A method for continuously producing medium- and long-chain triglycerides by enzymatic method and its product

Abstract

The present invention discloses a method for continuously producing medium- and long-chain triglycerides by enzymatic method and its product. By establishing a transesterification product prediction model, the usage ratio of substrates in the transesterification reaction is determined. Meanwhile, the activity of lipase is enhanced by the method of surfactant bio-imprinting. Using the imprinted enzyme as a catalyst, medium-chain triglycerides and edible oils are used as raw materials to carry out transesterification reaction to produce medium- and long-chain triglycerides. To obtain a higher concentration of medium- and long-chain triglycerides, after the transesterification reaction, medium-chain fatty acids are directly added to the transesterification reaction system, and then acidolysis reaction is carried out. After the acidolysis reaction, by applying a certain degree of vacuum to the system, the main reaction of the system is converted from acidolysis reaction to esterification reaction, so that the free fatty acids react with the by-products of the transesterification and acidolysis reactions, including monoglyceride and diglyceride, to carry out esterification reaction to generate triglyceride. Finally, the free fatty acids are removed by molecular distillation to obtain the final product.

Description

Technical Field

[0001] The invention belongs to the technical field of oils and fats, and specifically relates to a method for producing medium- and long-chain triglycerides by continuous enzymatic method and a product thereof. Background Art

[0002] Medium-chain triglycerides are structural triglycerides that contain both medium-chain fatty acids and long-chain fatty acids on the glycerol backbone. According to the length differences of the three fatty acid carbon chains acylated on the triglyceride molecular backbone, they can be divided into MLM type, MML type, LML type, and LLM type. Medium-chain triglycerides have many health functions, such as lowering blood lipids, inhibiting obesity, improving immunity, reducing inflammatory responses, reducing diabetes and cardiovascular diseases, and reducing the risk of cancer. The medium-chain fatty acids in medium-chain triglycerides are relatively water-soluble, can be quickly hydrolyzed in the stomach, and absorbed into the liver through the portal vein to supply immediate energy. At the same time, diglycerides or monoglycerides produced during gastric digestion can be used as emulsifiers to increase the solubility of lipids in the small intestine, assist pancreatic lipase in digesting lipids, and can make up for the defects of the digestive system to a certain extent. Therefore, this type of product has also been used as an important energy source and functional fatty acid supplement for patients with pancreatic lipase deficiency and bile salt deficiency.

[0003] At present, there are mainly chemical and enzymatic methods for synthesizing medium- and long-chain triglycerides. Due to the mild enzyme reaction conditions, fewer by-products, easier separation of enzymes and reactants, and the characteristics of certain selectivity, enzymes are widely used in the synthesis of medium- and long-chain triglycerides. In the enzymatic synthesis, the ester exchange method of directly reacting medium-chain triglycerides with edible oil to obtain medium- and long-chain triglycerides is widely used in industrial production because of its simple and easy process. However, the ester exchange reaction catalyzed by lipase is a reversible reaction. Medium-chain triglycerides and long-chain triglycerides are not only reactants, but also products. Therefore, after the reaction, the product contains a considerable amount of medium-chain triglycerides and long-chain triglycerides, and the content of medium- and long-chain triglycerides is relatively low. It is well known that the digestion and absorption rate of medium-chain triglycerides is too fast, which can easily cause a high osmotic pressure in the small intestinal lumen. Eating a large amount may cause diarrhea, vomiting, abdominal distension and other gastrointestinal discomfort symptoms. Therefore, the content of medium-chain triglycerides should be minimized in the production of medium- and long-chain triglycerides. However, obtaining products with high, medium and long-chain triglyceride content requires the reaction to be carried out at a high, medium and long-chain triglyceride ratio, and this high ratio will result in residual high, medium and long-chain triglycerides.

[0004] Therefore, how to make full use of the convenience of the transesterification reaction to efficiently obtain products with high-medium and long-chain triglyceride content and low-medium-chain triglyceride content is a technical problem that needs to be urgently solved in this field. Summary of the invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. 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.

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

[0007] Therefore, an object of the present invention is to overcome the deficiencies in the prior art and provide a method for continuously producing medium- and long-chain triglycerides by enzymatic method.

[0008] To solve the above technical problems, the present invention provides the following technical solution: A method for continuously producing medium- and long-chain triglycerides by enzymatic method, comprising:

[0009] Establish a transesterification product prediction model to determine the proportion of substrates in the transesterification reaction;

[0010] Improve the activity of lipase by the method of surfactant bioimprinting dissolved in isopropanol solution, and use the imprinted enzyme as a catalyst;

[0011] Use medium-chain triglycerides and edible oils as raw materials, and carry out transesterification reaction to produce medium- and long-chain triglycerides according to the substrate ratio predicted by the model;

[0012] Add medium-chain fatty acids to the transesterification reaction system for acidolysis reaction;

[0013] After the acidolysis reaction, apply a vacuum degree to the system to convert the reaction of the system into an esterification reaction to produce triglycerides;

[0014] Remove free fatty acids by molecular distillation to obtain the final product.

[0015] As a preferred embodiment of the method of the present invention, wherein: the molar ratio of the medium-chain triglycerides to the edible oil is less than 1:2, and the edible oil includes soybean oil, linseed oil, and sunflower oil.

[0016] As a preferred embodiment of the method of the present invention, wherein: the preparation method of the imprinted enzyme includes:

[0017] Dissolve a non-ionic surfactant in a mixed solution of isopropanol and water at a concentration of 20-100 mg / L, and add an immobilized lipase with a mass fraction of 10-30%;

[0018] Stir the mixture at 25 °C at 100-200 rpm for 30-45 min, and filter to obtain lipase;

[0019] Remove moisture by freeze-vacuum drying;

[0020] The surfactant imprinting template on the immobilized lipase Novozym 435 was eluted with a non-polar solvent. Subsequently, the lipase was filtered and the organic solvent was removed by vacuum drying to obtain the imprinted lipase.

[0021] As a preferred embodiment of the method of the present invention, wherein: the non-ionic surfactant includes Tween 20, Tween 60, and Tween 80.

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

[0023] As a preferred embodiment of the method of the present invention, wherein: the content of isopropanol in the mixed solution of isopropanol and water is greater than 70%.

[0024] As a preferred embodiment of the method of the present invention, wherein: for the transesterification reaction, the amount of the imprinted lipase added is 6-10 wt% based on the weight of the substrate, the temperature is 50-70 °C, the stirring speed is 600-800 rpm, and the reaction time is 6-10 h.

[0025] As a preferred embodiment of the method of the present invention, wherein: for the acidolysis reaction conditions, the molar ratio of medium-chain fatty acids to edible oils is 2-4:1, the temperature is 50-70 °C, the stirring speed is 600-800 rpm, the reaction time is 6-10 h, and the medium-chain fatty acids include caprylic acid, capric acid, and lauric acid.

[0026] As a preferred embodiment of the method of the present invention, wherein: the esterification reaction conditions after the acidolysis reaction are: the vacuum degree is 10-30 mbar, the temperature is 50-70 °C, the reaction time is 6-10 h, and the stirring rate is 600-800 rpm; the conditions for molecular distillation are the distillation temperature is 160-180 °C, the pressure is 2-5 Pa, and the condenser temperature is 20-30 °C.

[0027] Another object of the present invention is to overcome the deficiencies in the prior art and provide medium-chain triglycerides produced by a method for continuously producing medium- and long-chain triglycerides by an enzymatic method, with the content of medium-chain triglycerides less than 10%, the content of medium- and long-chain triglycerides greater than 75%, the content of glycidyl esters less than 0.4 mg / kg, and the content of chloropropanol esters less than 0.3 mg / kg.

[0028] Advantages of the present invention:

[0029] (1) The present invention enhances the activity and stability of an enzyme by using a nonionic surfactant bio-imprinted lipase dissolved in an isopropanol solution; by combining the methods of transesterification and acidolysis, on the one hand, a product with a relatively high content of medium- and long-chain triglycerides is obtained, and at the same time, the content of medium-chain triglycerides in the product is relatively low; after the transesterification and acidolysis reactions, by applying a vacuum degree in the same system, the main reaction is changed from acidolysis to esterification, so that the by-products in the reaction system, namely monoglyceride and diglyceride, react with fatty acids to produce triglycerides, improving the yield of triglycerides in the product and reducing the content of glycidyl esters and chloropropanol esters in the product after molecular distillation deacidification.

[0030] (2) The present invention determines the usage ratio of substrates in the transesterification reaction by establishing a transesterification product prediction model, avoiding the cumbersome work of optimizing the substrate ratio in actual experiments. Detailed implementation manners

[0031] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific implementation manners of the present invention will be given in conjunction with the embodiments of the specification.

[0032] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar generalizations without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0033] 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 with other embodiments.

[0034] Transesterification is a process of rearranging and interchanging the acyl groups between MCT and LCT. The balanced distribution of various fatty acids on the glycerol backbone is the essence of transesterification. Since transesterification is a reversible equilibrium reaction, when the reaction reaches equilibrium, various fatty acids reach a balanced distribution on the glycerol backbone. Therefore, the fatty acid distribution conforms to the random distribution principle at the equilibrium stage.

[0035] In random transesterification, due to the absence of position selectivity, the fatty acids are in a randomly distributed state on the glycerol backbone. Since the fatty acid composition is relatively complex in the oil and fat system, the types of fatty acids can be defined as n, different fatty acids are defined as Xi, and the amount of substance of different fatty acids in the system is M Xi mol. Therefore, in the system, the total amount of substance of all fatty acids is:

[0036]

[0037] According to the principle of random distribution of fatty acids, through permutation and combination, the probabilities of fatty acids on the glycerol backbone at reaction equilibrium are as follows:

[0038]

[0039] Therefore, the content of triglyceride molecule Xs-Xj-Xk at equilibrium is:

[0040]

[0041] For the reaction between long-chain triglycerides and medium-chain triglycerides, there are only two types of fatty acids in the system, long-chain fatty acids (L) and medium-chain fatty acids (M). Assume that the content of long-chain triglycerides in the system is r mol and the content of medium-chain triglycerides is h mol. At equilibrium in random transesterification, the probabilities of medium-chain fatty acids and long-chain fatty acids on the glycerol backbone are as follows:

[0042]

[0043] In directed transesterification, due to the presence of positional selectivity, without considering acyl transfer, only the fatty acids at the sn-1,3 positions on the glycerol backbone are in a state of random distribution.

[0044] Since the fatty acid composition in the oil and fat system is relatively complex, the types of fatty acids can be defined as n. Different fatty acids are defined as Xi, the amount of substance of different fatty acids in the system is MXi mol, and the amount of substance of different fatty acids at the sn-2 position is Msn-2Xi. Therefore, in the system, the amount of substance of fatty acids that can undergo random reactions is:

[0045]

[0046] According to the principle of random distribution of fatty acids, through permutation and combination, the probabilities of fatty acids on the glycerol backbone at reaction equilibrium are as follows:

[0047]

[0048] Therefore, the content of triglyceride molecule Xs-Xj-Xk at equilibrium is:

[0049]

[0050] In the reaction of long-chain triglycerides and medium-chain triglycerides, there are only two types of fatty acids in the system, long-chain fatty acids (L) and medium-chain fatty acids (M). Assuming that the content of long-chain triglycerides in the system is r mol and the content of medium-chain triglycerides is h mol, when random transesterification reaches equilibrium, the probabilities of medium-chain fatty acids and long-chain fatty acids appearing on the glycerol backbone are as follows:

[0051]

[0052] Therefore, in the transesterification reaction of long-chain triglycerides and medium-chain triglycerides, whether random reaction or directed reaction (without considering acyl transfer) is adopted, the composition of the obtained triglycerides is the same.

[0053] According to the distribution of medium-chain fatty acids and long-chain fatty acids on the glycerol backbone, they can be roughly divided into MLM, LMM, MML, LML, MLL, and LLM.

[0054] Therefore, after the reaction reaches equilibrium, the composition of various triglycerides in the system is shown in Table 1 below.

[0055] Table 1 Composition and proportion of triglycerides in random transesterification

[0056] Triglyceride species Relative molar content MML <![CDATA[h 2 r(h + r) 3 > LMM <![CDATA[rh 2 (h+r) 3 > MLM <![CDATA[h 2 r(h + r) 3 > LLM <![CDATA[r 2 h(h + r) 3 > MLL <![CDATA[r 2 h(h + r) 3 > LML <![CDATA[r 2 h(h + r) 3 > MMM <![CDATA[h 3 (h + r) 3 > LLL <![CDATA[r 3 (h + r) 3 > MML + LMM + LML + MMM h(h + r) MLM + LLM + MLL + LLL r(h+r) MML + LMM + LML + MLM + LLM + MLL + MMM + LLL 1

[0057] Therefore, when the reaction reaches equilibrium, the theoretical triglyceride composition at different ratios of long-chain triglycerides to medium-chain triglycerides is shown in the following table:

[0058] Table 2 Theoretical triglyceride composition

[0059]

[0060]

[0061] It can be seen from the theoretical calculated values that as the ratio of medium-chain triglycerides (MCT) to long-chain triglycerides (LCT) increases, the proportion of medium-long-chain triglycerides (MLCT) in the product first increases and then decreases. However, there will be a certain difference between the theoretical value and the actual value. The reason is that during the reaction, due to various reasons, the reaction cannot reach equilibrium. Therefore, in practice, the proportion of MLCT in the product is less than the theoretical value. Therefore, to further increase the content of MLCT and shorten the reaction time, choosing a catalyst with high activity is a better choice. On the one hand, it can shorten the reaction time and improve the reaction efficiency. On the other hand, a short reaction time is beneficial to reducing the oxidation of oils and fats.

[0062] Transesterification through the MMM and LCT reactions is an efficient reaction method for obtaining MLCT. However, if the substrate ratio is not paid attention to in the system, a relatively high proportion of MMM in the product will be caused. If the system contains a relatively large amount of MMM, after ingestion, the digestion and absorption rate of MCT is too fast, which is likely to cause a relatively high osmotic pressure in the small intestinal lumen, and a large amount of ingestion may cause gastrointestinal discomfort symptoms such as diarrhea, vomiting, and abdominal distension.

[0063] Therefore, the content of MCT should be minimized in the production of MLCT. It can be seen from the composition of triglycerides at different substrate ratios at the reaction equilibrium deduced by the model that when the ratio of MMM to LCT is less than or equal to 1:2, the content of MMM in the system is less than 5%.

[0064] Therefore, these ratios can be selected for the reaction to obtain MLCT products with a low MMM content. However, due to the relatively low proportion of MMM, the content of LCT in the system is relatively high after the reaction equilibrium. Therefore, a higher content of MLCT can be obtained by adding a small amount of medium-chain fatty acids.

[0065] Determination of transesterification activity: Soybean oil (2 g) was mixed with MCT at a molar ratio of 1:1. Subsequently, lipase accounting for 4 wt% of the total mass of the substrates was added to the mixture, and a round-bottom flask was used for the catalytic reaction. The reaction temperature was set at 50 °C, the stirring speed was 600 rpm. After reacting for 1 h, the product was taken out to analyze the triglyceride composition; the initial activity of the enzyme was evaluated according to the content (%) of medium- and long-chain triglycerides after 1 h of reaction.

[0066] Example 1

[0067] The activity of lipase was enhanced by the method of nonionic surfactant bioimprinting. The imprinting conditions were as follows: The nonionic surfactant Tween 60 was dissolved in a mixed solution of isopropanol and water (isopropanol content was 70%) at a concentration of 40 mg / L by mass fraction, and was fully dispersed. Immobilized lipase Novozym 435 with a mass fraction of 20% was added thereto, and the mixture was stirred at 200 rpm at 25 °C for 30 min, and the lipase was obtained by filtration. The water was removed by freeze-vacuum drying; The surfactant imprinting template on the immobilized lipase Novozym 435 was eluted with the nonpolar 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. The activity of the imprinted lipase Novozym435 was 38.7%, and the activity of the non-imprinted lipase Novozym 435 was 25.1%.

[0068] Medium-chain triglycerides (MCT) and soybean oil were added to a batch reactor at a molar ratio of 1:2 (MCT / soybean oil), and nitrogen was filled for protection. At the same time, the imprinted lipase Novozym 435 was added, and the addition amount of lipase was 10 wt% based on the weight of the substrate. The temperature was raised to 60 °C, and stirring was started at a stirring speed of 700 rpm for 8 h. After the reaction, the lipase was filtered to obtain the transesterification reaction product. The triglyceride composition and glyceride composition in the transesterification reaction product are shown as follows.

[0069] Table 3. Triglyceride composition and glyceride composition in the transesterification product

[0070] Triglyceride composition Content (%) LLL 33.6 MMM 6.7 MLL 39.3 MML 20.4 Glyceride composition Content (%) Monoglyceride 2.1 Diglyceride 3.7 Triglyceride 94.2

[0071] As can be seen from the above table, since the transesterification product contains a high content of LLL, the content of MLCT in the system is not very high. Therefore, the content of MLCT can be further increased by adding medium-chain fatty acids to the system for acidolysis reaction to make full use of the high efficiency of transesterification reaction and the orientation of acidolysis. Free medium-chain fatty acids were directly added to the transesterification reaction system, and the molar ratio of medium-chain fatty acid (capric acid) to soybean oil was 2:1, and then acidolysis reaction was carried out. The conditions of acidolysis reaction were: temperature 60 °C, stirring speed 700 rpm, reaction time 6 h. After the reaction, the triglyceride composition and glyceride composition of the reaction system are shown in the following table.

[0072] Table 4. Triglyceride composition and glyceride composition in the acidolysis reaction product

[0073] Triglyceride composition Content (%) LLL 14.3 MMM 8.4 MLL 34.6 MML 42.7 Glyceride composition Content (%) Monoglyceride 3.6 Diglyceride 4.2 Triglyceride 92.2

[0074] After the acidolysis reaction, it can be seen that the content of long-chain triglycerides in the system increases significantly, the content of LLL decreases significantly, and the contents of monoglyceride and diglyceride also increase to a certain extent. Therefore, by directly applying a vacuum degree to the acidolysis reaction system, the main reaction is changed from acidolysis reaction to esterification reaction, so that the free fatty acids in the system react with monoglyceride and diglyceride to generate triglyceride, and the yield of triglyceride is increased. The vacuum degree of esterification reaction is 10 mbar, the temperature is 60 °C, the reaction time is 6 h, and the stirring rate is 700 rpm. The triglyceride composition and glyceride of the obtained product are shown in the following table.

[0075] Table 5. Triglyceride composition and glyceride composition in the esterification reaction product

[0076] Triglyceride composition Content (%) LLL 14.1 MMM 8.1 MLL 35.3 MML 42.5 Glyceride composition Content (%) Monoglyceride 0.6 Diglyceride 0.7 Triglyceride 98.7

[0077] After the esterification reaction, both monoglycerides and diglycerides in the system were esterified to triglycerides. The yield of triglycerides increased to 98.7%, the content of MMM in the system was less than 10%, and the content of medium- and long-chain triglycerides was 77.8%.

[0078] Molecular distillation was used to remove free fatty acids from the esterification reaction system. The molecular distillation conditions were as follows: the distillation temperature was 160 °C, the pressure was 2 Pa, and the condenser temperature was 20 °C. The glycidyl ester content of the obtained product was 0.16 mg / kg, and the content of chloropropanol esters was 0.11 mg / kg.

[0079] Example 2

[0080] The activity of lipase was enhanced by the method of nonionic surfactant bio-imprinting. The imprinting conditions were as follows: the nonionic surfactant Tween 80 was dissolved in a mixed solution of isopropanol and water (isopropanol content was 80%) at a concentration of 100 mg / L by mass fraction, and fully dispersed. Then, 30% immobilized lipase Lipozyme RM IM by mass fraction was added thereto. The mixture was stirred at 150 rpm for 45 min at 25 °C, and the lipase was obtained by filtration. The water was removed by freeze-vacuum drying; the surfactant imprinting template on the immobilized lipase Lipozyme RM IM was eluted with the nonpolar 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. The activity of the imprinted lipase Lipozyme RM IM was 34.6%, and the activity of the non-imprinted lipase Lipozyme RM IM was 21.7%.

[0081] Medium-chain triglycerides (MCT) and linseed oil were added to the batch reactor in a molar ratio of 1:3 (MCT / linseed oil), and nitrogen was filled for protection. At the same time, the imprinted lipase Lipozyme RM IM was added, and the addition amount of the lipase was 8 wt% based on the weight of the substrate. The temperature was raised to 50 °C, and stirring was started at a stirring speed of 800 rpm for 10 h. After the reaction was completed, the lipase was filtered to obtain the transesterification reaction product. The triglyceride composition and glyceride composition in the transesterification reaction product are shown as follows.

[0082] Table 6. Triglyceride composition and glyceride composition in the transesterification product

[0083] Triglyceride composition Content (%) LLL 48.7 MMM 3.8 MLL 35.1 MML 12.4 Glyceride composition Content (%) Monoglyceride 1.8 Diglyceride 3.3 Triglyceride 94.9

[0084] As can be seen from the above table, since the transesterification product contains a high content of LLL, the content of MLCT in the system is not very high. Therefore, the content of MLCT can be further increased by adding medium-chain fatty acids to the system for acidolysis reaction, making full use of the high efficiency of the transesterification reaction and the orientation of the acidolysis reaction. Free medium-chain fatty acids were directly added to the transesterification reaction system, and the molar ratio of medium-chain fatty acid (lauric acid) to linseed oil was 3:1. Then, the acidolysis reaction was carried out. The conditions of the acidolysis reaction were as follows: temperature 50°C, stirring speed 800 rpm, reaction time 8 h. After the reaction, the triglyceride composition and glyceride composition of the reaction system are shown in the following table.

[0085] Table 7. Triglyceride composition and glyceride composition in the acidolysis reaction product

[0086] Triglyceride composition Content (%) LLL 16.6 MMM 5.7 MLL 40.4 MML 37.3 Glyceride composition Content (%) Monoglyceride 3.1 Diglyceride 4.5 Triglyceride 92.4

[0087] After the acidolysis reaction, it can be seen that the content of long-chain triglycerides in the system increased significantly, the content of LLL decreased significantly, and the contents of monoglyceride and diglyceride also increased to a certain extent. Therefore, by directly applying a vacuum to the acidolysis reaction system, the main reaction was changed from the acidolysis reaction to the esterification reaction, so that the free fatty acids in the system reacted with monoglyceride and diglyceride to form triglycerides, and the yield of triglycerides was increased. The vacuum degree of the esterification reaction was 20 mbar, the temperature was 50°C, the reaction time was 8 h, and the stirring rate was 800 rpm. The triglyceride composition and glyceride of the obtained product are shown in the following table.

[0088] Table 8. Triglyceride composition and glyceride composition in the esterification reaction product

[0089] Triglyceride composition Content (%) LLL 15.4 MMM 5.9 MLL 40.6 MML 38.1 Glyceride composition Content (%) Monoglyceride 0.6 Diglyceride 0.8 Triglyceride 98.6

[0090] After the esterification reaction, both monoglyceride and diglyceride in the system were esterified to triglycerides, the yield of triglycerides was increased to 98.6%, the content of MMM in the system was less than 10%, and the content of medium- and long-chain triglycerides was 78.7%.

[0091] Molecular distillation was used to remove the free fatty acids in the esterification reaction system. The conditions of molecular distillation were as follows: distillation temperature 170°C, pressure 3 Pa, condenser temperature 25°C. The glycidyl ester content of the obtained product was 0.21 mg / kg, and the content of chloropropanol ester was 0.16 mg / kg.

[0092] Example 3

[0093] The activity of lipase was improved by nonionic surfactant bioimprinting. The imprinting conditions were as follows: The nonionic surfactant Tween 20 was dissolved in a mixed solution of isopropanol and water (isopropanol content was 90%) at a concentration of 20 mg / L by mass fraction, and was fully dispersed. Then, 10% immobilized lipase NS40086 by mass fraction was added thereto, and the mixture was stirred at 100 rpm for 30 min at 25 °C. The lipase was obtained by filtration, and the water was removed by freeze-vacuum drying; The surfactant imprinting template on the immobilized lipase NS40086 was eluted with the nonpolar 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. The activity of the imprinted lipase NS40086 was 34.2%, and the activity of the non-imprinted lipase NS40086 was 22.1%.

[0094] Medium-chain triglycerides (MCT) and sunflower oil were added to the batch reactor in a molar ratio of 1:4 (MCT / sunflower oil), and nitrogen was charged for protection. At the same time, the imprinted lipase NS40086 was added, and the addition amount of the lipase was 6 wt% based on the weight of the substrate. The temperature was raised to 70 °C, and stirring was started at a stirring speed of 600 rpm for 6 h. After the reaction, the lipase was filtered to obtain the transesterification reaction product. The triglyceride composition and glyceride composition in the transesterification reaction product are shown as follows.

[0095] Table 9. Triglyceride composition and glyceride composition in the transesterification product

[0096] Triglyceride composition Content (%) LLL 55.6 MMM 2.3 MLL 34.4 MML 7.7 Glyceride composition Content (%) Monoglyceride 1.3 Diglyceride 3.1 Triglyceride 95.6

[0097] As can be seen from the above table, since the transesterification product contains a high content of LLL, the content of MLCT in the system is not very high. Therefore, the content of MLCT can be further increased by adding medium-chain fatty acids to the system for acidolysis reaction, making full use of the high efficiency of the transesterification reaction and the orientation of the acidolysis reaction. Free medium-chain fatty acids were directly added to the transesterification reaction system, and the molar ratio of medium-chain fatty acids (caprylic acid) to sunflower oil was 4:1. Then, the acidolysis reaction was carried out. The acidolysis reaction conditions were as follows: temperature 70 °C, stirring speed 600 rpm, reaction time 10 h. After the reaction, the triglyceride composition and glyceride composition of the reaction system are shown in the following table.

[0098] Table 10. Triglyceride composition and glyceride composition in the acidolysis reaction product

[0099]

[0100]

[0101] After the acidolysis reaction, it can be seen that the content of long-chain triglycerides in the system increases significantly, the content of LLL decreases significantly, and the contents of monoglycerides and diglycerides also increase to a certain extent. Therefore, by directly applying a vacuum to the acidolysis reaction system, the main reaction is changed from acidolysis reaction to esterification reaction, so that the free fatty acids in the system react with monoglycerides and diglycerides to form triglycerides, thereby increasing the yield of triglycerides. The vacuum degree for the esterification reaction is 30 mbar, the temperature is 70 °C, the reaction time is 10 h, and the stirring rate is 600 rpm. The triglyceride composition and glycerides of the obtained product are shown in the following table.

[0102] Table 11. Triglyceride composition and glyceride composition in the esterification reaction product

[0103] Triglyceride composition Content (%) LLL 17.4 MMM 4.3 MLL 42.7 MML 35.6 Glyceride composition Content (%) Monoglyceride 0.5 Diglyceride 0.7 Triglyceride 98.8

[0104] After the esterification reaction, both monoglycerides and diglycerides in the system are esterified to triglycerides, and the yield of triglycerides is increased to 98.8%. The content of MMM in the system is less than 10%, and the content of medium- and long-chain triglycerides is 78.3%.

[0105] Molecular distillation is used to remove the free fatty acids in the esterification reaction system. The conditions for molecular distillation are: the distillation temperature is 180 °C, the pressure is 5 Pa, and the condenser temperature is 30 °C. The content of glycidyl esters in the obtained product is 0.33 mg / kg, and the content of chloropropanol esters is 0.25 mg / kg.

[0106] Comparative Example 1

[0107] Referring to the conditions of Example 1, the lipase is not imprinted. The specific steps are as follows:

[0108] Medium-chain triglycerides (MCT) and soybean oil are added to the batch reactor in a molar ratio of 1:2 (MCT / soybean oil), nitrogen is filled for protection, and at the same time, lipase Novozym 435 is added. The addition amount of lipase is 10 wt% based on the weight of the substrate. The temperature is raised to 60 °C, and stirring is started. The stirring speed is 700 rpm, and the reaction time is 8 h.

[0109] After the reaction is completed, the lipase is filtered to obtain the transesterification reaction product. The triglyceride composition and glyceride composition in the transesterification reaction product are shown as follows.

[0110] Table 12. Triglyceride composition and glyceride composition in the transesterification product

[0111] Triglyceride composition Content (%) LLL 38.2 MMM 10.3 MLL 34.8 MML 16.7 Glyceride composition Content (%) Monoglyceride 1.8 Diglyceride 3.1 Triglyceride 95.1

[0112] As can be seen from the above table, since the transesterification product contains a high content of LLL, the content of MLCT in the system is not very high.

[0113] Therefore, the content of MLCT can be further increased by further adding medium-chain fatty acids to the system for acidolysis reaction, making full use of the high efficiency of transesterification reaction and the orientation of acidolysis.

[0114] Free medium-chain fatty acids were directly added to the transesterification reaction system, and the molar ratio of medium-chain fatty acid (capric acid) to soybean oil was 2:1. Then, acidolysis reaction was carried out. The conditions of acidolysis reaction were as follows: temperature 60 °C, stirring speed 700 rpm, reaction time 6 h. After the reaction, the triglyceride composition and glyceride composition of the reaction system are shown in the following table.

[0115] Table 13. Triglyceride composition and glyceride composition in the acidolysis reaction product

[0116] Triglyceride composition Content (%) LLL 23.7 MMM 9.6 MLL 31.3 MML 35.4 Glyceride composition Content (%) Monoglyceride 2.7 Diglyceride 3.5 Triglyceride 93.8

[0117] After the acidolysis reaction, it can be seen that the content of long-chain triglycerides in the system increased significantly, the content of LLL decreased significantly, and the contents of monoglyceride and diglyceride also increased to a certain extent. Therefore, by directly applying a vacuum to the acidolysis reaction system, the main reaction was changed from acidolysis reaction to esterification reaction, so that the free fatty acids in the system reacted with monoglyceride and diglyceride to generate triglyceride, improving the yield of triglyceride. The vacuum degree of the esterification reaction was 10 mbar, the reaction time was 6 h, and the stirring rate was 700 rpm. The triglyceride composition and glyceride of the obtained product are shown in the following table.

[0118] Table 14. Triglyceride composition and glyceride composition in the esterification reaction product

[0119] Triglyceride composition Content (%) LLL 23.3 MMM 9.8 MLL 31.5 MML 35.4 Glyceride composition Content (%) Monoglyceride 0.8 Diglyceride 1.1 Triglyceride 98.1

[0120] It can be known from the comparative example that the lipase activity without bio-imprinting is relatively low. At the end of the catalytic transesterification reaction, the contents of LLL and MMM in the reactant of Comparative Example 1 are higher than those in Example 1, and at the same time, it also leads to the content of MLCT in Comparative Example 1 being lower than that in Example 1. In the acidolysis reaction, on the one hand, the enzyme activity is relatively lower than that in Example 1, and on the other hand, due to the higher contents of LLL and MMM in the comparative example, after the acidolysis reaction, the MLCT product in Comparative Example 1 is lower than that in Example 1, and finally it also leads to the lower content of MLCT in Comparative Example 1 after the esterification reaction.

[0121] Comparative Example 2

[0122] The reaction conditions of Example 2 were adopted, and esterification reaction was not carried out, and molecular distillation was directly used for deacidification.

[0123] The activity of lipase was improved by bio-imprinting with non-ionic surfactants. The imprinting conditions were as follows: the non-ionic surfactant Tween 80 was dissolved in a mixed solution of isopropanol and water (with an isopropanol content of 80%) at a concentration of 100 mg / L, fully dispersed, and 30% of immobilized lipase Lipozyme RM IM was added thereto. The mixture was stirred at 150 rpm for 45 min at 25 ° C, filtered to obtain lipase, and the water was removed by freeze vacuum drying; the surfactant imprinting 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 h to remove the organic solvent to obtain the imprinted lipase. The activity of the imprinted lipase Lipozyme RM IM was 34.6%, and the activity of the non-imprinted lipase Lipozyme RM IM was 21.7%.

[0124] Medium chain triglyceride (MCT) and linseed oil were added to a batch reactor at a molar ratio of 1:3 (MCT / linseed oil), nitrogen was filled for protection, and imprinted lipase Lipozyme RM IM was added at the same time, the amount of lipase added was 8wt% of the weight of the substrate, the temperature was raised to 50°C, and stirring was started at a stirring speed of 800rpm, and the reaction time was 10h. After the reaction was completed, the lipase was filtered to obtain an ester exchange reaction product.

[0125] Since the transesterification product contains a high amount of LLL, the content of MLCT in the system is not very high. Therefore, the content of MLCT can be further increased by further adding medium-chain fatty acids to the system for acidolysis reaction, making full use of the high efficiency and directionality of the transesterification reaction. Free medium-chain fatty acids are directly added to the transesterification reaction system, and the molar ratio of medium-chain fatty acids (lauric acid) to linseed oil is 3:1, and then acidolysis reaction is carried out. The acidolysis reaction conditions are: temperature 50°C, stirring speed 800rpm, reaction time 8h, and acidolysis products are obtained. Molecular distillation is used to remove free fatty acids in the acidolysis reaction system. The molecular distillation conditions are: distillation temperature 170°C, pressure 3Pa, condenser temperature 25°C, and the glycidyl ester content of the obtained product is 0.51mg / kg, and the chloropropanol ester content is 0.32mg / kg. The composition of glycerides in the product is: triglyceride content is 93.1%, diglyceride is 4.2%, and monoglyceride is 2.7%.

[0126] Since the system has not undergone esterification reaction, a certain amount of monoglyceride and diglyceride exists in the system, which increases the content of glycidyl ester and chloropropanol ester in the molecular distillation stage and reduces the yield of product triglyceride.

[0127] It can be seen from the theoretically derived values that when the ratio of MCT to LCT is 1:1, the content of MLCT in the product can reach the theoretical maximum of 75%. However, the content of MMM in the system is 12.5%. Therefore, it can be theoretically deduced that it is basically impossible to achieve an MLCT content of more than 75% and an MMM content of less than 10% through transesterification reaction.

[0128] In the acidolysis reaction, if only the acidolysis reaction is used for the reaction and a relatively high content of medium-chain fatty acids is incorporated into triglycerides, an extremely high substrate ratio is required, that is, the medium-chain fatty acids need to be highly excessive. On the one hand, this causes waste of fatty acid raw materials and increases production costs. On the other hand, it will cause difficulties in subsequent deacidification.

[0129] Therefore, by combining transesterification and acidolysis reactions, first generating partial MLCT through transesterification and then carrying out an acid reaction with a small amount of fatty acids as raw materials, the usage amount of free fatty acids can be reduced on the basis of obtaining a relatively high purity of MLCT.

[0130] Comparative Example 3

[0131] Referring to the conditions in Example 3, without using isopropanol as the solvent and using methanol, ethanol, butanol, n-hexane, and octane as solvents, the effects of different solvents on the activity of the imprinted lipase NS40086 were compared. Other conditions were the same as in Example 3.

[0132] Table 15 Effects of different solvents on lipase activity

[0133]

[0134]

[0135] In Example 3, isopropanol has good solubility for non-ionic surfactants and good interaction with lipase. Therefore, it can promote the imprinting of non-ionic surfactants and lipase, enhance the imprinting effect, and thus improve the enzyme activity.

[0136] Comparative Example 4

[0137] Referring to the conditions in Example 1, a mixed solution with an isopropanol content of 65% and 55% was used to dissolve the surfactant, and lipase Novozym 435 was imprinted. Other conditions were the same as in Example 1.

[0138] Table 16 Effects of mixed solutions with different isopropanol contents on lipase activity

[0139] Species Activity 65% Isopropanol 30.2％ 55% Isopropanol 28.5％ Example 1 38.7％ Unimprinted lipase 25.1％

[0140] When the isopropanol in the mixed solution of isopropanol and water is less than 70%, the dissolution ability of the mixed solution for the surfactant drops sharply. Therefore, the surfactant cannot act well with lipase, and finally the imprinting effect is reduced.

[0141] Comparative Example 5

[0142] Referring to the conditions in Example 2, lipase Lipozyme RM IM was imprinted, and the reaction was carried out to produce medium and long-chain triglycerides. For continuous multi-batch reactions, the enzyme activity of the imprinted lipase and the non-imprinted lipase was compared in different batches, and the enzyme stability was compared.

[0143] Table 17 Comparison of the activities of the imprinted enzyme and the non-imprinted enzyme and the enzyme inactivation rate in different reaction batches

[0144]

[0145]

[0146] Non-ionic surfactants and lipase are combined with each other through hydrophobic interactions. The polar head of the surfactant acts with the hydrophilic group of the lipase, and the non-polar head acts with the hydrophobic group of the lipase. Since lipase itself is water-soluble and has more hydrophilic groups on its surface, through the action with non-ionic surfactants, the surface hydrophilicity and hydrophobicity of lipase are changed, that is, the surface of lipase becomes more hydrophobic. Therefore, during the reaction process, excessive contact between lipase and polar substances can be avoided, preventing the water on the surface of lipase from being taken away, resulting in the inactivation of lipase. At the same time, the hydrophobic interaction force of non-ionic surfactants is weak and will not cause a drastic change in the conformation of lipase. Therefore, it will not inhibit the enzyme activity. However, ionic surfactants interact through ionic bonds, which will change the surface charge distribution of lipase, resulting in a drastic change in the conformation of lipase, thereby inhibiting the lipase activity.

[0147] To efficiently obtain a structured lipid product with a high content of medium and long-chain triglycerides, the present patent technology determines the usage ratio of substrates in the transesterification reaction by establishing a transesterification product prediction model, uses surfactants to modify and enhance the enzyme activity, first obtains a product with a certain content of medium and long-chain triglycerides through transesterification reaction, then further improves the content of medium and long-chain triglycerides through acidolysis reaction, and finally by applying a vacuum degree, the reaction system is changed from acidolysis to esterification, so that monoglycerides and diglycerides in the system are converted into triglycerides, improving the triglyceride yield of the product and reducing the content of harmful substances in the product after post-treatment.

[0148] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than 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 continuously producing medium- and long-chain triglycerides by enzymatic method, characterized in that: Including, Establish a transesterification product prediction model to determine the ratio of substrates in the transesterification reaction; Improve the activity and stability of lipase by the method of bioimprinting with a nonionic surfactant dissolved in an isopropanol solution to obtain an imprinted enzyme; Among them, the preparation method of the imprinted enzyme is to dissolve the nonionic surfactant in a mixed solution of isopropanol and water at a concentration of 20 - 100 mg / L by mass, add immobilized lipase with a mass fraction of 10 - 30%; stir the mixture at 25 °C at 100 - 200 rpm for 30 - 45 min, filter to obtain lipase; remove moisture by freeze-drying under vacuum; use a non-polar solvent to elute the excess surfactant imprinting template on the immobilized lipase, then filter the lipase and dry it under vacuum to remove the organic solvent to obtain the imprinted lipase; the content of isopropanol in the mixed solution of isopropanol and water is greater than 70%, the nonionic surfactant is selected from Tween 20, Tween 60, Tween 80, and the lipase is selected from Novozym 435, Lipozyme RM IM and NS40086; Using the imprinted enzyme as a catalyst, medium-chain triglycerides and edible oils are used as raw materials, and medium- and long-chain triglycerides are produced by transesterification reaction with the substrate ratio predicted by the model. Among them, the molar ratio of medium-chain triglycerides to edible oils is less than 1:2, the edible oils are selected from soybean oil, linseed oil, sunflower oil, the addition amount of the imprinted lipase is 6 - 10 wt% based on the weight of the substrate, the reaction temperature is 50 - 70 °C, and the reaction time is 6 - 10 h; Add medium-chain fatty acids to the transesterification reaction system for acidolysis reaction. Among them, the molar ratio of medium-chain fatty acids to edible oils is 2 - 4:1, the reaction temperature is 50 - 70 °C, the stirring speed is 600 - 800 rpm, and the reaction time is 6 - 10 h. The medium-chain fatty acids are selected from caprylic acid, capric acid, lauric acid; After the acidolysis reaction, apply a vacuum to the system to convert the reaction in the system into an esterification reaction to generate triglycerides. Among them, the esterification reaction conditions after the acidolysis reaction are: the vacuum degree is 10 - 30 mbar, the temperature is 50 - 70 °C, the reaction time is 6 - 10 h, and the stirring rate is 600 - 800 rpm; Remove free fatty acids by molecular distillation to obtain the final product. Among them, the content of medium-chain triglycerides in the product is less than 10%, the content of medium- and long-chain triglycerides is greater than 75%, the content of glycidyl esters is less than 0.4 mg / kg, and the content of chloropropanol esters is less than 0.3 mg / kg.

2. The method according to claim 1, wherein: For the transesterification reaction, the stirring speed is 600 - 800 rpm.

3. The method according to claim 1, characterized in that: The conditions of the molecular distillation are that the distillation temperature is 160 - 180 °C, the pressure is 2 - 5 Pa, and the condenser temperature is 20 - 30 °C.

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