.e., 93.65%. The triglyceride yield was lower than that of the final product in Example 3. The content of glycidyl ester in the product was 0.52 mg / kg, and that of chloropropanol ester was 0.31 mg / kg.

[0120] Before vacuum esterification, there are still a certain amount of monoglyceride and diglyceride in the system, which ultimately affects the yield of triglyceride. At the same time, in the molecular distillation deacidification stage, more glycidyl esters and chloropropanol esters will be generated due to the presence of monoglyceride and diglyceride.

[0121] Comparative Example 3

[0122] Referring to Example 3, the lipase was not double-imprinted, and the fatty acid was not imprinted, but only the surfactant was used to imprint the lipase.

[0123] The nonionic surfactant Tween 80 is mixed with a mixed solution of isopropanol and water (the isopropanol content is 70%). The mixed solution has good solubility for fatty acids and surfactants and can achieve a better imprinting effect. The concentration of Tween 80 is 80 mg / L and is fully dispersed to obtain a mixed solution 1.

[0124] Then, immobilized lipase NS40086 was added to the mixed solution 1, and the immobilized enzyme accounted for 20 wt % of the mixed solution 1;

[0125] The mixture was stirred at 100 rpm for 60 min at 25°C, the lipase was filtered, and the water and isopropanol were removed by vacuum freeze drying. The surfactant imprinting template on the immobilized lipase was eluted with a non-polar solvent octane, and the lipase was placed in a vacuum dryer and dried at room temperature for 24 h to remove the organic solvent to obtain the imprinted lipase. The lipase activity was measured as shown in the following table.

[0126] Table 12 Comparison of the imprinted enzyme of Comparative Example 3, Example 3 and the non-imprinted enzyme

[0127] type Enzyme activity (%) EPA selectivity (%) DHA selectivity (%) Comparative Example 5 4.16 30.2 23.5 Example 3 5.88 9.77 8.12 Non-imprinted NS40086 3.13 23.56 17.87

[0128] It can be seen from the experimental results that the activity of the lipase imprinted only with nonionic surfactants is improved, but the selectivity for EPA and DHA is also improved, indicating that the activity of EPA and DHA is also improved, and this activity improvement is not selective. Therefore, during the reaction, on the one hand, the access rate of medium-chain fatty acids is improved, and on the other hand, the content of EPA and DHA in the product is reduced. In Example 3, the enzyme activity is high, but the selectivity for EPA and DHA is low, indicating that while improving the activity of the lipase, the activity of the enzyme to EPA and DHA is reduced. Therefore, it is explained that the imprinted lipase without fatty acid imprinting and only using surfactants only improves the activity of the lipase, but has little effect on the selectivity of the lipase to fatty acids.

[0129] Comparative Example 4

[0130] Referring to Example 3, the double imprinting of lipase was not performed, and only the imprinting of lipase with fatty acid was performed, and the imprinting of lipase without surfactant was performed.

[0131] The fatty acid C10:0 is mixed with a mixed solution of isopropanol and water (the isopropanol content is 70%). The mixed solution has good solubility for the fatty acid and can achieve a better imprinting effect. The concentration of the fatty acid is 100 mg / L and is fully dispersed to obtain a mixed solution 1;

[0132] Then, immobilized lipase NS40086 was added to the mixed solution 1, and the immobilized enzyme accounted for 20 wt % of the mixed solution 1;

[0133] The mixture was stirred at 100 rpm for 60 min at 25°C, the lipase was filtered, and the water and isopropanol were removed by vacuum freeze drying. The fatty acid and surfactant imprinting template on the immobilized lipase were eluted with a non-polar solvent octane. The lipase was placed in a vacuum dryer and dried at room temperature for 24 h to remove the organic solvent to obtain the imprinted lipase.

[0134] Table 13 Comparison of the imprinted enzymes of Comparative Example 4 and Example 3 and the unimprinted enzymes

[0135] type Enzyme activity (%) EPA selectivity (%) DHA selectivity (%) Comparative Example 5 4.46 20.9 12.7 Example 3 5.88 9.77 8.12 Non-imprinted NS40086 3.13 23.56 17.87

[0136] Compared with the non-imprinted lipase, the activity of the lipase only imprinted with fatty acids was improved, while the selectivity for EPA and DHA was reduced, indicating that its activity for EPA and DHA was reduced. The activity of the enzyme in Comparative Example 5 was lower than that in Example 3, and its selectivity for EPA and DHA was also higher than that in Example 3.

[0137] The advantage of double imprinting of fatty acids and nonionic surfactants is that, due to the surface activity of fatty acids, the surface activity of nonionic surfactants is used to open the lid of the active center of lipase, and then the fatty acid substrate properties are used to combine the fatty acid with the active center. By removing water and eluting the imprinting template, the active center is imprinted, thereby improving the activity of the enzyme on the one hand and the selectivity of the enzyme for fatty acids on the other hand. Therefore, when fatty acids and nonionic surfactants exist at the same time and within a certain concentration range, the effect of improving both activity and selectivity can be achieved.

[0138] Comparative Example 5

[0139] Referring to Example 2, isopropanol was not used as the solvent, and ethanol, methanol, n-hexane, octane and butanol were used as the solvent respectively.

[0140] The fatty acid C8:0 and the nonionic surfactant Tween 40 are mixed with the mixed solution of the above-mentioned solvent and water (the above-mentioned solvent content is 80%), respectively. The mixed solution has good solubility for the fatty acid and the surfactant, and can achieve a relatively good imprinting effect. The concentration of the fatty acid is 60 mg / L, and the concentration of Tween 40 is 50 mg / L. They are fully dispersed to obtain a mixed solution 1;

[0141] Then, immobilized lipase Lipozyme RM IM is added to the mixed solution 1, and the immobilized enzyme accounts for 10 wt % of the mixed solution 1;

[0142] The mixture was stirred at 200 rpm for 30 min at 25°C, the lipase was filtered, and the water and isopropanol were removed by vacuum freeze drying. The fatty acid and surfactant imprinting template on the immobilized lipase were eluted with a non-polar solvent n-hexane. The lipase was placed in a vacuum dryer and dried at room temperature for 24 h to remove the organic solvent to obtain the imprinted lipase.

[0143] Table 14 Effect of different solvents on lipase properties

[0144] type Enzyme activity (%) EPA selectivity (%) DHA selectivity (%) Ethanol 2.39 11.75 13.40 Methanol 1.27 6.19 6.32 Butanol 4.89 8.53 14.29 n-Hexane 3.68 20.62 18.20 Octane 3.71 19.66 16.88 Example 2 5.52％ 4.73％ 8.22％ Non-imprinted LipozymeRMIM 3.47％ 25.68％ 20.35％

[0145] As can be seen from the table, ethanol and methanol are used as solvents, which have a higher polarity, resulting in a decrease in enzyme activity and a lower selectivity for EPA and DHA. This is actually due to the decrease in their activity. Butanol has a lower solubility in surfactants than isopropanol, so its enzyme activity and selectivity for EPA and DHA are not as good as isopropanol; n-hexane and octane are non-polar organic solvents. In this system, an emulsified system is formed by stirring, and fatty acids are dissolved in organic solvents. The surfactants are at the interface, so many of them do not react with lipase, resulting in a reduced imprinting effect.

[0146] Comparative Example 6

[0147] Referring to Example 2, multiple batches of reactions were carried out using dual-imprinted Lipozyme RM IM and non-imprinted Lipozyme RM IM, and the catalytic activities of the two enzymes in different batches were compared to evaluate their catalytic stability. Other conditions were the same as in Example 2.

[0148] Table 15 Enzyme activity and enzyme inactivation rate of double-imprinted and non-imprinted Lipozyme RM IM in different batches

[0149]

[0150]

[0151] Bioprotein macromolecules such as enzymes are flexible in aqueous solution, rigid in organic solution, and have memory function. Lipase is different from other enzymes in that it acts on the oil-water interface and exhibits unique interfacial activity. The active site of lipase is completely buried under a "lid"-like structure, and the "lid" is composed of one or two α-helices. Therefore, by using a certain method to open the "lid" of lipase, and at the same time using substrates or substrate analogs to bind to the enzyme active center, the enzyme is in a catalytic conformation, and the conformation is maintained in a certain way, which will significantly improve the reaction activity of the enzyme in the catalytic reaction and the substrate specificity of the enzyme. At the same time, since lipase is water-soluble, the hydrophilic groups on its surface are much higher than the hydrophobic groups, and the non-ionic surfactant interacts hydrophobically with the surface of the lipase, and the polar head of the non-ionic surfactant interacts with the hydrophilic groups on the surface of the lipase, and the non-polar head interacts with the hydrophobic groups, and the hydrophilicity and hydrophobicity of the surface of the lipase change, thereby making the surface of the lipase more hydrophobic, thereby avoiding excessive contact with polar substances in the system, further maintaining the moisture on the surface of the lipase, maintaining the catalytic structure of the lipase, and finally improving the catalytic stability of the lipase. Therefore, the technology of the present invention improves the activity and substrate selectivity of the enzyme by using fatty acids and non-ionic surfactants to double-imprint lipase, and after the ester exchange reaction is completed, the reaction mode is changed by vacuuming, and the acyl donor and acceptor in the system are used for esterification reaction, so that the monoglyceride and diglyceride in the system are converted into triglycerides, thereby improving the product triglyceride yield and reducing the generation of harmful substances in post-treatment.

[0152] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for preparing medium- and long-chain triglycerides enriched in n-3 polyunsaturated fatty acids, characterized in that: include, A nonionic surfactant and a fatty acid are added to a mixed solution of isopropanol and water to prepare a blotting solution; Add immobilized lipase to the imprinting solution, stir at 100-200 rpm for 30-60 min at 25°C, filter the lipase, and remove water and isopropanol by vacuum freeze drying; The imprinted template on the immobilized lipase is eluted with a non-polar solvent, and dried in a vacuum dryer at room temperature to obtain a double imprinted lipase; wherein the concentration of the fatty acid is 60 mg / L, and the concentration of the surfactant is 50 mg / L; in the mixed solution of isopropanol and water, the mass concentration of isopropanol is 80%, and the immobilized lipase is added to the imprinted solution, wherein the content of the immobilized enzyme is 10wt%, and the fatty acid is C8:0; the nonionic surfactant is Tween 40, and the lipase is lipase Lipozyme RM IM; Using double imprinted lipase as a catalyst, medium-chain fatty acids and fish oil rich in n-3 polyunsaturated fatty acids are subjected to acid hydrolysis to obtain medium- and long-chain triglycerides rich in n-3 polyunsaturated fatty acids; The vacuum degree of the reaction system of medium-chain triglycerides rich in n-3 polyunsaturated fatty acids is increased, and the reaction is reversed to an esterification reaction, and the fatty acids in the acidolysis reaction are used to carry out an esterification reaction with glycerides, wherein the vacuum degree of the reaction system is 10-30 mbar, the reaction time is 5-10 hours, and the stirring rate is 600-800 rpm; After the fatty acids are removed by molecular distillation, triglyceride products are obtained.

2. The preparation method according to claim 1, characterized in that: Conditions of acid hydrolysis catalyzed by double imprinted lipase: The molar ratio of medium-chain fatty acids to fish oil is 2-5:1, the temperature is 40-70°C, the amount of enzyme added is 6-15wt% of the weight of the fish oil, the stirring rate is 600-800rpm, and the reaction time is 6-10h.

3. The preparation method according to claim 1, characterized in that: The molecular distillation conditions are: distillation temperature is 160°C, pressure is 3Pa, and condenser temperature is 30°C.

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