Medium-long carbon chain triglyceride edible oil and preparation method thereof

Abstract

The invention relates to the field of medium-long-chain triglyceride, and particularly discloses medium-long-carbon-chain triglyceride edible oil and a preparation method thereof. The preparation method of the medium-long carbon chain triglyceride edible oil comprises the following steps: carrying out reaction activation on sialic acid and an oxidizing agent to obtain an activated sialic acid derivative, adding the activated sialic acid derivative and lipase into a phosphate buffer solution, carrying out stirring reaction at 4 DEG C for 12-24 hours, then adding sorbitan monooleate, carrying out stirring reaction at 30-40 DEG C for 20-40 minutes, and carrying out centrifugation to obtain the medium-long carbon chain triglyceride edible oil. Freezing and drying to obtain the functionalized lipase; and adding purple perilla seed oil, medium-chain triglyceride and the functionalized lipase into a reaction kettle for reaction, removing free fatty acid in a crude product by using a molecular distillation technology, and extracting and purifying by using ethanol to obtain the medium-chain and long-chain triglyceride edible oil. The medium-long carbon chain triglyceride edible oil has the advantages of being high in yield, rich in alpha-linolenic acid and high in nutritive value.

Description

Technical Field

[0001] The present application relates to the field of medium- and long-chain triglycerides, and more specifically, to a medium- and long-chain triglyceride edible oil and a preparation method thereof. Background Art

[0002] Structured lipids, i.e. reconstructed lipids that change the fatty acid composition or positional distribution in natural oils and fats through enzymatic or chemical ester exchange, are a special type of triglycerides. Medium-chain triglycerides (MCT) are a new type of functional structural substance synthesized by combining medium-chain fatty acids (MCFA, C6-C12) and long-chain fatty acids (LCFA, C16-C22) on a glycerol backbone. MCT is superior to traditional LCFA and MCFA in nutritional metabolism. MCT can reduce the body's serum triglyceride and cholesterol levels and inhibit the accumulation of body fat; in addition, MCT can overcome the shortcomings of MCFA, not only providing the body with essential fatty acids and energy supply, but also avoiding poisoning caused by excessive ketone bodies in the body.

[0003] The synthesis methods of MCT mainly include chemical synthesis and lipase method. Chemical synthesis is the most common process for producing MCT, but the preparation process requires the use of a large amount of organic solvents, high energy consumption, and serious environmental pollution, which cannot meet the requirements of green and environmentally friendly production of MCT. Compared with traditional chemical methods, the lipase method effectively avoids the use of organic compounds and has the advantages of mild reaction conditions, low energy consumption, high efficiency, and selectivity, but there are problems such as poor miscibility of oil-soluble substrates.

[0004] Perilla seed oil is a natural oil, belonging to long-chain fatty acids, and its α-linolenic acid content is as high as 60%. α-linolenic acid is a precursor of ω-3 long-chain polyunsaturated fatty acids, which are very important to the human body. It can be converted into eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the human body, which are the main components of brain cells and human nerve cells.

[0005] With respect to the above-mentioned related technologies, it is an urgent problem to be solved at present to prepare edible oil rich in α-linolenic acid and having high nutritional value by using perilla seed oil as a reaction substrate. Summary of the invention

[0006] In order to prepare an edible oil with a high MCT content and rich in α-linolenic acid, the present application provides a medium- and long-chain triglyceride edible oil and a preparation method thereof.

[0007] In the first aspect, the present application provides a method for preparing medium- and long-chain triglyceride edible oil, using the following technical solution:

[0008] A method for preparing medium- and long-chain triglyceride edible oil comprises the following steps:

[0009] After sialic acid is activated by reacting with an oxidant, an activated sialic acid derivative is obtained, the activated sialic acid derivative and lipase are added to a phosphate buffer, stirred at 4° C. for reaction for 12-24 hours, sorbitan monooleate is added, stirred at 30-40° C. for reaction for 20-40 minutes, centrifuged, and freeze-dried to obtain a functionalized lipase; the mass ratio of sialic acid, sorbitan monooleate, and lipase is (5-15):(1-5):10;

[0010] Perilla seed oil and medium-chain triglyceride are added into a reaction kettle in a mass ratio of (0.6:0.4)-(0.8:0.2), and the functionalized lipase is added in an amount of 2-20% of the mass of the reactants to carry out the reaction, and a crude product is obtained after the reaction is completed;

[0011] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0012] By adopting the above technical scheme, the inventors creatively discovered that lipase modified with sialic acid can promote the ester exchange reaction between perilla seed oil and medium-chain triglycerides, thereby increasing the yield of medium- and long-chain triglycerides while increasing the α-linolenic acid content in the product; further modification with non-ionic surfactant sorbitan monooleate improves the miscibility of lipase with oil reactants, and further promotes the improvement of the catalytic activity and reaction efficiency of lipase; under the action of the above-mentioned functionalized lipase, medium- and long-chain triglyceride edible oil is produced by the ester exchange method, the reaction is simple, and there are few by-products, and edible oil with high medium- and long-chain triglyceride content, rich in α-linolenic acid, and high nutritional value can be obtained.

[0013] Preferably, the sialic acid activation step specifically comprises:

[0014] Sialic acid and an oxidant are dissolved in water for oxidation reaction, and then purified by dialysis to obtain an activated sialic acid derivative; the molar ratio of sialic acid to the oxidant is 1:(3.5-4.5).

[0015] Preferably, the oxidizing agent is periodate.

[0016] Preferably, the sialic acid is selected from one or both of N-acetylneuraminic acid and N-glycolylneuraminic acid.

[0017] By adopting the above technical scheme, sialic acid is oxidized to generate sialic acid derivatives containing aldehyde groups. The sialic acid derivatives can undergo coupling reactions with amino groups and other groups on the surface of lipase through the aldehyde groups, thereby realizing the covalent modification of lipase by sialic acid. The inventors have found through experiments that after lipase is modified with sialic acid, the content of α-linolenic acid in the catalytic reaction product is higher.

[0018] In addition, the inventors found that compared with conventional physical mixing modification, the functional lipase prepared by covalent modification with sialic acid and then combined with sorbitan monooleate has better catalytic effect. This may be because sorbitan monooleate not only improves the miscibility of the catalyst, but also further participates in the construction of the hydrogen bond system between lipase and sialic acid to change the spatial structure. The covalent modification and the change of the spatial structure of the lipase based on hydrogen bonds further provide a more stable and more active catalytic system for enzymatic reactions.

[0019] Preferably, the lipase is selected from one or more of Lipozyme TL IM, Lipozyme RM IM, Lipozyme 435, Novozym 435, and NS40086.

[0020] Preferably, the enzymatic reaction is carried out in a supercritical carbon dioxide medium.

[0021] Preferably, the reaction process specifically includes:

[0022] Perilla seed oil, medium-chain triglyceride and functionalized lipase are put into a reactor. In a sealed state, carbon dioxide is introduced to replace the air in the reactor. Then, carbon dioxide is introduced into the reactor at 40-60°C until the pressure is 7.5-8MPa to ensure that the reaction process is in a supercritical state. Magnetic stirring is performed at 200-500r / min and the reaction is carried out at a constant temperature for 3-6h. After the reaction is completed, the reactor is cooled to room temperature, the gas is released, and the catalyst is removed by centrifugation to obtain a crude product.

[0023] By adopting the above technical scheme, the supercritical carbon dioxide fluid has the advantages of good solubility and non-toxicity, which helps to promote the miscibility of oil reactants and the mass transfer performance of the reaction medium, promote the improvement of reaction efficiency, and help to isolate oxygen, improve the antioxidant properties and acid value of the reaction products; the functionalized lipase prepared by the above method can better maintain a stable and highly active catalytic structure in supercritical carbon dioxide compared with conventional solvents, thereby further improving the efficiency of the enzymatic reaction.

[0024] Preferably, the amount of the functionalized lipase added is 2-10% of the amount of the reactants.

[0025] Preferably, the medium chain triglyceride is one or more of caprylic triglyceride, nonanoic triglyceride, capric triglyceride and caprylic / capric triglyceride.

[0026] In a second aspect, the present application provides a medium- and long-chain triglyceride edible oil, which adopts the following technical solution:

[0027] A medium- and long-carbon-chain triglyceride edible oil is prepared by the above-mentioned method for preparing the medium- and long-carbon-chain triglyceride edible oil.

[0028] Preferably, in the medium- and long-chain triglyceride edible oil, the content of medium- and long-chain triglycerides is ≥85%, and the content of α-linolenic acid is ≥40%.

[0029] In summary, this application has the following beneficial effects:

[0030] 1. The present application uses sialic acid and sorbitan monooleate to modify lipase, thereby improving the catalytic activity of lipase and the miscibility of lipase with oil substrates, improving the reaction efficiency, and promoting the increase of MCT content and α-linolenic acid content in the reaction product;

[0031] 2. In the present application, it is preferred to carry out the reaction under supercritical carbon dioxide, which further improves the miscibility of the reaction substrates and the catalyst activity, improves the reaction efficiency, and helps to improve the antioxidant properties and acid value of the reaction products. DETAILED DESCRIPTION

[0032] To further help understand the technical solution of the present invention, the technical solution of the present invention is described in more detail below by providing several specific implementation examples. All of these described embodiments are only partial embodiments of the present invention, not all of them.

[0033] The following specific embodiments may be combined with each other, and the same or similar concepts or processes may not be described in detail in some embodiments; and the reaction devices, monomer compounds, etc. involved in the following embodiments are all commercially available.

[0034] The following specific embodiments may be combined with each other, and the same or similar concepts or processes therein may not be described in detail in some embodiments.

[0035] The following examples are further explanations of the present invention, but the present invention is not limited thereto. Unless otherwise specified in the examples, the percentages are all by mass.

[0036] Example

[0037] Example 1

[0038] This embodiment discloses a medium- and long-chain triglyceride edible oil, and the preparation method is as follows:

[0039] Dissolve N-acetylneuraminic acid in 10 times the mass of distilled water, add potassium periodate in a molar ratio of 1:4, mix well, and react with magnetic stirring at 30°C for 4 hours. After the reaction, dialysis purification is performed, and vacuum drying is performed after concentration to obtain the aldehyde-modified sialic acid derivative.

[0040] Lipase Novozym 435 (ester exchange activity: 10000 PLU / g) and sialic acid derivatives were added to sodium phosphate buffer (pH=7.5) at a mass ratio of 10:5, and magnetically stirred at 4°C for 12 hours. Sorbitan monooleate was then added thereto at a mass ratio of lipase Novozym 435: sorbitan monooleate of 10:1, and the mixture was magnetically stirred at 36°C for 30 minutes, followed by centrifugation and freeze-drying to obtain a functionalized lipase.

[0041] The transesterification activity of the functionalized lipase of this example was tested: lauric acid and glycerol were added to a round-bottom flask in a mass ratio of 10:16, tert-butanol and 5% functionalized lipase were added, heated in a water bath at 52°C, the stirring speed was controlled to 350r / min, and the reaction was carried out for 90min; after separating the product, the lauric acid conversion rate and the yield of monolaurin were detected. The transesterification activity of the functionalized lipase Novozym 435 was expressed as propyl laurate units (PLU / g) generated per 1 gram of enzyme per unit time.

[0042] In this example, the transesterification activity of the functionalized lipase was 12952 PLU / g, and the transesterification activity of the modified functionalized lipase was increased by 29.52% compared with that of the unmodified lipase.

[0043] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor at a mass ratio of 6:4, and functionalized lipase of 6% by mass of the oil substrate was added thereto. Nitrogen was filled into the reactor, and the reaction was stirred at 50°C for 4 hours. After the reaction was completed, a crude product was obtained.

[0044] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0045] Example 2

[0046] The only difference between this embodiment and embodiment 1 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0047] Dissolve N-acetylneuraminic acid in 10 times the mass of distilled water, add potassium periodate in a molar ratio of 1:4, mix well, and react with magnetic stirring at 30°C for 4 hours. After the reaction, dialysis purification is performed, and vacuum drying is performed after concentration to obtain the aldehyde-modified sialic acid derivative.

[0048] Lipase Novozym 435 (transesterification activity: 10000 PLU / g) and sialic acid derivatives were added to sodium phosphate buffer (pH=7.5) at a mass ratio of 10:5, and magnetic stirring was performed at 4°C for 12 hours. Sorbitol monooleate was then added at a mass ratio of lipase Novozym 435: sorbitan monooleate of 10:1, and magnetic stirring was performed at 36°C for 30 minutes, and then centrifuged and freeze-dried to obtain functionalized lipase. The lauric acid-glycerol transesterification method was used for detection, and the modified functionalized lipase had a 29.52% higher transesterification activity than the unmodified lipase.

[0049] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor in a mass ratio of 6:4, and functionalized lipase of 6% by mass of the oil substrate was added thereto. Carbon dioxide was introduced to replace the air in the reactor. Carbon dioxide was introduced into the reactor at 50°C until the pressure was 7.6 MPa to ensure that the reaction process was in a supercritical state. The reactor was stirred magnetically at a speed of 200 r / min and reacted at a constant temperature for 4 hours. After the reaction was completed, it was cooled to room temperature, the gas was released, and the catalyst was removed by centrifugation to obtain a crude product.

[0050] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0051] Example 3

[0052] The only difference between this embodiment and embodiment 2 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0053] Dissolve N-acetylneuraminic acid in 10 times the mass of distilled water, add potassium periodate in a molar ratio of 1:4, mix well, and react with magnetic stirring at 30°C for 4 hours. After the reaction, dialysis purification is performed, and vacuum drying is performed after concentration to obtain the aldehyde-modified sialic acid derivative.

[0054] Lipase Novozym 435 (transesterification activity: 10000 PLU / g) and sialic acid derivatives were added to sodium phosphate buffer (pH=7.5) at a mass ratio of 10:8, and magnetic stirring was performed at 4°C for 12 hours. Sorbitol monooleate was then added at a mass ratio of 10:1 for lipase Novozym 435: sorbitan monooleate, and magnetic stirring was performed at 36°C for 30 minutes, and then centrifuged and freeze-dried to obtain functionalized lipase. The lauric acid-glycerol transesterification method was used for detection, and the modified functionalized lipase had a 33.20% higher transesterification activity than the unmodified lipase.

[0055] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor in a mass ratio of 6:4, and functionalized lipase of 6% by mass of the oil substrate was added thereto. Carbon dioxide was introduced to replace the air in the reactor. Carbon dioxide was introduced into the reactor at 50°C until the pressure was 7.6 MPa to ensure that the reaction process was in a supercritical state. The reactor was stirred magnetically at a speed of 200 r / min and reacted at a constant temperature for 4 hours. After the reaction was completed, it was cooled to room temperature, the gas was released, and the catalyst was removed by centrifugation to obtain a crude product.

[0056] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0057] Example 4

[0058] The only difference between this embodiment and embodiment 2 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0059] Dissolve N-acetylneuraminic acid in 10 times the mass of distilled water, add potassium periodate in a molar ratio of 1:4, mix well, and react with magnetic stirring at 30°C for 4 hours. After the reaction, dialysis purification is performed, and vacuum drying is performed after concentration to obtain the aldehyde-modified sialic acid derivative.

[0060] Lipase Novozym 435 (transesterification activity: 10000 PLU / g) and sialic acid derivatives were added to sodium phosphate buffer (pH=7.5) at a mass ratio of 10:10, and magnetic stirring was performed at 4°C for 12 hours. Sorbitol monooleate was then added at a mass ratio of lipase Novozym 435: sorbitan monooleate of 10:1, and magnetic stirring was performed at 36°C for 30 minutes, and then centrifuged and freeze-dried to obtain functionalized lipase. The lauric acid-glycerol transesterification method was used for detection, and the modified functionalized lipase had a 34.08% higher transesterification activity than the unmodified lipase.

[0061] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor in a mass ratio of 6:4, and functionalized lipase of 6% by mass of the oil substrate was added thereto. Carbon dioxide was introduced to replace the air in the reactor. Carbon dioxide was introduced into the reactor at 50°C until the pressure was 7.6 MPa to ensure that the reaction process was in a supercritical state. The reactor was stirred magnetically at a speed of 200 r / min and reacted at a constant temperature for 4 hours. After the reaction was completed, it was cooled to room temperature, the gas was released, and the catalyst was removed by centrifugation to obtain a crude product.

[0062] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0063] Example 5

[0064] The only difference between this embodiment and embodiment 2 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0065] Dissolve N-acetylneuraminic acid in 10 times the mass of distilled water, add potassium periodate in a molar ratio of 1:4, mix well, and react with magnetic stirring at 30°C for 4 hours. After the reaction, dialysis purification is performed, and vacuum drying is performed after concentration to obtain the aldehyde-modified sialic acid derivative.

[0066] Lipase Novozym 435 (transesterification activity: 10000 PLU / g) and sialic acid derivatives were added to sodium phosphate buffer (pH=7.5) at a mass ratio of 10:14, and magnetic stirring was performed at 4°C for 12 hours. Sorbitol monooleate was then added at a mass ratio of 10:1 for lipase Novozym 435: sorbitan monooleate, and magnetic stirring was performed at 36°C for 30 minutes, and then centrifuged and freeze-dried to obtain functionalized lipase. The lauric acid-glycerol transesterification method was used for detection, and the modified functionalized lipase had a 34.16% higher transesterification activity than the unmodified lipase.

[0067] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor in a mass ratio of 6:4, and functionalized lipase of 6% by mass of the oil substrate was added thereto. Carbon dioxide was introduced to replace the air in the reactor. Carbon dioxide was introduced into the reactor at 50°C until the pressure was 7.6 MPa to ensure that the reaction process was in a supercritical state. The reactor was stirred magnetically at a speed of 200 r / min and reacted at a constant temperature for 4 hours. After the reaction was completed, it was cooled to room temperature, the gas was released, and the catalyst was removed by centrifugation to obtain a crude product.

[0068] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0069] Example 6

[0070] The only difference between this embodiment and embodiment 2 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0071] Dissolve N-acetylneuraminic acid in 10 times the mass of distilled water, add potassium periodate in a molar ratio of 1:4, mix well, and react with magnetic stirring at 30°C for 4 hours. After the reaction, dialysis purification is performed, and vacuum drying is performed after concentration to obtain the aldehyde-modified sialic acid derivative.

[0072] Lipase Novozym 435 (transesterification activity: 10000 PLU / g) and sialic acid derivatives were added to sodium phosphate buffer (pH=7.5) at a mass ratio of 10:8, and magnetic stirring was performed at 4°C for 12 hours. Sorbitol monooleate was then added at a mass ratio of lipase Novozym 435: sorbitan monooleate of 10:3, and magnetic stirring was performed at 36°C for 30 minutes, and then centrifuged and freeze-dried to obtain functionalized lipase. The lauric acid-glycerol transesterification method was used for detection, and the modified functionalized lipase had a 35.12% higher transesterification activity than the unmodified lipase.

[0073] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor in a mass ratio of 6:4, and functionalized lipase of 6% by mass of the oil substrate was added thereto. Carbon dioxide was introduced to replace the air in the reactor. Carbon dioxide was introduced into the reactor at 50°C until the pressure was 7.6 MPa to ensure that the reaction process was in a supercritical state. The reactor was stirred magnetically at a speed of 200 r / min and reacted at a constant temperature for 4 hours. After the reaction was completed, it was cooled to room temperature, the gas was released, and the catalyst was removed by centrifugation to obtain a crude product.

[0074] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0075] Example 7

[0076] The only difference between this embodiment and embodiment 2 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0077] Dissolve N-acetylneuraminic acid in 10 times the mass of distilled water, add potassium periodate in a molar ratio of 1:4, mix well, and react with magnetic stirring at 30°C for 4 hours. After the reaction, dialysis purification is performed, and vacuum drying is performed after concentration to obtain the aldehyde-modified sialic acid derivative.

[0078] Lipase Novozym 435 (transesterification activity: 10000 PLU / g) and sialic acid derivatives were added to sodium phosphate buffer (pH=7.5) at a mass ratio of 10:8, and magnetic stirring was performed at 4°C for 12 hours. Sorbitol monooleate was then added at a mass ratio of lipase Novozym 435: sorbitan monooleate of 10:5, and magnetic stirring was performed at 36°C for 30 minutes, and then centrifuged and freeze-dried to obtain functionalized lipase. The lauric acid-glycerol transesterification method was used for detection, and the modified functionalized lipase had a 35.50% higher transesterification activity than the unmodified lipase.

[0079] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor in a mass ratio of 6:4, and functionalized lipase of 6% by mass of the oil substrate was added thereto. Carbon dioxide was introduced to replace the air in the reactor. Carbon dioxide was introduced into the reactor at 50°C until the pressure was 7.6 MPa to ensure that the reaction process was in a supercritical state. The reactor was stirred magnetically at a speed of 200 r / min and reacted at a constant temperature for 4 hours. After the reaction was completed, it was cooled to room temperature, the gas was released, and the catalyst was removed by centrifugation to obtain a crude product.

[0080] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0081] Example 8

[0082] The only difference between this embodiment and embodiment 2 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0083] Dissolve N-acetylneuraminic acid in 10 times the mass of distilled water, add potassium periodate in a molar ratio of 1:4, mix well, and react with magnetic stirring at 30°C for 4 hours. After the reaction, dialysis purification is performed, and vacuum drying is performed after concentration to obtain the aldehyde-modified sialic acid derivative.

[0084] Lipase Novozym 435 (transesterification activity: 10000 PLU / g) and sialic acid derivatives were added to sodium phosphate buffer (pH=7.5) at a mass ratio of 10:8, and magnetic stirring was performed at 4°C for 12 hours. Sorbitol monooleate was then added at a mass ratio of lipase Novozym 435: sorbitan monooleate of 10:3, and magnetic stirring was performed at 36°C for 30 minutes, and then centrifuged and freeze-dried to obtain functionalized lipase. The lauric acid-glycerol transesterification method was used for detection, and the modified functionalized lipase had a 35.12% higher transesterification activity than the unmodified lipase.

[0085] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor in a mass ratio of 8:2, and functionalized lipase of 6% by mass of the oil substrate was added thereto. Carbon dioxide was introduced to replace the air in the reactor. Carbon dioxide was introduced into the reactor at 50°C until the pressure was 7.6 MPa to ensure that the reaction process was in a supercritical state. The reactor was stirred magnetically at a speed of 200 r / min and reacted at a constant temperature for 4 hours. After the reaction was completed, it was cooled to room temperature, the gas was released, and the catalyst was removed by centrifugation to obtain a crude product.

[0086] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0087] Example 9

[0088] The only difference between this embodiment and embodiment 2 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0089] Dissolve N-acetylneuraminic acid in 10 times the mass of distilled water, add potassium periodate in a molar ratio of 1:4, mix well, and react with magnetic stirring at 30°C for 4 hours. After the reaction, dialysis purification is performed, and vacuum drying is performed after concentration to obtain the aldehyde-modified sialic acid derivative.

[0090] Lipase Novozym 435 (transesterification activity: 10000 PLU / g) and sialic acid derivatives were added to sodium phosphate buffer (pH=7.5) at a mass ratio of 10:8, and magnetic stirring was performed at 4°C for 12 hours. Sorbitol monooleate was then added at a mass ratio of lipase Novozym 435: sorbitan monooleate of 10:3, and magnetic stirring was performed at 36°C for 30 minutes, and then centrifuged and freeze-dried to obtain functionalized lipase. The lauric acid-glycerol transesterification method was used for detection, and the modified functionalized lipase had a 35.12% higher transesterification activity than the unmodified lipase.

[0091] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor in a mass ratio of 6:4, and functionalized lipase of 3% by mass of the oil substrate was added thereto. Carbon dioxide was introduced to replace the air in the reactor. Carbon dioxide was introduced into the reactor at 50°C until the pressure was 7.6 MPa to ensure that the reaction process was in a supercritical state. The reactor was stirred magnetically at a speed of 200 r / min and reacted at a constant temperature for 4 hours. After the reaction was completed, it was cooled to room temperature, the gas was released, and the catalyst was removed by centrifugation to obtain a crude product.

[0092] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0093] Example 10

[0094] The only difference between this embodiment and embodiment 2 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0095] Dissolve N-acetylneuraminic acid in 10 times the mass of distilled water, add potassium periodate in a molar ratio of 1:4, mix well, and react with magnetic stirring at 30°C for 4 hours. After the reaction, dialysis purification is performed, and vacuum drying is performed after concentration to obtain the aldehyde-modified sialic acid derivative.

[0096] Lipase Novozym 435 (transesterification activity: 10000 PLU / g) and sialic acid derivatives were added to sodium phosphate buffer (pH=7.5) at a mass ratio of 10:8, and magnetic stirring was performed at 4°C for 12 hours. Sorbitol monooleate was then added at a mass ratio of lipase Novozym 435: sorbitan monooleate of 10:3, and magnetic stirring was performed at 36°C for 30 minutes, and then centrifuged and freeze-dried to obtain functionalized lipase. The lauric acid-glycerol transesterification method was used for detection, and the modified functionalized lipase had a 35.12% higher transesterification activity than the unmodified lipase.

[0097] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor in a mass ratio of 6:4, and functionalized lipase of 10% of the mass of the oil substrate was added thereto. Carbon dioxide was introduced to replace the air in the reactor. Carbon dioxide was introduced into the reactor at 50°C until the pressure was 7.6 MPa to ensure that the reaction process was in a supercritical state. The reactor was stirred magnetically at a speed of 200 r / min and reacted at a constant temperature for 4 hours. After the reaction was completed, it was cooled to room temperature, the gas was released, and the catalyst was removed by centrifugation to obtain a crude product.

[0098] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0099] Embodiment 11

[0100] The only difference between this embodiment and embodiment 2 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0101] Dissolve N-acetylneuraminic acid in 10 times the mass of distilled water, add potassium periodate in a molar ratio of 1:4, mix well, and react with magnetic stirring at 30°C for 4 hours. After the reaction, dialysis purification is performed, and vacuum drying is performed after concentration to obtain the aldehyde-modified sialic acid derivative.

[0102] Lipase Lipozyme RM IM (ester exchange activity: 275IUN / g) and sialic acid derivatives were added to sodium phosphate buffer (pH=7.5) at a mass ratio of 10:5, and magnetic stirring was performed at 4°C for 12h, and then sorbitan monooleate was added thereto at a mass ratio of 10:1 for lipase Novozym 435: sorbitan monooleate, and magnetic stirring was performed at 36°C for 30min, and then centrifuged and freeze-dried to obtain functionalized lipase. The olive oil-stearate exchange method was used for detection, and the amount of enzyme required to exchange 1μmol of stearic acid per minute was defined as 1 lipase activity unit (U). The ester exchange activity of the functionalized lipase in this embodiment was 351IUN / g, and the modified functionalized lipase had an ester exchange activity of 27.64% higher than that of the unmodified lipase.

[0103] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor in a mass ratio of 6:4, and functionalized lipase of 6% by mass of the oil substrate was added thereto. Carbon dioxide was introduced to replace the air in the reactor. Carbon dioxide was introduced into the reactor at 50°C until the pressure was 7.6 MPa to ensure that the reaction process was in a supercritical state. The reactor was stirred magnetically at a speed of 200 r / min and reacted at a constant temperature for 4 hours. After the reaction was completed, it was cooled to room temperature, the gas was released, and the catalyst was removed by centrifugation to obtain a crude product.

[0104] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0105] Example 12

[0106] The only difference between this embodiment and embodiment 2 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0107] Dissolve N-acetylneuraminic acid in 10 times the mass of distilled water, add potassium periodate in a molar ratio of 1:4, mix well, and react with magnetic stirring at 30°C for 4 hours. After the reaction, dialysis purification is performed, and vacuum drying is performed after concentration to obtain the aldehyde-modified sialic acid derivative.

[0108] Lipase NS40086 (ester exchange activity: 275 IUN / g) and sialic acid derivatives were added to sodium phosphate buffer (pH = 7.5) at a mass ratio of 10:5, and magnetic stirring was performed at 4°C for 12 hours. Sorbitol monooleate was then added thereto at a mass ratio of 10:1 for lipase Novozym 435: sorbitan monooleate, and magnetic stirring was performed at 36°C for 30 minutes, and then centrifuged and freeze-dried to obtain functionalized lipase. The olive oil-stearate exchange method was used for detection, and the ester exchange activity of the functionalized lipase in this embodiment was 355 IUN / g, and the modified functionalized lipase was 29.09% higher than that of the unmodified lipase.

[0109] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor in a mass ratio of 6:4, and functionalized lipase of 6% by mass of the oil substrate was added thereto. Carbon dioxide was introduced to replace the air in the reactor. Carbon dioxide was introduced into the reactor at 50°C until the pressure was 7.6 MPa to ensure that the reaction process was in a supercritical state. The reactor was stirred magnetically at a speed of 200 r / min and reacted at a constant temperature for 4 hours. After the reaction was completed, it was cooled to room temperature, the gas was released, and the catalyst was removed by centrifugation to obtain a crude product.

[0110] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0111] Embodiment 13

[0112] The only difference between this embodiment and embodiment 2 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0113] Dissolve N-glycolylneuraminic acid in 10 times the mass of distilled water, add potassium periodate in a molar ratio of 1:4, mix well, and react with magnetic stirring at 30°C for 4 hours. After the reaction, dialysis purification is performed, and vacuum drying is performed after concentration to obtain the aldehyde-modified sialic acid derivative.

[0114] Lipase Novozym 435 (transesterification activity: 10000 PLU / g) and sialic acid derivatives were added to sodium phosphate buffer (pH=7.5) at a mass ratio of 10:5, and magnetic stirring was performed at 4°C for 12 hours. Sorbitol monooleate was then added at a mass ratio of lipase Novozym 435: sorbitan monooleate of 10:1, and magnetic stirring was performed at 36°C for 30 minutes, and then centrifuged and freeze-dried to obtain functionalized lipase. The lauric acid-glycerol transesterification method was used for detection, and the transesterification activity of the modified functionalized lipase was increased by 28.11% compared with that of the unmodified lipase.

[0115] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor at a mass ratio of 6:4, and functionalized lipase of 6% by mass of the oil substrate was added thereto. Nitrogen was filled into the reactor, and the reaction was stirred at 50°C for 4 hours. After the reaction was completed, a crude product was obtained.

[0116] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0117] Embodiment 14

[0118] The only difference between this embodiment and embodiment 2 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0119] Dissolve polysialic acid (molecular weight of about 20 kDa) in 10 times the mass of distilled water, add potassium periodate in a molar ratio of 1:4, mix well, and react with magnetic stirring at 30°C for 4 hours. After the reaction is completed, dialysis is performed for purification, and the product is concentrated and vacuum dried to obtain an aldehyde-modified sialic acid derivative.

[0120] Lipase Novozym 435 (transesterification activity: 10000 PLU / g) and sialic acid derivatives were added to sodium phosphate buffer (pH=7.5) at a mass ratio of 10:5, and magnetic stirring was performed at 4°C for 12 hours. Sorbitol monooleate was then added at a mass ratio of lipase Novozym 435: sorbitan monooleate of 10:1, and magnetic stirring was performed at 36°C for 30 minutes, and then centrifuged and freeze-dried to obtain functionalized lipase. The lauric acid-glycerol transesterification method was used for detection, and the modified functionalized lipase had a transesterification activity increased by 18.50% compared with the unmodified lipase.

[0121] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor at a mass ratio of 6:4, and functionalized lipase of 6% by mass of the oil substrate was added thereto. Nitrogen was filled into the reactor, and the reaction was stirred at 50°C for 4 hours. After the reaction was completed, a crude product was obtained.

[0122] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0123] Embodiment 15

[0124] The only difference between this embodiment and embodiment 2 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0125] Dissolve N-acetylneuraminic acid in 10 times the mass of distilled water, add potassium periodate in a molar ratio of 1:4, mix well, and react with magnetic stirring at 30°C for 4 hours. After the reaction, dialysis purification is performed, and vacuum drying is performed after concentration to obtain the aldehyde-modified sialic acid derivative.

[0126] Lipase Novozym 435 (transesterification activity: 10000 PLU / g) and sialic acid derivatives were added to sodium phosphate buffer (pH=7.5) at a mass ratio of 10:5, and magnetic stirring was performed at 4°C for 12 hours. Sorbitol monopalmitate was then added at a mass ratio of lipase Novozym 435: sorbitan monopalmitate of 10:1, and magnetic stirring was performed at 36°C for 30 minutes, and then centrifuged and freeze-dried to obtain functionalized lipase. The lauric acid-glycerol transesterification method was used for detection, and the modified functionalized lipase had a 22.15% higher transesterification activity than the unmodified lipase.

[0127] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor at a mass ratio of 6:4, and functionalized lipase of 6% by mass of the oil substrate was added thereto. Nitrogen was filled into the reactor, and the reaction was stirred at 50°C for 4 hours. After the reaction was completed, a crude product was obtained.

[0128] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0129] Example 16

[0130] The only difference between this embodiment and embodiment 2 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0131] Dissolve N-acetylneuraminic acid in 10 times the mass of distilled water, add potassium periodate in a molar ratio of 1:4, mix well, and react with magnetic stirring at 30°C for 4 hours. After the reaction, dialysis purification is performed, and vacuum drying is performed after concentration to obtain the aldehyde-modified sialic acid derivative.

[0132] Lipase Novozym 435 (transesterification activity: 10000 PLU / g) and sialic acid derivatives were added to sodium phosphate buffer (pH=7.5) at a mass ratio of 10:5, and magnetic stirring was performed at 4°C for 12 hours. Then, linoleic acid monoglyceride was added thereto at a mass ratio of 10:1 of lipase Novozym 435: linoleic acid monoglyceride, and magnetic stirring was performed at 36°C for 30 minutes, and then centrifuged and freeze-dried to obtain functionalized lipase. The lauric acid-glycerol transesterification method was used for detection, and the transesterification activity of the modified functionalized lipase was increased by 23.62% compared with that of the unmodified lipase.

[0133] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor at a mass ratio of 6:4, and functionalized lipase of 6% by mass of the oil substrate was added thereto. Nitrogen was filled into the reactor, and the reaction was stirred at 50°C for 4 hours. After the reaction was completed, a crude product was obtained.

[0134] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0135] Comparative Example

[0136] Comparative Example 1

[0137] The difference between this comparative example and Example 1 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0138] Perilla seed oil and caprylic acid / capric acid triglyceride were added into a closed reactor at a mass ratio of 6:4, and lipase Novozym 435 was added thereto at a mass ratio of 6% of the mass of the oil substrate. Nitrogen was filled into the reactor, and the reaction was stirred at 50° C. for 4 hours. After the reaction was completed, a crude product was obtained.

[0139] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0140] Comparative Example 2

[0141] The difference between this comparative example and Example 1 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0142] Lipase Novozym 435 (transesterification activity: 10000 PLU / g) and sorbitan monooleate were added to sodium phosphate buffer (pH=7.5) at a mass ratio of 10:6, and magnetic stirring was performed at 36°C for 30 minutes, followed by centrifugation and freeze drying to obtain functionalized lipase. The lauric acid-glycerol transesterification method was used for detection, and the transesterification activity of the modified functionalized lipase was increased by 21.40% compared with that of the unmodified lipase.

[0143] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor at a mass ratio of 6:4, and functionalized lipase of 6% by mass of the oil substrate was added thereto. Nitrogen was filled into the reactor, and the reaction was stirred at 50°C for 4 hours. After the reaction was completed, a crude product was obtained.

[0144] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0145] Comparative Example 3

[0146] The difference between this comparative example and Example 1 is that the preparation method of medium- and long-chain triglyceride edible oil is as follows:

[0147] Dissolve N-acetylneuraminic acid in 10 times the mass of distilled water, add potassium periodate in a molar ratio of 1:4, mix well, and react with magnetic stirring at 30°C for 4 hours. After the reaction, dialysis purification is performed, and vacuum drying is performed after concentration to obtain the aldehyde-modified sialic acid derivative.

[0148] Lipase Novozym 435 (transesterification activity: 10000 PLU / g) and sialic acid derivatives were added to sodium phosphate buffer (pH=7.5) at a mass ratio of 10:6, magnetically stirred at 4°C for 12 hours, centrifuged and freeze-dried to obtain functionalized lipase. The lauric acid-glycerol transesterification method was used for detection, and the modified functionalized lipase had a transesterification activity increased by 18.21% compared with the unmodified lipase.

[0149] Perilla seed oil and caprylic / capric triglyceride were added into a closed reactor at a mass ratio of 6:4, and functionalized lipase of 6% by mass of the oil substrate was added thereto. Nitrogen was filled into the reactor, and the reaction was stirred at 50°C for 4 hours. After the reaction was completed, a crude product was obtained.

[0150] The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

[0151] Performance testing

[0152] Test 1: MCT yield:

[0153] The MCT yield in triglycerides was determined by gas chromatography. The triglycerides obtained by transesterification reaction include the following configurations: MMM, MLM, LMM, LLM, LML and LLL. Among them, MLM, LMM, LLM and LML are MCT. The total carbon number (TCN) of fatty acids on triglycerides was determined by gas chromatography. The TCN of MMM is 24-30, MLM and LMM are 32-38, LLM and LML are 40-46, and LLL is 48-54. The calculation formula for MCT yield is:

[0154] r=((W MLM +W MML +W LLM +W LML ) / (W MMM +W MLM +W MML +W LLM +W LML +W LLL ))×100%.

[0155] Test 2: α-linolenic acid content test

[0156] The test is carried out in accordance with GB5009.168-2016 "National Food Safety Standard - Determination of Fatty Acids in Foods".

[0157] The test and calculation results are summarized in Table 1.

[0158] Table 1

[0159]

[0160]

[0161] It can be seen from Examples 1-16 and Comparative Examples 1-3 and Table 1 that by chemically modifying lipase with sialic acid and sorbitan monooleate using the method disclosed in the present application, the yield of MCT and the content of α-linolenic acid in the product are increased, and an edible oil rich in α-linolenic acid and high in nutritional value can be produced.

[0162] Combining Example 1, Comparative Examples 1-3 and Table 1, it can be seen that after sialic acid is activated by aldehyde formation, the lipase is covalently modified with a sialic acid derivative, and the content of α-linolenic acid in the product is increased, but the improvement rate of the total yield of MCT is poor; further, the sialic acid-modified lipase is modified with sorbitan monooleate, and the yield of MCT is further improved. This may be due to the poor hydrophobicity of the lipase after sialic acid modification and insufficient compatibility with the substrate. Sorbitan monooleate helps to improve the miscibility of lipase with the oil reaction substrate, and helps to increase the yield of MCT.

[0163] It can be seen from Example 1, Example 2 and Table 1 that carrying out the reaction under supercritical carbon dioxide is helpful to promote the reaction and improve the MCT yield.

[0164] Combining Example 2, Example 11-12 and Table 1, it can be seen that the MCT content and α-linoleic acid content in the edible oil product catalyzed by the functionally modified lipase Novozym 435 are higher. This may be because the lipase Novozym 435 is a non-positionally selective lipase that can act on three sites of triglycerides, thereby obtaining a higher MCT yield.

[0165] It can be seen from Example 1, Example 13, Example 14 and Table 1 that the modification of lipase with small molecule sialic acid can promote the increase of MCT content and α-linolenic acid content in edible oil products. This may be due to the better binding performance of small molecule sialic acid with lipase, which can modify the amino acid residues of lipase, and will not adversely affect the conformation of lipase while improving the catalytic activity and stability of lipase; at the same time, compared with N-glycolylneuraminic acid, N-acetylneuraminic acid has a better modification effect, and the hydrogen bonds and covalent modifications between N-acetylneuraminic acid, lipase and sorbitan monooleate realize a lipase spatial structure that is more conducive to enzymatic reaction, and the catalytic effect is better.

[0166] In combination with Example 1, Example 15, and Example 16 and Table 1, it can be seen that the use of sorbitan monooleate as a nonionic surfactant to further modify the sialic acid-modified lipase has the best transesterification activity of the lipase, and the MCT yield and α-linolenic acid content are better. This may be because the chain length structure or active group of sorbitan monooleate is more conducive to forming a stable and highly active catalytic system compared to sorbitan monopalmitate and linoleic acid monoglyceride.

[0167] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for preparing medium- and long-chain triglyceride edible oil, characterized in that: The following steps are involved: After sialic acid is activated by reacting with an oxidant, an activated sialic acid derivative is obtained, the activated sialic acid derivative and lipase are added to a phosphate buffer, stirred at 4° C. for reaction for 12-24 hours, sorbitan monooleate is added, stirred at 30-40° C. for reaction for 20-40 minutes, centrifuged, and freeze-dried to obtain a functionalized lipase; the mass ratio of sialic acid, sorbitan monooleate, and lipase is (5-15):(1-5):10; Perilla seed oil and medium-chain triglyceride are added into a reaction kettle at a mass ratio of (0.6:0.4)-(0.8:0.2), and the functionalized lipase is added at 2-20% of the mass of the reactants to carry out an ester exchange reaction, and a crude product is obtained after the reaction is completed; The free fatty acids in the crude product are removed by molecular distillation technology, and then the crude product is extracted and purified with ethanol to obtain medium- and long-chain triglyceride edible oil.

2. The method for preparing medium- and long-chain triglyceride edible oil according to claim 1, characterized in that: The sialic acid activation step specifically comprises: Sialic acid and an oxidant are dissolved in water for oxidation reaction, and then purified by dialysis to obtain an activated sialic acid derivative; the molar ratio of sialic acid to the oxidant is 1:(3.5-4.5).

3. The method for preparing medium- and long-chain triglyceride edible oil according to claim 1, characterized in that: The sialic acid is selected from one or both of N-acetylneuraminic acid and N-glycolylneuraminic acid.

4. The method for preparing medium- and long-chain triglyceride edible oil according to claim 1, characterized in that: The lipase is selected from one or more of Lipozyme TLIM, Lipozyme RMIM, Lipozyme 435, Novozym 435, and NS40086.

5. The method for preparing medium- and long-chain triglyceride edible oil according to claim 1, characterized in that: The transesterification reaction is carried out under supercritical carbon dioxide medium.

6. The method for preparing medium- and long-chain triglyceride edible oil according to claim 5, characterized in that: The reaction process specifically includes: Perilla seed oil, medium-chain triglyceride and functionalized lipase are put into a reactor. In a sealed state, carbon dioxide is introduced to replace the air in the reactor. Then, carbon dioxide is introduced into the reactor at 40-60°C until the pressure is 7.5-8MPa to ensure that the reaction process is in a supercritical state. Magnetic stirring is performed at 200-500r / min and the reaction is carried out at a constant temperature for 3-6h. After the reaction is completed, the reactor is cooled to room temperature, the gas is released, and the functionalized lipase is removed by centrifugation to obtain a crude product.

7. The method for preparing medium- and long-chain triglyceride edible oil according to claim 6, characterized in that: The added amount of the functionalized lipase is 2-10% of the total mass of the perilla seed oil and medium-chain triglyceride.

8. The method for preparing medium- and long-chain triglyceride edible oil according to claim 1, characterized in that: The medium chain triglyceride is one or more of caprylic triglyceride, nonanoic triglyceride, capric triglyceride and caprylic-decanoic triglyceride.

9. A medium- and long-chain triglyceride edible oil, characterized in that: The edible oil is prepared by the method for preparing the medium- and long-chain triglyceride edible oil according to any one of claims 1 to 8.

10. The medium- and long-chain triglyceride edible oil according to claim 9, wherein the medium- and long-chain triglyceride edible oil has a medium- and long-chain triglyceride content of ≥85% and an α-linolenic acid content of ≥40%.

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