A process for the preparation of a medium-long chain triglyceride-enriched margarine / shortening base oil and products thereof

Abstract

The application discloses a preparation method of a medium-long-chain triglyceride-rich artificial cream / shortening base oil and a product thereof, and belongs to the technical field of food processing. The preparation method comprises the following steps: taking medium-chain triglyceride and palm stearin as raw materials, adding imprint lipase, and obtaining an ester exchange product and imprint lipase rich in medium-long-chain triglyceride through enzyme catalysis of ester exchange reaction and filtration; separating unreacted medium-chain triglyceride and simultaneously fractionating the medium-long-chain triglyceride through programmed cooling, and obtaining high-melting-point medium-long-chain triglyceride; adding medium-chain fatty acid, adding imprint lipase for ester exchange reaction, and catalyzing reaction under vacuum; and removing the fatty acid through molecular distillation, so that the product is obtained. The imprint lipase of the non-ionic surfactant improves the activity and stability of the lipase, improves the reaction efficiency, and reduces the production cost. The unreacted medium-chain triglyceride is removed through programmed cooling fractionation after the ester exchange reaction, and the high-melting-point medium-long-chain triglyceride solid fat is obtained through fractionation at the same time.

Description

Technical Field

[0001] This invention belongs to the field of oil preparation technology, specifically relating to a method for preparing margarine / shortening base oil rich in medium and long chain triglycerides and its products. Background Technology

[0002] Margarine / shortening base oils are plastic fats with a certain melting point. Currently, most base oils are made from natural animal and vegetable oils and their hydrogenated oils, which contain high levels of saturated fatty acids or trans fatty acids, which can affect serum cholesterol levels and lead to chronic heart disease. With the increasing Westernization of dietary habits, the market demand for margarine / shortening is increasing year by year, making the development of margarine / shortening base oils with health benefits extremely urgent.

[0003] Medium- and long-chain triglycerides possess numerous health benefits, including lowering blood lipids, inhibiting obesity, enhancing immunity, reducing inflammation, lowering the risk of diabetes and cardiovascular disease, and reducing the risk of cancer. They are a superior choice as a base oil for margarine / shortening. Medium- and long-chain triglycerides are hydrolyzed in the small intestine by lipases into medium- and long-chain fatty acids and sn-2 monoglycerides. Medium-chain fatty acids, with their short carbon chains, are directly transported to the liver via the portal vein after absorption in the digestive tract. They do not require the formation of chylomicrons or the transport by carnitine acyltransferase, and directly enter the mitochondria of hepatocytes for β-oxidation. They are less prone to accumulation in adipose tissue and liver tissue, exhibiting rapid digestion, absorption, and metabolism. sn-2 monoglycerides and long-chain fatty acids dissolve in bile acids to form micro-macromolecules, which are then re-esterified in the epithelial cells of the small intestine to form triglycerides. These triglycerides are then transported via the lymphatic system in the form of chylomicrons and stored in the liver and peripheral tissues. Because some medium-chain fatty acids in medium- and long-chain triglycerides are directly metabolized and do not participate in triglyceride synthesis, the level of triglycerides in the blood can be reduced, and to some extent, fat accumulation can be reduced. Currently, the main methods for synthesizing medium- and long-chain triglycerides are chemical and enzymatic methods. Among them, the enzymatic method is widely used due to its advantages such as mild conditions, fewer byproducts, and easy separation.

[0004] However, a major problem with enzymatic methods is the high price of enzymes, their relatively low activity, and their relatively long catalytic time. Furthermore, enzymatic reactions are two-step processes, producing monoglycerides and diglycerides during the reaction. These substances reduce the yield of triglycerides and can increase the content of harmful substances during subsequent deacidification treatment.

[0005] The preparation of margarine / shortening requires base oils with high melting points. Palm stearin, due to its wide availability and adjustable melting point, is widely used in margarine / shortening production. Therefore, producing high-melting-point margarine / shortening base oils through enzymatic transesterification of palm stearin with medium-chain triglycerides is the most convenient method. However, because medium-chain triglycerides have low melting points, the resulting product has a lower melting point, sometimes failing to meet the required melting point for margarine / shortening. Furthermore, since medium-chain triglycerides are both reactants and products in the transesterification reaction, obtaining a product with high to medium carbon chain triglyceride content requires a high ratio of high to medium carbon chain triglycerides to long chain triglycerides. This high ratio leads to high to medium chain triglyceride residue. High to medium chain triglyceride residue affects the product's melting point, and because the digestion and absorption rate of medium chain triglycerides is too rapid, it can easily create high osmotic pressure in the small intestine, potentially causing gastrointestinal discomfort such as diarrhea, vomiting, and bloating if consumed in large quantities.

[0006] Therefore, it is necessary to find ways to increase the content of medium and long chain triglycerides in products and minimize the content of medium chain triglycerides. Summary of the Invention

[0007] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0008] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0009] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing margarine / shortening base oil rich in medium and long chain triglycerides.

[0010] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing a margarine / shortening base oil rich in medium- and long-chain triglycerides, comprising,

[0011] Using medium-chain triglycerides and palm stearin as raw materials, imprinted lipase was added, and the transesterification reaction was catalyzed by enzymes. After filtration, transesterification products rich in medium and long-chain triglycerides and imprinted lipase were obtained.

[0012] The transesterification product rich in medium and long chain triglycerides is cooled by programmed cooling to separate unreacted medium chain triglycerides and simultaneously extract medium and long chain triglycerides to obtain high melting point medium and long chain triglycerides.

[0013] Medium-chain fatty acids are added to high-melting-point medium- and long-chain triglycerides, and imprinted lipase for transesterification is added to catalyze the reaction under vacuum.

[0014] High-melting-point medium- and long-chain triglycerides, which can be used to prepare margarine base oil, are obtained by removing fatty acids through molecular distillation.

[0015] As a preferred embodiment of the preparation method described in this invention, the method for preparing the imprinted lipase includes:

[0016] A nonionic surfactant is dissolved in a mixed solution of isopropanol and water at a concentration of 20–100 mg / L, and an immobilized lipase at a mass fraction of 10–30% is added to the solution.

[0017] The mixture was stirred at 100–200 rpm for 30–60 min at 25°C and then filtered to obtain lipase.

[0018] Moisture was removed by freeze-drying, and the surfactant imprint template on the immobilized lipase was eluted with a non-polar solvent.

[0019] The lipase was filtered and vacuum dried to remove the organic solvent, yielding imprinted lipase.

[0020] In a preferred embodiment of the preparation method described in this invention, the immobilized lipase includes Novozym435, Lipozyme RM IM, and NS40086; and the isopropanol mass concentration in the mixed solution of isopropanol and water is greater than 70%.

[0021] The nonionic surfactant is from the Tween series, including Tween 20, Tween 60 and Tween 80.

[0022] As a preferred embodiment of the preparation method described in this invention, the enzyme-catalyzed transesterification reaction includes,

[0023] Medium-chain triglycerides and palm stearin were added to a batch reactor, which was then purged with nitrogen for protection. Simultaneously, imprinted lipase was added at a rate of 6–10 wt% of the substrate weight.

[0024] Raise the temperature to 60–80°C, stir at 600–800 rpm, and react for 6–10 hours.

[0025] The palm stearin is obtained by separating palm oil, and its melting point includes, but is not limited to, 44°C, 52°C, and 58°C.

[0026] In a preferred embodiment of the preparation method described in this invention, the molar ratio of the medium-chain triglyceride to palm stearin is greater than or equal to 1:1.

[0027] In a preferred embodiment of the preparation method described in this invention, the step of cooling the transesterification product rich in medium- and long-chain triglycerides by a programmed temperature procedure includes:

[0028] The transesterification product is kept at 60-80℃ for 1-2 hours, and the temperature is reduced to 40-50℃ at a rate of 6-10℃ / h and kept for 6-24 hours. Liquid oil is removed by centrifugation or filtration to obtain solid ester.

[0029] As a preferred embodiment of the preparation method described in this invention, the catalytic reaction under vacuum includes,

[0030] The solid fat is heated to 60-80°C and medium-chain fatty acids are added. The molar ratio of the added medium-chain fatty acids to the solid fat triglycerides is 1-3:1.

[0031] Add the imprinted lipase filtered during the transesterification reaction, apply a vacuum of 10–30 mbar, react for 6–10 h, and stir at 600–800 rpm.

[0032] As a preferred embodiment of the preparation method described in this invention, the molecular distillation conditions are: distillation temperature of 160–180°C, pressure of 2–5 Pa, and condenser temperature of 20–30°C.

[0033] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a margarine / shortening base oil rich in medium- and long-chain triglycerides. The resulting margarine base oil contains less than 10% medium- and long-chain triglycerides, more than 70% medium- and long-chain triglycerides, less than 0.4 mg / kg of glycidyl esters, and less than 0.3 mg / kg of chloropropanol esters.

[0034] Beneficial effects of this invention:

[0035] (1) In this invention, the nonionic surfactant imprinted lipase dissolved in isopropanol solution is used to enhance the activity and stability of lipase and improve the reaction efficiency. After the transesterification reaction, unreacted medium-chain triglycerides are removed by programmed cooling and fractionation. At the same time, high-melting-point medium- and long-chain triglyceride solid lipids are obtained by fractionation, thereby reducing the content of medium-chain triglycerides and increasing the content and melting point of medium- and long-chain triglycerides in the final product.

[0036] (2) This invention adds medium-chain fatty acids to high-melting-point solid fats and applies a certain vacuum to cause monoglycerides and diglycerides in the reaction system to undergo esterification and partial acid hydrolysis, thereby increasing the yield of triglycerides, reducing the content of long-chain triglycerides, and also reducing the content of glycidyl esters and chloropropanol esters during molecular distillation deacidification. Detailed Implementation

[0037] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0038] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0039] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0040] Transesterification is a process in which the acyl groups between MCT and LCT are rearranged and their positions interchanged. The essence of transesterification lies in the equilibrium distribution of various fatty acids on the glycerol backbone. Since transesterification is a reversible equilibrium reaction, when the reaction reaches equilibrium, all fatty acids are distributed in equilibrium on the glycerol backbone. Therefore, the distribution of fatty acids in the equilibrium stage conforms to the principle of random distribution.

[0041] In random transesterification, due to the lack of positional selectivity, fatty acids are randomly distributed along the glycerol backbone. Since the fatty acid composition in lipid systems is relatively complex, we can define the number of fatty acid types as n, the different fatty acids as Xi, and the amount of each fatty acid in the system as M. Xi Therefore, the total amount of all fatty acids in the system is: mol.

[0042]

[0043] Based on the principle of random distribution of fatty acids, the probability of fatty acids being on the glycerol backbone at reaction equilibrium can be determined through permutations and combinations as follows:

[0044]

[0045] Therefore, the content of triglyceride molecules Xs-Xj-Xk at equilibrium is:

[0046]

[0047] In the reaction between long-chain and medium-chain triglycerides, only two types of fatty acids exist in the system: long-chain fatty acids (L) and medium-chain fatty acids (M). Assuming the content of long-chain triglycerides in the system is r mol and the content of medium-chain triglycerides is h mol, the probabilities of medium-chain and long-chain fatty acids appearing on the glycerol backbone at the point of random transesterification equilibrium are as follows:

[0048]

[0049] In directed transesterification, due to position selectivity, and without considering acyl transfer, only the fatty acids at positions sn-1 and sn-3 on the glycerol backbone are randomly distributed. Since the fatty acid composition in lipid systems is complex, we can define the number of fatty acid types as n, each fatty acid as Xi, the amount of each fatty acid in the system as MXi mol, and the amount of each fatty acid at position sn-2 as Msn-2Xi. Therefore, the amount of fatty acid that can be used for random reactions in the system is:

[0050]

[0051] Based on the principle of random distribution of fatty acids, the probability of fatty acids being on the glycerol backbone at reaction equilibrium can be determined through permutations and combinations as follows:

[0052]

[0053] Therefore, the content of triglyceride molecules Xs-Xj-Xk at equilibrium is:

[0054]

[0055] In the reaction between long-chain and medium-chain triglycerides, only two types of fatty acids exist in the system: long-chain fatty acids (L) and medium-chain fatty acids (M). Assuming the content of long-chain triglycerides in the system is r mol and the content of medium-chain triglycerides is h mol, the probabilities of medium-chain and long-chain fatty acids appearing on the glycerol backbone at the point of random transesterification equilibrium are as follows:

[0056]

[0057] Therefore, in the transesterification reaction between long-chain triglycerides and medium-chain triglycerides, the composition of the resulting triglycerides is consistent regardless of whether a random reaction or a directional reaction is used (without considering acyl transfer).

[0058] Based on the distribution of medium-chain and long-chain fatty acids on the glycerol backbone, they can be roughly classified into MLM, LMM, MML, LML, MLL, and LLM. Therefore, after the reaction reaches equilibrium, the composition of various triglycerides in the system is shown in Table 1 below.

[0059] Table 1. Composition and proportion of triglycerides in random transesterification

[0060]

[0061]

[0062] Therefore, when the reaction reaches equilibrium, the theoretical triglyceride compositions for different ratios of long-chain to medium-chain triglycerides are shown in the table below:

[0063] Table 2 Theoretical triglyceride composition

[0064]

[0065] Theoretical calculations show that as the ratio of medium-chain triglycerides (MCT) to long-chain triglycerides (LCT) increases, the proportion of medium- and long-chain triglycerides (MLCT) in the product first increases and then decreases. However, there will be a certain difference between theoretical and actual values. This is because, during the reaction process, various factors may prevent the reaction from reaching equilibrium. Therefore, the actual proportion of MLCT in the product is lower than the theoretical value. Thus, to further increase the MLCT content while shortening the reaction time, selecting a highly active catalyst is a better choice. On the one hand, it can shorten the reaction time and improve the reaction efficiency; on the other hand, a shorter reaction time is beneficial for reducing the oxidation of oils.

[0066] For the production of highly saturated medium- and long-chain triglycerides, the difference in melting points between oils and fats can be utilized to separate high-melting-point medium- and long-chain triglycerides from incompletely reacted MCTs in the system through fractionation, thereby obtaining high-purity high-melting-point medium- and long-chain triglycerides. Theoretical model derivation shows that, with the increase of the MCT ratio in the substrate, the amount of LLL in the reactants at equilibrium gradually decreases, the amount of MMM gradually increases, while the amount of MLCT initially increases and then decreases. However, if MMM is removed through a certain method, the amount of MLCT in the reactants gradually increases. When the MCT to LCT ratio is greater than 1:1, the proportion of MLCT in the reactants after MMM removal is greater than 85%.

[0067] Determination of transesterification activity: Soybean oil (2g) was mixed with MCT at a 1:1 molar ratio. Then, 4wt% of the total substrate mass of lipase was added to the mixture. The reaction was carried out in a round-bottom flask at 50℃ with a stirring speed of 600 rpm. After 1 hour of reaction, the product was collected and the triglyceride composition was analyzed. The initial enzyme activity was assessed based on the percentage (%) of medium- and long-chain triglycerides after 1 hour of reaction.

[0068] Example 1

[0069] The activity and stability of lipase were enhanced by bioimprinting with nonionic surfactants. The imprinting conditions were as follows: Tween 60 (100 mg / L) was dissolved in a mixture of isopropanol and water (80% isopropanol), and the mixture was thoroughly dispersed. Immobilized lipase Novozym 435 (30% by mass) was added, and the mixture was stirred at 200 rpm for 30 min at 25°C. The lipase was obtained by filtration and then dehydrated by freeze-drying. Excess surfactant imprinting template on the immobilized lipase Novozym 435 was eluted with the nonpolar solvent n-hexane. The lipase was then filtered and dried at room temperature for 24 h in a vacuum desiccator to remove organic solvents, yielding the imprinted lipase. The activity of the imprinted lipase Novozym 435 was 38.4%, while the activity of the unimprinted lipase Novozym 435 was 24.5%.

[0070] Medium-chain triglycerides (MCT) and palm stearin (melting point 58℃) ​​were added to a batch reactor at a molar ratio of 2:1 (MCT / palm stearin). The reactor was purged with nitrogen for protection, and imprinted lipase Novozym 435 was added simultaneously at a concentration of 10 wt% of the substrate. The temperature was raised to 80℃, and stirring was initiated at 800 rpm for 6 hours. After the reaction, the lipase was filtered to obtain the transesterification product. The composition of the triglycerides and glycerides in the transesterification product is shown below.

[0071] Table 3. Composition of triglycerides and glycerides in transesterification products

[0072] Triglyceride composition content(%) LLL 10.2 MMM 31.5 MLL 26.7 MML 31.6 Glyceryl ester composition content(%) monoglycerides 2.1 diglycerides 3.3 Triglycerides 94.6

[0073] Since the product contains a certain amount of MMM, most of the MMM is removed by utilizing the difference in melting points. Simultaneously, MMM can also be used as a solvent to further fractionate medium- and long-chain triglycerides, increasing their melting points. The resulting transesterification product is held at 80°C for 1 hour, then the temperature is lowered to 50°C at a rate of 10°C / hour and maintained at this temperature for 12 hours. Liquid fats are removed by centrifugation or filtration to obtain solid lipids. The triglyceride and glyceride composition of the solid lipids is shown below.

[0074] Table 4. Composition of triglycerides and glycerides in solid fats

[0075] Triglyceride composition content(%) LLL 13.2 MMM 8.5 MLL 37.1 MML 41.3 Glyceryl ester composition content(%) monoglycerides 2.7 diglycerides 3.8 Triglycerides 93.5

[0076] As shown in the table, after fractionation, the content of MMM (MCT) in the product decreased to 8.5%, while the content of MCLT increased to 78.4%. Since the system also contains monoglycerides and diglycerides byproducts generated from the transesterification reaction, the presence of these byproducts will increase the formation of harmful substances during the deacidification stage and reduce the triglyceride yield of the product. Therefore, the solid fat was heated to 80℃, and medium-chain fatty acids were added. The molar ratio of medium-chain fatty acids to solid fat triglycerides was 2:1 (medium-chain fatty acids / solid fat, mol / mol). Imprinted lipase Novozym 435 filtered during the transesterification reaction was added. The reaction was carried out under vacuum at 10 mbar for 6 hours with a stirring speed of 800 rpm for esterification, while also undergoing partial acidolysis. After the reaction, the lipase was filtered, and the triglyceride and glycerol composition of the obtained esterification product is shown below.

[0077] Table 5. Composition of triglycerides and glycerides in esterification reactants

[0078] Triglyceride composition content(%) LLL 12.1 MMM 8.3 MLL 37.9 MML 41.7 Glyceryl ester composition content(%) monoglycerides 0.5 diglycerides 0.7 Triglycerides 98.8

[0079] Free fatty acids in the esterification reaction system were removed by molecular distillation. The molecular distillation conditions were: distillation temperature 160℃, pressure 2Pa, and condenser temperature 20℃. The obtained product contained 0.14 mg / kg of glycidyl ester and 0.12 mg / kg of chloropropanol ester. The melting point of the obtained medium- and long-chain triglycerides was determined to be 64℃.

[0080] Example 2

[0081] The activity and stability of lipase were enhanced by nonionic surfactant bioimprinting. The imprinting conditions were as follows: Tween 80 (60 mg / L) was dissolved in a mixture of isopropanol and water (70% isopropanol), and the mixture was thoroughly dispersed. 20% (w / w) of immobilized lipase Lipozyme RMIM was added, and the mixture was stirred at 150 rpm for 45 min at 25 °C. The lipase was obtained by filtration and then dehydrated by freeze-drying. Excess surfactant imprinting template on the immobilized lipase Lipozyme RMIM was eluted with the nonpolar solvent octane. The lipase was then filtered and dried at room temperature for 36 h in a vacuum desiccator to remove organic solvents, yielding the imprinted lipase. The activity of the imprinted lipase Lipozyme RMIM was 34.1%, while the activity of the unimprinted lipase Lipozyme RMIM was 21.1%.

[0082] Medium-chain triglycerides (MCT) and palm stearin (melting point 52℃) were added to a batch reactor at a molar ratio of 3:1 (MCT / palm stearin). The reactor was purged with nitrogen for protection, and simultaneously, imprinted lipase Lipozyme RM IM was added at 8 wt% of the substrate weight. The temperature was raised to 70℃, and stirring was initiated at 700 rpm for 8 hours. After the reaction, the lipase was filtered to obtain the transesterification product. The composition of the triglycerides and glycerides in the transesterification product is shown below.

[0083] Table 6. Composition of triglycerides and glycerides in transesterification products

[0084]

[0085]

[0086] Since the product contains a certain amount of MMM, most of the MMM is removed by utilizing the difference in melting points. Simultaneously, MMM can also be used as a solvent to further fractionate medium- and long-chain triglycerides, increasing their melting points. The resulting transesterification product is held at 70°C for 2 hours, then the temperature is lowered to 45°C at a rate of 6°C / hour and maintained at this temperature for 24 hours. Liquid oil is removed by centrifugation or filtration to obtain solid lipids. The triglyceride and glycerol ester composition of the solid lipids is shown below.

[0087] Table 7. Composition of triglycerides and glycerides in solid fats

[0088] Triglyceride composition content(%) LLL 10.1 MMM 9.1 MLL 30.7 MML 50.2 Glyceryl ester composition content(%) monoglycerides 3.3 diglycerides 4.5 Triglycerides 92.2

[0089] As shown in the table, after fractionation, the content of MMM (MCT) in the product decreased to 9.1%, while the content of MCLT increased to 80.9%. Since the system also contains monoglycerides and diglycerides byproducts generated from the transesterification reaction, the presence of these byproducts will increase the formation of harmful substances during the deacidification stage and reduce the triglyceride yield of the product. Therefore, the solid fat was heated to 70℃, and medium-chain fatty acids were added. The molar ratio of added medium-chain fatty acids to solid fat triglycerides was 1:1 (medium-chain fatty acids / solid fat, mol / mol). The imprinted lipase Lipozyme RM IM filtered during the transesterification reaction was added. The reaction was carried out under vacuum at 20 mbar for 8 hours with a stirring speed of 700 rpm for esterification, while also undergoing partial acidolysis. After the reaction, the lipase was filtered, and the triglyceride and glycerol composition of the obtained esterification product is shown below.

[0090] Table 8. Composition of triglycerides and glycerides in esterification reactants

[0091]

[0092]

[0093] Free fatty acids in the esterification reaction system were removed by molecular distillation. The molecular distillation conditions were: distillation temperature 170℃, pressure 3Pa, and condenser temperature 25℃. The resulting product contained 0.22 mg / kg of glycidyl ester and 0.17 mg / kg of chloropropanol ester. The melting point of the obtained medium- and long-chain triglycerides was determined to be 57℃.

[0094] Example 3

[0095] The activity and stability of lipase were enhanced by bioimprinting with nonionic surfactants. The imprinting conditions were as follows: Tween 20 (20 mg / L) was dissolved in a mixture of isopropanol and water (90% isopropanol), and thoroughly dispersed. Immobilized lipase NS40086 (10% by mass) was added, and the mixture was stirred at 100 rpm for 60 min at 25°C. The lipase was obtained by filtration and dehydrated by freeze-drying. Excess surfactant imprinting template on the immobilized lipase NS40086 was eluted with the nonpolar solvent octane. The lipase was then filtered and dried at room temperature for 12 h in a vacuum desiccator to remove organic solvents, yielding the imprinted lipase. The activity of the imprinted lipase NS40086 was 33.7%, while the activity of the unimprinted lipase NS40086 was 21.8%.

[0096] Medium-chain triglycerides (MCT) and palm stearin (melting point 44℃) were added to a batch reactor at a molar ratio of 1:1 (MCT / palm stearin). The reactor was purged with nitrogen for protection, and Western blot lipase NS40086 was added simultaneously at a concentration of 6 wt% of the substrate. The temperature was raised to 60℃, and stirring was initiated at 600 rpm for 10 h. After the reaction, the lipase was filtered to obtain the transesterification product. The composition of the triglycerides and glycerides in the transesterification product is shown below.

[0097] Table 9. Composition of triglycerides and glycerides in transesterification products

[0098] Triglyceride composition content(%) LLL 16.2 MMM 17.7 MLL 34.5 MML 31.6 Glyceryl ester composition content(%) monoglycerides 2.6 diglycerides 3.1 Triglycerides 94.3

[0099] Since the product contains a certain amount of MMM, most of the MMM is removed by utilizing the difference in melting points. Simultaneously, MMM can also be used as a solvent to further fractionate medium- and long-chain triglycerides, increasing their melting points. The resulting transesterification product is held at 60°C for 1.5 hours, then the temperature is lowered to 40°C at a rate of 8°C / hour, and held at this temperature for 6 hours. Liquid fats are removed by centrifugation or filtration to obtain solid lipids. The triglyceride and glyceride composition of the solid lipids is shown below.

[0100] Table 10. Composition of triglycerides and glycerides in solid fats

[0101] Triglyceride composition content(%) LLL 19.7 MMM 7.2 MLL 39.4 MML 33.7 Glyceryl ester composition content(%) monoglycerides 3.3 diglycerides 3.8 Triglycerides 92.9

[0102] As shown in the table, after fractionation, the content of MMM (MCT) in the product decreased to 7.1%, while the content of MCLT increased to 73.1%. Since the system also contains monoglycerides and diglycerides byproducts generated from the transesterification reaction, the presence of these byproducts will increase the formation of harmful substances during the deacidification stage and reduce the triglyceride yield of the product. Therefore, the solid fat was heated to 60℃, and medium-chain fatty acids were added. The molar ratio of medium-chain fatty acids to solid fat triglycerides was 3:1 (medium-chain fatty acids / solid fat, mol / mol). Imprinted lipase NS40086 filtered during the transesterification reaction was added, and the reaction was carried out under vacuum at 30 mbar for 10 h with a stirring speed of 600 rpm. Partial acidolysis was also performed. After the reaction, the lipase was filtered, and the triglyceride and glycerol composition of the obtained esterification product is shown below.

[0103] Table 11. Composition of triglycerides and glycerides in esterification reactants

[0104] Triglyceride composition content(%) LLL 13.6 MMM 8.3 MLL 41.5 MML 36.6 Glyceryl ester composition content(%) monoglycerides 0.9 diglycerides 1.2 Triglycerides 97.9

[0105] Free fatty acids in the esterification reaction system were removed by molecular distillation. The distillation conditions were: distillation temperature 180℃, pressure 5 Pa, and condenser temperature 30℃. The resulting product contained 0.31 mg / kg of glycidyl ester and 0.21 mg / kg of chloropropanol ester. The melting point of the obtained medium- and long-chain triglycerides was determined to be 51℃.

[0106] Comparative Example 1

[0107] The reaction was carried out under the conditions of Example 1, without fractionation to remove unreacted MCT.

[0108] The activity of lipase was enhanced by bioimprinting with nonionic surfactants. The imprinting conditions were as follows: Tween 60, a nonionic surfactant, was dissolved at a concentration of 100 mg / L in a mixed solution of isopropanol and water (isopropanol content was 80%) and fully dispersed. Immobilized lipase Novozym 435 (30% by mass) was added to the solution. The mixture was stirred at 200 rpm for 30 min at 25 °C, and the lipase was obtained by filtration. The moisture was removed by freeze-drying. The surfactant imprint template on the immobilized lipase Novozym 435 was eluted with the nonpolar solvent n-hexane. The lipase was then filtered and dried at room temperature in a vacuum dryer for 24 h to remove organic solvents, yielding the imprinted lipase.

[0109] Medium-chain triglycerides (MCT) and palm stearin (melting point 58℃) ​​were added to a batch reactor at a molar ratio of 2:1 (MCT / palm stearin). The reactor was purged with nitrogen for protection, and imprinted lipase Novozym 435 was added simultaneously at a concentration of 10 wt% of the substrate. The temperature was raised to 80℃, and stirring was initiated at 800 rpm for 6 hours. After the reaction was complete, the lipase was filtered to obtain the transesterification product.

[0110] Since the system also contains monoglycerides and diglycerides byproducts generated by transesterification, the presence of these byproducts will increase the generation of harmful substances during the deacidification stage and reduce the yield of triglycerides in the product.

[0111] Therefore, the transesterification product was heated to 80°C, and medium-chain fatty acids were added. The molar ratio of the added medium-chain fatty acids to the triglycerides in the transesterification product was 2:1 (medium-chain fatty acids / solid lipids, mol / mol). Imprinted lipase Novozym 435 filtered during the transesterification reaction was added, and the reaction was carried out under vacuum at a degree of 10 mbar for 6 hours with a stirring speed of 800 rpm. After the esterification reaction, the lipase was filtered out, and the composition of the triglycerides and glycerides in the obtained esterification product is shown below.

[0112] Table 12. Composition of triglycerides and glycerides in esterification reactants

[0113] Triglyceride composition content(%) LLL 10.9 MMM 30.7 MLL 27.3 MML 31.2 Melting point 40℃

[0114] As shown in the table above, compared with Example 1, the product that has not undergone fractionation has a higher MCT content and a relatively lower MLCT content. At the same time, the melting point of the product is much lower than that of the product in Example 1.

[0115] Comparative Example 2

[0116] The reaction was carried out under the conditions of Example 3, without esterification or partial acidolysis.

[0117] The activity of lipase was enhanced by bioimprinting with nonionic surfactants. The imprinting conditions were as follows: Tween 20 (20 mg / L) was dissolved in a mixture of isopropanol and water (90% isopropanol), and the mixture was thoroughly dispersed. Immobilized lipase NS40086 (10% by mass) was added, and the mixture was stirred at 100 rpm for 60 min at 25°C. The lipase was obtained by filtration and then dehydrated by freeze-drying. Excess surfactant on the immobilized lipase NS40086 was eluted with the nonpolar solvent octane. The lipase was then filtered and dried at room temperature for 12 h in a vacuum desiccator to remove organic solvents, yielding the imprinted lipase. The activity of the imprinted lipase NS40086 was 14.8%, while the activity of the unimprinted lipase NS40086 was 8.1%.

[0118] Medium-chain triglycerides (MCT) and palm stearin (melting point 44℃) were added to a batch reactor at a molar ratio of 1:1 (MCT / palm stearin). The reactor was purged with nitrogen for protection, and imprinted lipase NS40086 was added simultaneously at a concentration of 6 wt% of the substrate. The temperature was raised to 60℃, and stirring was initiated at 600 rpm for 10 hours. After the reaction was complete, the lipase was filtered to obtain the transesterification product.

[0119] Since the product contains a certain amount of MMM, most of the MMM is removed by utilizing the difference in melting points. Simultaneously, MMM can also be used as a solvent to further fractionate medium- and long-chain triglycerides, increasing their melting points. The resulting transesterification product is held at 60°C for 1.5 hours, then the temperature is lowered to 40°C at a rate of 8°C / hour, and held at this temperature for 6 hours. Liquid fats are removed by centrifugation or filtration to obtain solid fats.

[0120] Molecular distillation was used to remove free fatty acids generated from transesterification in solid fats. The distillation conditions were: 180℃, 5 Pa, and condenser temperature 30℃. The resulting product contained 2.9% monoglycerides, 3.9% diglycerides, and 93.2% triglycerides. The product also contained 0.47 mg / kg of glycidyl esters and 0.33 mg / kg of chloropropanol esters. Because monoglycerides and diglycerides were not converted to triglycerides, the triglyceride yield in Comparative Example 2 was lower than that in Example 2. Furthermore, due to the absence of simultaneous acid hydrolysis, the system contained a higher content of long-chain triglycerides. The triglyceride composition is shown in the table below.

[0121] Table 13. Composition of triglycerides in the product of Comparative Example 2

[0122]

[0123]

[0124] During the vacuum reaction with medium-chain fatty acids, the system primarily undergoes esterification, but also some acidolysis. Esterification reacts medium-chain fatty acids with monoglycerides and diglycerides to form new medium- and long-chain triglycerides. Simultaneously, long-chain triglycerides can undergo acidolysis with medium-chain fatty acids to obtain new medium- and long-chain triglycerides. Therefore, this process increases the content of medium- and long-chain triglycerides in the product while decreasing the content of long-chain triglycerides.

[0125] Comparative Example 3

[0126] Referring to Example 1, instead of using a mixed solution of isopropanol and water, a mixed solution of methanol, ethanol, butanol, n-hexane, octane, and water was selected to compare the blotting effect on lipase Novozym 435. Other conditions were the same as in Example 1.

[0127] Table 14. Effects of different solubility types on enzyme activity

[0128] Solvent type Enzyme activity methanol 19.1％ ethanol 26.2％ Butanol 34.3％ n-Hexane 29.1％ Octane 26.6％ Isopropanol 38.4％ No trace 24.5％

[0129] Because isopropanol has good solubility for surfactants and its suitable polarity does not damage lipase activity, while also having a better interaction with lipases, it improves the imprinting effect of surfactants, thereby giving the enzyme better catalytic activity.

[0130] Comparative Example 4

[0131] Referring to Example 1, the concentration of isopropanol in the isopropanol-water mixture was changed, and the effect of the blot on the lipase Novozym435 was compared. Other conditions were the same as in Example 1.

[0132] Table 15. Effect of different isopropanol concentrations on enzyme activity

[0133]

[0134]

[0135] When the concentration of isopropanol in a mixed solution of isopropanol and water is greater than 70%, the mixed solution still has good solubility for the surfactant. However, when the concentration is too low, the solubility of the surfactant decreases, the imprinting effect decreases, and the enzyme activity decreases.

[0136] Comparative Example 5

[0137] Referring to Example 2, the cooling program was changed, and the composition of the product was compared, while other conditions remained the same as in Example 2.

[0138] According to the conditions of Example 2, imprinted lipase was obtained by imprinting, and intermediate products were obtained by transesterification. The programmed cooling conditions were as follows: the obtained transesterification product was kept at 85°C for 0.5 h, the temperature was reduced to 55°C at a rate of 12°C / h, and kept at this temperature for 24 h. Liquid oil was removed by centrifugation or filtration to obtain solid lipids. The triglyceride composition of the solid lipids is shown below.

[0139] Table 16. Triglyceride composition in the solid lipids after fractionation

[0140] Triglyceride composition content(%) LLL 8.2 MMM 21.7 MLL 26.5 MML 43.6

[0141] Due to unsuitable fractionation conditions, the content of medium-chain triglycerides in Comparative Example 5 was much higher than that in Example 2, while the content of medium- and long-chain triglycerides was much lower than that in Example 2.

[0142] Comparative Example 6

[0143] Under the conditions of Example 3, multiple batches of reactions were carried out using imprinted and non-imprinted lipases to compare the catalytic stability of imprinted and non-imprinted lipases.

[0144] Table 17. Enzyme activity and enzyme activity loss rate of imprinted and non-imprinted lipase NS40086 in different reaction batches.

[0145] batch Imprint NS40086 Non-imprinted NS40086 1 33.7％ 21.8％ 2 33.1％ 21.2％ 3 32.6％ 20.5％ 4 32.1％ 19.3％ 5 31.5％ 18.3％ 6 30.3％ 17.4％ 7 29.7％ 16.2％ 8 29.1％ 14.5％ 9 28.2％ 13.3％ 10 26.5％ 11.6％ Enzyme activity loss rate 21.4％ 46.8％

[0146] The catalytic stability of lipases plays a crucial role in ensuring consistent product quality. Furthermore, maintaining good catalytic stability allows for increased batch production and further cost reduction. After nine consecutive batches of reaction, the imprinted lipase showed an enzyme activity loss rate of only 28.4%, significantly lower than that of unimprinted lipase, while still maintaining higher activity than the original activity of unimprinted lipase. This is primarily due to the interaction between the nonionic surfactant and the lipase via surface hydrophobic forces. Specifically, the polar head of the nonionic surfactant binds to the hydrophilic groups of the lipase, while the nonpolar head binds to the hydrophobic groups on the lipase surface. Since lipase itself is water-soluble, its surface has a much larger proportion of hydrophilic groups than hydrophobic groups. Therefore, the nonionic surfactant alters the hydrophilic-hydrophobic properties of the lipase surface, changing it from hydrophilic to hydrophobic. This prevents excessive contact between polar substances in the system and the lipase, preventing water loss and disruption of the enzyme's catalytic conformation, thus maintaining good catalytic stability.

[0147] Nonionic surfactants have weaker hydrophobic interactions, unlike ionic surfactants which interact through ionic bonds. They do not change the catalytic conformation of enzymes and therefore do not lead to a loss of enzyme activity. However, they can interact with the active site of lipases, causing the lipase to open the cap of the active site and remove water, thus maintaining the active catalytic conformation of the lipase and enhancing its activation activity.

[0148] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for preparing a margarine / shortening base oil rich in medium- and long-chain triglycerides, characterized in that: include, Using medium-chain triglycerides and palm stearin as raw materials, lipase imprinted with a nonionic surfactant dissolved in isopropanol solution was added. Through enzymatic catalytic transesterification reaction, and filtration, transesterification products rich in medium and long-chain triglycerides and imprinted lipase were obtained. The transesterification product rich in medium and long chain triglycerides is cooled by programmed cooling to separate unreacted medium chain triglycerides and simultaneously extract medium and long chain triglycerides to obtain high melting point medium and long chain triglycerides. Medium-chain fatty acids are added to high-melting-point medium- and long-chain triglycerides, and imprinted lipase for transesterification is added. The reaction is catalyzed under vacuum, and the catalytic reaction includes esterification and acidolysis. Fatty acids are removed by molecular distillation to obtain high-melting-point medium- and long-chain triglycerides for use in the preparation of margarine base oil. The imprinted lipase is prepared by dissolving a nonionic surfactant in a mixed solution of isopropanol and water at a concentration of 20-100 mg / L by mass, and adding 10-30% immobilized lipase by mass to the solution. The mixture was stirred at 100-200 rpm for 30-60 minutes at 25°C and then filtered to obtain lipase. Moisture was removed by freeze-drying, and excess surfactant imprinted template on the immobilized lipase was eluted with a non-polar solvent. The lipase was filtered and vacuum dried to remove organic solvent, thus obtaining imprinted lipase. The immobilized lipase is a commercially available lipase including Novozym 435, Lipozyme RM IM, and NS40086; the isopropanol-water mixture has an isopropanol mass concentration greater than 70%; and the nonionic surfactant includes Tween 20, Tween 60, and Tween 80. The enzyme-catalyzed transesterification reaction includes adding medium-chain triglycerides and palm stearin to a batch reactor, purging with nitrogen for protection, and simultaneously adding imprinted lipase at an amount of 6-10 wt% of the substrate weight; raising the temperature to 60-80°C, stirring at 600-800 rpm, and reacting for 6-10 hours. The process of cooling the transesterification product rich in medium and long chain triglycerides by programmed cooling includes maintaining the transesterification product at 60-80°C for 1-2 hours, reducing the temperature to 40-50°C at a rate of 6-10°C / h and maintaining it for 6-24 hours, and removing the liquid oil by centrifugation or filtration to obtain solid lipids. The catalytic reaction under vacuum includes heating the solid fat to 60-80°C and adding medium-chain fatty acids, wherein the molar ratio of the amount of medium-chain fatty acids added to the solid fat triglycerides is 1-3:

1. Add the imprinted lipase filtered during the transesterification reaction, apply a vacuum of 10-30 mbar, react for 6-10 hours, and stir at 600-800 rpm.

2. The preparation method according to claim 1, characterized in that: The palm stearin is obtained by palm oil fractionation and has melting points of 44°C, 52°C and 58°C.

3. The preparation method according to claim 1, characterized in that: The molar ratio of the medium-chain triglyceride to palm stearin is greater than or equal to 1:

1.

4. The preparation method according to claim 1, characterized in that: The molecular distillation conditions are as follows: distillation temperature of 160~180℃, pressure of 2~5Pa, and condenser temperature of 20~30℃.

5. The margarine / shortening base oil rich in medium- and long-chain triglycerides prepared by the preparation method according to any one of claims 1 to 4.

6. The margarine / shortening base oil as described in claim 5, characterized in that: The margarine / shortening base oil contains less than 10% medium-chain triglycerides, more than 70% medium- and long-chain triglycerides, less than 0.4 mg / kg glycidyl esters, and less than 0.3 mg / kg chloropropanol esters.

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