Method for preparing medium-chain fatty glyceride by double-enzyme method

Abstract

The invention belongs to the technical field of biochemical engineering, and particularly relates to a method for preparing medium-chain fatty glyceride through double-enzyme catalysis. The method provided by the invention comprises the following steps: firstly, catalyzing raw materials such as coconut oil to be completely hydrolyzed by utilizing lipase, and separating medium-chain fatty acid with the purity of more than or equal to 99.5% by adopting a molecular distillation cooling crystallization technology; then, high-purity fatty acid and glycerol are used as substrates, immobilized lipase is used as a catalyst, directional esterification is performed in a reactor provided with a dehydration membrane block, and the water activity of the system is controlled to be 0.1-0.3. The content of medium-chain fatty glyceride in the final product is higher than 98%, and the final product does not contain long-chain fatty acid impurities. The process is green and energy-saving, the catalyst can be recycled, the economical efficiency is good, and an innovative solution is provided for large-scale production of the high-purity structured lipid.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological chemical industry, and particularly relates to a method for catalytically preparing medium-chain fatty acid glyceride by a double-enzyme method. BACKGROUND

[0002] Medium-chain triglycerides (MCTs) are a kind of lipid substances formed by esterification of medium-chain fatty acids (generally referring to C6-C12 fatty acids such as hexanoic acid, octanoic acid, decanoic acid and lauric acid) and glycerol molecules, and have excellent physicochemical properties and physiological functions. MCTs are rapidly metabolized in the human body, have high oxidation utilization rate, are not easy to accumulate in the body, and can be rapidly converted into energy, and are therefore widely used in food nutrition enhancers, sports nutrition supplements, infant formula foods, enteral and parenteral nutritional preparations, medical special diet foods, and cosmetics, pharmaceutical preparations and other fields. Meanwhile, with the further promotion of functional lipid research, MCTs also show potential application value in improving intestinal absorption disorders, lipid metabolism disorders, and auxiliary treatment of nervous system diseases, and thus the market demand for MCTs is growing.

[0003] However, the preparation of medium-chain fatty acid glyceride still has certain technical bottlenecks at present. The traditional industrial production usually adopts a chemical esterification method, which needs to be carried out under high temperature (180-250℃), strong acid catalyst (such as sulfuric acid and p-toluenesulfonic acid) and strict dehydration conditions. Although this method has fast reaction speed, it inevitably has the following disadvantages: harsh reaction conditions and high energy consumption; the acid catalyst is corrosive, and the equipment requirement is high; side reactions such as fatty acid polymerization, ester exchange and byproduct formation are easy to occur in the reaction system, leading to a decrease in product purity; the post-processing steps are complex, and neutralization, washing and refining are needed, which not only increases the production cost, but also produces a large amount of acid wastewater and solid waste residues, and does not meet the green and sustainable production requirements. In addition, the chemical method is difficult to obtain glycerides with specific structures, and is not suitable for nutrition or pharmaceutical grade products which are sensitive to product structure.

[0004] With the rapid development of biocatalysis technology, the preparation of fatty acid glyceride by enzyme catalysis has gradually become a research hotspot due to its advantages such as mild reaction conditions, high regioselectivity, few byproducts and environmental friendliness. The commonly used enzyme catalysis methods at present mainly include esterification and ester exchange. Among them, the ester exchange method usually uses natural plant oils such as coconut oil and palm kernel oil as raw materials, and obtains medium-chain glycerides by fatty acid exchange under the catalysis of lipase. This method is limited by the source of raw materials and the composition of fatty acids, and it is difficult to accurately control the content and distribution of medium-chain fatty acids in the final product. In addition, the ester exchange process often needs to use excessive medium-chain fatty acids or specific oils, resulting in increased cost.

[0005] Direct esterification typically involves the enzymatic esterification of medium-chain fatty acids to obtain glycerides. However, in existing technologies, medium-chain fatty acids are often sourced externally or obtained through chemical hydrolysis of oils, resulting in high overall costs and significant environmental impact. Furthermore, chemical hydrolysis also requires high temperatures, high pressures, or strong acid / alkali conditions, which are not conducive to green production.

[0006] To address the problems of high raw material costs, complex processes, numerous byproducts, and high energy consumption in existing technologies, researchers have begun exploring an integrated process route combining enzymatic hydrolysis and enzymatic esterification. This approach involves first using biocatalytic hydrolytic enzymes to gently hydrolyze triglycerides in natural vegetable oils into medium-chain fatty acids, and then combining them with glycerol under the catalysis of esterifying enzymes to convert them into medium-chain fatty acid glycerides. This significantly reduces environmental pollution, lowers energy consumption, and improves product quality. However, most existing technologies suffer from insufficient hydrolysis efficiency, cumbersome fatty acid purification steps, low esterification efficiency, glycerol-induced enzyme activity inhibition, and low yields. For example, some hydrolytic enzymes have limited activity in high-oil-concentration systems, leading to incomplete hydrolysis; while some esterifying enzymes have insufficient affinity for glycerol, making efficient condensation difficult, and sometimes requiring the use of solvent systems or the addition of dehydrating agents, further complicating the operation. Simultaneously, balancing enzyme stability, reaction condition coordination, and overall economic efficiency in both steps remains a key challenge for achieving large-scale industrial production.

[0007] Therefore, there is an urgent need to develop a new dual-enzyme process for preparing medium-chain fatty acid glycerides, which can achieve efficient hydrolysis and esterification under mild conditions, avoid complex post-processing, improve production efficiency and product purity, reduce environmental pollution, and achieve green and sustainable production. Summary of the Invention

[0008] To address the problems of existing technologies, this invention provides a method for preparing medium-chain fatty acid glycerides using a dual-enzyme method, comprising the following steps:

[0009] A method for preparing medium-chain fatty acid glycerides using a two-enzyme method, characterized by comprising the following steps:

[0010] (1) Mix natural oils rich in medium-chain fatty acids with water and carry out hydrolysis under the catalysis of the first lipase A; then, purify the hydrolysis products by molecular distillation and programmed cooling crystallization to obtain high-purity medium-chain fatty acids.

[0011] (2) The high-purity medium-chain fatty acids obtained in step (1) are mixed with glycerol at a molar ratio of (3.0-3.5):1 and esterified under the catalysis of a second lipase. The esterification reaction is carried out in a reactor equipped with a built-in dehydration module. During the reaction, the generated water is removed in real time through the dehydration module, and the water activity of the system is maintained at 0.1-0.3.

[0012] (3) After the reaction is completed, the second lipase is separated and recovered, and the resulting reaction solution is purified by distillation to obtain a high-purity medium-chain fatty acid triglyceride product.

[0013] Furthermore, the natural oil rich in medium-chain fatty acids mentioned in step (1) is coconut oil or palm kernel oil.

[0014] Further, the first lipase mentioned in step (1) is selected from lipase FM-10, lipase 5000MM, Transform 2.0, Lipura Split, Advance, TL 100L One or more lipases of 20000L Lipozyme CALB.

[0015] Furthermore, the operating conditions for molecular distillation in step (1) are: system pressure ≤100Pa, heating plate temperature 80~110℃, and condenser plate temperature ≤40℃.

[0016] Furthermore, the endpoint temperature of the programmed cooling crystallization described in step (1) is 0-5℃, the cooling rate is 0.5-1.5℃ / min, and seed crystals are added during the programmed cooling crystallization process to induce crystallization.

[0017] Further, in step (2), the second type of lipase is an immobilized lipase modified by site-directed mutagenesis, selected from one or more of the immobilized lipases IM-NE100, Novozym 435, Lipura Select, Lipozyme RM, Lipozyme TL IM, Lipozyme 435, Novozym 435, and Lipura Flex.

[0018] Furthermore, the esterification reaction in step (2) is carried out at a temperature of 55-65°C under negative pressure with a vacuum of -0.8-1 kPa. The water activity of the reaction system is maintained at 0.15-0.25 by controlling the negative pressure dehydration module.

[0019] Furthermore, in step (2), the amount of the second lipase added is 0.5% to 8% of the total substrate mass.

[0020] Furthermore, the distillation purification described in step (3) is short-path distillation, and the operating conditions are: temperature 120-150℃, pressure 0.1-1.0kPa.

[0021] Compared with the prior art, the preparation method provided by the present invention has the following beneficial effects:

[0022] The dual-enzyme process provided by this invention brings significant technological advancements and economic improvements. The final product consistently maintains an MCT content of over 98%. The entire process is conducted under mild conditions, without the use of any strong acids, strong alkalis, or toxic solvents, resulting in minimal emissions and adhering to green chemistry principles. Through system integration and unit operation optimization, overall production costs are effectively controlled. The process parameters are clearly defined, facilitating engineering scale-up and automation, thus laying a solid foundation for large-scale production. Detailed Implementation

[0023] The present invention will be further described in conjunction with specific implementation examples, which will enable those skilled in the art to better understand and master the invention, rather than limiting the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0024] The acid value determination method described in this embodiment of the invention is as follows:

[0025] Referring to GB 5009.229-2016, take a clean 250mL Erlenmeyer flask and weigh 5g of the prepared oil sample using a balance; the mass m is in grams. Take another clean 250mL Erlenmeyer flask, add 50mL of 95% ethanol, and then add 0.5mL of phenolphthalein indicator. Then, place this Erlenmeyer flask in a 100℃ water bath and heat until the ethanol just begins to boil. Remove the Erlenmeyer flask, and while the ethanol temperature is still above 70℃, immediately titrate the ethanol with a 0.5mol / L potassium hydroxide solution containing standard titrant. When the ethanol initially turns a faint red color and there is no obvious fading within 15 seconds, immediately stop the titration; the acidity of the ethanol has been neutralized. Immediately pour this neutralized ethanol solution, while still hot, into the Erlenmeyer flask containing the sample, and then place it in a 100℃ water bath and heat until the ethanol just begins to boil, vigorously shaking the Erlenmeyer flask during this process to form a suspension. Finally, remove the conical flask and immediately titrate the hot ethanol suspension of the sample with a 0.5 mol / L potassium hydroxide solution containing the standard titrant while it is still hot. When the sample solution initially turns slightly red and there is no obvious fading within 15 seconds, the titration is at its endpoint. Stop the titration immediately and record the number of milliliters of standard titrant solution consumed in this titration. This value is V.

[0026] Acid value calculation formula:

[0027]

[0028] In the formula X AVV represents the acid value of the sample; V represents the volume of standard titration solution consumed in the sample determination (mL); V0 represents the volume of standard titration solution consumed in the corresponding blank determination (mL); c represents the molar concentration of the standard titration solution (mol / L); 56.1 represents the molar mass of potassium hydroxide (g / mol); and m represents the weight of the oil sample (g).

[0029] The method for determining the glyceride content in this embodiment of the invention is as follows:

[0030] The content of glycerides in the product was determined by high performance liquid chromatography. 25 mg of sample was dissolved in 1 mL of mobile phase, mixed evenly, filtered through a membrane, and then loaded onto the sample.

[0031] Chromatographic conditions: Sepax HP-Silica column (4.6 mm × 250 mm × 5 μm);

[0032] Detector: Evaporative light scattering detector;

[0033] Column temperature: 30℃;

[0034] Sample concentration: 25 mg / mL;

[0035] Injection volume: 15 μL;

[0036] The ratio of the mobile phase n-hexane:isopropanol:formic acid is 15:1:0.03 (v / v / v);

[0037] The flow rate is 1 mL / min.

[0038] Example 1: Preparation of medium-chain fatty acid glycerides using the dual-enzyme method of the present invention.

[0039] 1000g of refined coconut oil was added to 300g of deionized water and 50g of lipase FM-10. The mixture was reacted at 45℃ and 300rpm for 8 hours until complete hydrolysis. The reaction mixture was subjected to molecular distillation at a pressure of 10Pa, a heating plate temperature of 95℃, and a condenser plate temperature of 35℃ to obtain approximately 780g of crude medium-chain fatty acids. The crude product was then cooled to 2℃ at a programmed rate of 0.8℃ / min, crystallized, and separated to obtain 760g of a refined mixture of octanoic acid and capric acid (C8:C10≈65:35). The total purity was determined to be 98.7% by liquid chromatography.

[0040] 700g of the above-mentioned refined fatty acids and glycerol (molar ratio 3.2:1) were added to the reactor, along with 35g of immobilized lipase IM-NE100. The reaction was carried out at 60℃, slight negative pressure (-0.05MPa), and 300rpm for 10 hours, while maintaining the water activity of the system at 0.18.

[0041] After the reaction was completed, the acid value of the product was determined to be 1.2 mg KOH / g. The immobilized lipase IM-NE100 was recovered by filtration, and the filtrate was subjected to short-path distillation at 120℃ and 1 kPa to obtain 665 g of colorless and transparent medium-chain fatty acid glycerides.

[0042] The content of medium-chain fatty acid glycerides in the product was determined to be 98.5% by high performance liquid chromatography.

[0043] Example 2: Preparation of medium-chain fatty acid glycerides by conventional acid catalysis

[0044] In a high-pressure reactor, equal amounts of unpurified, hydrolyzed mixed fatty acids (C8+C10 content approximately 85%) from the same source were mixed with glycerol (molar ratio 3.2:1). A catalyst was added, and the reaction temperature was raised to 200°C. The reaction system was maintained in a liquid phase by pressurization at 1.0 MPa. The reaction time was set to 4 hours. After the reaction was completed, the mixture was cooled and neutralized with an aqueous sodium carbonate solution. The reaction mixture was then washed several times with a large amount of hot water (60-70°C) to remove residual salts and catalyst until the aqueous phase was neutral. The oil phase was separated, and the target product was separated by vacuum distillation at 120°C and 1.0 kPa. The acid value was determined to be 2.2 mg KOH / g. The content of medium-chain fatty acid glycerides was determined to be 82.5% by high-performance liquid chromatography (HPLC), and a large amount of monoglycerides and diglycerides were generated, indicating low purity of the target product.

[0045] Example 3: Preparation of medium-chain fatty acid glycerides using the conventional single-enzyme method

[0046] 700g of unpurified coconut oil hydrolysate fatty acid mixture (purity approximately 85%, containing approximately 12% long-chain fatty acids) was mixed with glycerol in the same proportion, and an equal amount of immobilized lipase Novozym 435 was added. The mixture was reacted at the same temperature. No special dehydration equipment was used during the reaction; moisture was removed only by purging with dry nitrogen. After reacting for 12 hours, the product was obtained after post-treatment under the same conditions. The acid value was determined to be 20 mg KOH / g, the content of medium-chain fatty acid glycerides was determined to be 88.3%, and approximately 5% of medium- and long-chain triglyceride impurities were detected in the product.

[0047] Example 4: Verification of the number of cycles of immobilized lipase

[0048] This embodiment simulates an industrial continuous production scenario, and conducts long-term batch recycling tests on the immobilized lipase IM-NE100 of the present invention to evaluate its operational stability and the economy of the entire process.

[0049] The same batch of immobilized lipase IM-NE100 was used for the synthesis of medium-chain fatty acid glycerides in 20 consecutive batches. Each batch was processed under standard conditions (same substrate, 60°C, water activity 0.18, reaction time 10 h). After each batch, the enzyme was recovered by simple filtration, washed with a small amount of tert-butanol, vacuum dried, and used directly in the next batch. The conversion rate of each batch was recorded. Samples were taken from batches 1, 5, 10, 15, and 20 to analyze the content of medium-chain fatty acid glycerides in the product.

[0050] Experimental results showed that the conversion rate remained very stable (97.5% ± 0.5%) in the first 15 batches, decreased slightly in batches 16-20, and reached 95.0% in batch 20. The content of medium-chain fatty acid glycerides in the product quality indicators was >98%, and remained stable in all batches.

[0051] Example 5: Adaptability verification of different raw material sources

[0052] To demonstrate the broad adaptability of the process of this invention to raw materials, we selected refined coconut oil from three different origins—the Philippines, Indonesia, and Vietnam—and palm kernel oil from Malaysia as starting materials. All raw materials were processed according to the standard process flow of Example 1 of this invention: first, complete hydrolysis with lipase FM-10 at 45°C; then, purification of the hydrolysis products through the same molecular distillation (95°C, 10 Pa) and programmed cooling crystallization (final temperature 2°C) to obtain the corresponding medium-chain fatty acids. Then, directional esterification was uniformly performed using immobilized lipase IM-NE100 at 60°C and a water activity of 0.18. Analysis results showed that although there were slight differences in the initial fatty acid composition of the raw materials from different origins (e.g., the C8:C10 ratio fluctuated between 62:38 and 68:32), the purity of the obtained fatty acid substrates after the purification steps of this invention all reached over 99.5%. The final synthesized medium-chain fatty acid glycerides exhibited a high degree of consistency in their core quality indicators: the medium-chain fatty acid glyceride content ranged from 98.5% to 99.0%, and no C12 or higher long-chain fatty acid impurities were detected in any of the products. This demonstrates that the process provided by this invention is not sensitive to the source of raw materials, possesses universality and stable product quality output capabilities, and is applicable to raw material supplies from different production regions worldwide.

[0053] In summary, the dual-enzyme process provided by this invention brings significant technological advancements and economic improvements. The final product consistently maintains a medium-chain fatty acid glyceride content of over 98%. The entire process is conducted under mild conditions, without the use of any strong acids, strong alkalis, or toxic solvents, resulting in minimal emissions and adhering to green chemistry principles. Through system integration and unit operation optimization, overall production costs are effectively controlled. The process parameters are clearly defined, facilitating engineering scale-up and automation, thus laying a solid foundation for large-scale production.

Claims

1. A process for the production of medium chain fatty acid glycerides by a two enzyme method, characterized in that, The method comprises the following steps: (1) mixing natural oil rich in medium-chain fatty acids with water, and performing hydrolysis reaction under the catalysis of a first lipase A; then, performing purification treatment on the obtained hydrolysis product by molecular distillation and programmed cooling crystallization in sequence to obtain high-purity medium-chain fatty acids; (2) mixing the high-purity medium-chain fatty acids obtained in step (1) with glycerol according to a molar ratio of (3.0-3.5):1, and performing esterification reaction under the catalysis of a second lipase, wherein the esterification reaction is performed in a reactor equipped with a built-in dehydration module, and water generated in the reaction process is removed in real time through the dehydration module, and the water activity of the system is maintained at 0.1-0.3; (3) after the reaction is completed, the second lipase is separated and recovered, and the obtained reaction liquid is subjected to distillation purification to obtain high-purity medium-chain fatty acid triglyceride product.

2. The method of claim 1, wherein, The natural oil rich in medium-chain fatty acids in step (1) is coconut oil or palm kernel oil.

3. The method of claim 1, wherein, The first lipase in step (1) is selected from one or more of lipase FM-10, lipase 5000 MM, Eversa Transform 2.0, Lipura Split, Eversa Advance, Lipozyme TL 100 L, Palatase 20000 L Lipozyme CALB.

4. The method of claim 1, wherein, The operating conditions of the molecular distillation in step (1) are as follows: system pressure ≤100 Pa, heating plate temperature 80-110 °C, and condensing plate temperature ≤40 °C.

5. The method of claim 1, wherein, The end point temperature of the programmed cooling crystallization in step (1) is 0-5 °C, the cooling rate is 0.5-1.5 °C / min, and seed crystals are added to induce crystallization during the programmed cooling crystallization.

6. The method of claim 1, wherein, The second lipase in step (2) is a fixed lipase modified by site-directed mutagenesis, and is selected from one or more of fixed lipase IM-NE100, Novozym 435, Lipura Select, Lipozyme RM, Lipozyme TL IM, Lipozyme 435, Novozym 435, Lipura Flex.

7. The method of claim 1, wherein, The temperature of the esterification reaction in step (2) is 55-65 °C, the reaction is performed under negative pressure, the vacuum degree is negative 0.8-1 kPa, the water activity of the reaction system is maintained at 0.15-0.25 through the control of the negative pressure dehydration module.

8. The method of claim 1, wherein, The addition amount of the second lipase in step (2) is 0.5%-8% of the total mass of the substrate.

9. The method of claim 1, wherein, The distillation purification in step (3) is short-path distillation, and the operating conditions are as follows: temperature 120-150 °C, and pressure 0.1-1.0 kPa.

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