A method for enzymatic synthesis of monoglycerides in an ionic liquid system

Abstract

The application discloses a method for synthesizing monoglyceride by using an enzyme in an ionic liquid system. Glycerin and fatty acid are mixed, a lipase is used as a catalyst, a molecular sieve is used as a dehydrating agent, and stirring reaction is carried out, and finally, monoglyceride is obtained through centrifugal separation. The application uses ionic liquids with different polarities, and selectively synthesizes monoglyceride by using ionic liquids with relatively large polarities, so that the yield of monoglyceride is improved, and the defects, such as easy volatilization of organic solvents, toxic gas, harm to human body and environmental pollution, are effectively avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of structured glyceride synthesis, and particularly relates to a method for synthesizing monoglyceride by an enzymatic method in an ionic liquid system. Background Art

[0002] Fatty acid glycerides (MAGs) are polyol-based nonionic surfactants. The varying carbon numbers of fatty acids result in varying hydrophilic-lipophilic balances (HLBs), resulting in diverse industrial applications. In recent years, the use of high-purity MAGs has grown in various industries, with usage increasing annually. Based on the name of the constituent fatty acids, MAGs can be categorized as monostearate, monopalmitate, monolaurate, and monooleate. Monostearate is the most produced and widely used MAG. MAG synthesis methods include chemical catalysis and enzymatic catalysis. Chemical catalysis often requires high temperatures, resulting in serious issues such as darkening of the unsaturated fatty acid-rich oils and fatty acid isomerization. It also consumes high energy, produces numerous byproducts, and poses environmental risks. Enzymatic catalysis, on the other hand, offers milder conditions, lower energy consumption, and is environmentally friendly. Furthermore, the high selectivity of the enzymes makes the reaction more efficient, minimizes side effects, and produces high-purity products. Therefore, enzymatic synthesis of glycerides remains a hot topic of research. Enzymatic synthesis of MAGs involves hydrolysis, alcoholysis, glycerolysis, and esterification of fatty acids with glycerol. Currently, the most common method for producing MAG is through the glycerolysis of edible oils. While glycerolysis offers high yields, it struggles to produce high-purity monoglycerides, which can only be obtained through the esterification of fatty acids with glycerol. Furthermore, both esterification and glycerolysis reactions are multi-step processes, ultimately producing MAG as well as DAG and TAG as byproducts, making selective enrichment of MAG difficult. Previous studies have reported that increasing the polarity of organic solvents through solvent engineering can adjust the reaction equilibrium, favoring MAG enrichment.

[0003] In recent years, enzyme-catalyzed methods have become a hot topic in ester synthesis due to their advantages, including mild reaction conditions, low energy consumption, high selectivity, efficient reactions, few side reactions, and high product purity. While organic solvents as reaction media in current lipase-catalyzed reactions offer advantages such as increased reaction rate, milder reaction conditions, and improved enzyme stability, they also suffer from disadvantages such as numerous side reactions, low selectivity, and the generation of toxic gases that can harm humans and pollute the environment. Therefore, the search for superior reaction media for enzymatic ester synthesis is urgent. Summary of the Invention

[0004] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0005] The present invention is proposed in view of the above-mentioned problems and / or problems existing in the existing synthetic MAG.

[0006] Therefore, the purpose of the present invention is to solve the shortcomings of the prior art chemical synthesis and synthesis of MAG in organic solvents, which have environmental and safety issues, and to provide a method for MAG synthesis in ionic liquids.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] As a preferred embodiment of the method for enzymatic synthesis of monoglycerides in an ionic liquid system according to the present invention, wherein:

[0009] The beneficial effects of the present invention are:

[0010] 1. In the method for enzymatic synthesis of monoglycerides in an ionic liquid system provided by the present invention, compared with the harm caused to human body and environment by organic solvents, ionic liquids have a wide liquid range and no vapor pressure, and are a truly clean, pollution-free green solvent, which is increasingly valued in the field of organic synthesis.

[0011] 2. In the method for enzymatic synthesis of monoglycerides in an ionic liquid system provided by the present invention, the ionic liquid can improve the activity and stability of lipase and can be recycled and reused, thereby reducing costs to a certain extent.

[0012] 3. In the enzymatic synthesis of monoglycerides using an ionic liquid system provided by the present invention, ionic liquids act as a designable solvent. By varying the types of anions and cations in the ionic liquids, their polarity, hydrophobicity, hydrogen bonding properties, and viscosity can be modified. This increases the contact area between fatty acids and glycerol, improving mass transfer. Simultaneously, the ionic liquids retain an appropriate amount of water to maintain enzyme activity and increase the esterification reaction rate. Furthermore, the polarity and hydrogen bonding properties of the ionic liquids adjust the reaction equilibrium, selectively enriching MAG, reaching a concentration of 60.1% in the reaction product.

[0013] 4. Traditional MAG synthesis requires the addition of excessive amounts of glycerol. In the present invention, the use of a highly polar ionic liquid modulates the reaction, producing more MAG while maintaining an optimal glycerol / oleic acid molar ratio of only 3 / 1, effectively saving the amount of glycerol added as a raw material.

[0014] 5. After the reaction is completed, the product and the ionic liquid can be separated by simple centrifugation, which is easy to scale up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0016] Figure 1-5 The effect of ionic liquid properties on MAG selectivity and fatty acid conversion was shown in Figure 2. The reaction conditions were as follows: 1.049 g of oleic acid (5 mmol) and 0.460 g of glycerol (5 mmol) were mixed, 30% by weight of the ionic liquid (0.5608 g) was added as the reaction medium, and after stirring, 4% by weight of immobilized lipase Novozyme 435 was added. The reaction was incubated at 60°C with magnetic stirring for 8 hours.

[0017] Figure 1 The effect of polarity on MAG selectivity and fatty acid conversion. Figure 2 The effect of hydrogen bond basicity on MAG selectivity and fatty acid conversion rate. Figure 3 This is the hydrogen bonding mechanism between ionic liquid and MAG. Figure 4 is the effect of log P on MAG selectivity and fatty acid conversion. Figure 5 Effect of viscosity on MAG selectivity and fatty acid conversion

[0018] Figure 6 The effect of lipase type on MAG synthesis. The reaction conditions were: temperature 60°C, substrate molar ratio (glycerol:oleic acid) 1:1, enzyme addition amount 4%, ionic liquid addition amount 30%, and reaction time 8 hours. Novozymes 435, Lipozyme RM IM, and Lipozyme TL IM were selected for the reaction.

[0019] Figure 7 Effect of reaction time on MAG synthesis: The reaction conditions were: reaction temperature 50°C, substrate molar ratio (glycerol:oleic acid) 3:1, enzyme addition amount 4% and ionic liquid addition amount 30%, and reaction time was selected from 1 to 12 hours.

[0020] Figure 8 Effect of lipase addition on MAG synthesis. The reaction conditions were: reaction time 8 h, reaction temperature 50 ° C, substrate molar ratio (glycerol: oleic acid) 3:1 and ionic liquid addition amount 30%, and the reaction was carried out at 2%-10%.

[0021] Figure 9 Effect of temperature on MAG synthesis. Reaction conditions: reaction time 8h, lipase addition amount 4%, substrate molar ratio (glycerol:oleic acid) 3:1 and ionic liquid addition amount 30%, select 30-70°C to react

[0022] Figure 10 Effect of ionic liquid addition amount on MAG synthesis. Reaction conditions: reaction time 8h, lipase addition amount 4%, substrate molar ratio (glycerol:oleic acid) 3:1 and temperature 50°C, select 20%-60% to react

[0023] Figure 11 Effect of substrate molar ratio on MAG synthesis. Reaction conditions: reaction time 8h, lipase addition amount 4%, ionic liquid addition amount 30% and temperature 50°C, select glycerol, molar ratio of which to fatty acid is 1:0.2-1 to react. DETAILED DESCRIPTION

[0024] In order to make the above objectives, features and advantages of the present application more apparent, the specific embodiments of the present application will be described in detail below.

[0025] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without the specific details given below. In other instances, well-known methods have not been described in detail in order to avoid obscuring the present application. Therefore, the present application is not intended to be limited by the specific embodiments disclosed below.

[0026] Secondly, the "one embodiment" or "embodiment" referred to herein means that the specific features, structures or characteristics can be included in at least one implementation of the present application. "In one embodiment" appearing in different places in the specification does not mean the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments.

[0027] The reaction system of the present application is a mixed reaction system, and the ionic liquid favorable to MAG synthesis is screened, and the optimization of the enzyme species, reaction temperature, time, enzyme addition amount, ionic liquid addition amount and substrate molar ratio and other factors is determined to determine the optimal synthesis process of the mixed reaction system.

[0028] Example 1

[0029] 1.1 Screening of ionic liquid

[0030] 1.049g oleic acid (5mmol) and 0.460g glycerol (5mmol) were mixed, and an ionic liquid (0.5608g) accounting for 30% of the total mass of the substrate was added as a reaction medium. After stirring, an immobilized lipase Novozyme435 accounting for 4% of the total mass of the substrate was added. After magnetic stirring at 60°C for 8 hours, the ionic liquid (containing glycerol) and lipase were removed by centrifugation to obtain a product containing monoglycerides. The content of MAG in the system after the reaction was detected using a normal phase high performance liquid chromatography evaporative light detector (NP-HPLC-ELSD) and quantified using the external standard method. The change in fatty acid content was detected by acid-base titration. Esterification rate = (1-molar amount of fatty acid after reaction / molar amount of fatty acid before reaction) × 100%

[0031] NP-HPLC-ELSD analysis of MAG content

[0032] At regular intervals, 20 μl of the reaction mixture was removed and dissolved in phase B solvent, and 5 μl of the sample was injected. The glycerolipid content in the reaction system was determined by normal-phase high-performance liquid chromatography with evaporative light detection (NP-HPLC-ELSD). Chromatographic conditions: Luna silica columns (4.6 mm × 250 mm × 5 μm, Phenomenex, USA), column temperature 30°C; ELSD conditions: drift tube temperature 55°C, gain 1, gas flow rate 1.8 mL / min; sample concentration 0.5 mg / mL, injection volume 5 μL; binary gradient elution system: (Phase A) n-hexane / isopropanol = 98 / 2 (v / v), (Phase B) n-hexane / isopropanol / glacial acetic acid = 1 / 1 / 0.01 (v / v / v), flow rate 1.0 mL / min. The gradient elution program is shown in Table 2.

[0033] Table 1 - Binary gradient elution program for determination of glyceride content by HPLC-ELSD

[0034]

[0035] Determination of glyceride selectivity: Establish standard curves for monoglyceride, diglyceride, and triglyceride, respectively, and use the external standard method to measure the absolute content of monoglyceride, diglyceride, and triglyceride in the reaction system. The selectivity can be expressed as:

[0036]

[0037] Table 2 shows the results of MAG selectivity and fatty acid conversion in different ionic liquids. By conducting correlation studies with the properties of different solvents for ionic liquids, ionic liquids suitable for the selective synthesis of MAG were screened.

[0038] Table 2 - MAG selectivity, fatty acid conversion, and properties of ionic liquids

[0039]

[0040] (1) Effect of polarity on MAG selectivity,

[0041] For most ionic liquids, the selectivity of monoglycerides increases with increasing polarity, while the conversion rate of fatty acids decreases. Among them, [HO(CH2)2MIM]NTf2 achieves the highest monoglyceride selectivity (50.47%). This pattern is also found in [Tf2N] - The extraction in ionic liquids is more significant. Through curve fitting, we can get [Tf2N] - Monoglyceride selectivity, fatty acid conversion and polarity E in ionic liquids N T The correlation curves of the values ​​are: y = -593.231 + 2730.38x - 3932.44x 2 +1872.421x 3 R 2 =0.89012, y=199.80+571.198x-946.6537x 2 +572.427x 3 R 2 =0.89012. Polarity and monoglyceride selectivity show a strong positive correlation. The inventors discovered that increasing solvent polarity decreases the thermodynamic activity of highly hydrophobic MAGs in the reaction system, while increasing the activity of highly hydrophobic DAGs and TAGs, shifting the reaction equilibrium toward the production of more MAGs. However, the [BMIM]N(CN)2 ionic liquid, while not as polar as [HO(CH2)2MIM]NTf2, exhibits a high MAG selectivity (57.31%). [PF6] - and [BF4] - However, ionic liquids show a trend opposite to the polarity rule.

[0042] (2) Effect of hydrogen bonding properties on MAG selectivity

[0043] At present, the parameters related to hydrogen bonding of ionic liquids can be expressed by Kamlet–Taft parameters (α, β, π*). α is the hydrogen bond acidity, which indicates the hydrogen bond donor ability; β is the hydrogen bond basicity, which indicates the hydrogen bond acceptor ability; and π* is the ratio of dipole to polarizability. As can be seen from Table 2, α is the same polarity as E N T The values ​​have the same trend, and thus the influence on MAG selectivity and FFA conversion is also similar. [HO(CH2)2MIM]NTf2 has a very high α value (1.17) probably because the cation has a hydroxyl group, which is a strong hydrogen bond donor. Figure 3The ionic liquid shown in A forms hydrogen bonds with the hydroxyl groups on MAG, thereby reducing the activity of MAG and increasing the selectivity. In fact, since the calculation formula of α is the same as E N T There is a certain relationship as shown below,

[0044] α=0.0649E T (30)-2.03-0.27π *

[0045]

[0046] For most ionic liquids, the value of π* does not change much, so α and E N T This shows a positive correlation. This explains the effect of polarity on the selectivity of monoglycerides from another aspect.

[0047] The β value shows a clear positive correlation with the MAG selectivity. In particular, [BMIM]N(CN)2 has the highest β value (0.71). Although its α value (0.54) is not high, it has a high monoglyceride selectivity (57.31%). Figure 3 As shown in B, this may be due to the fact that [BMIM]N(CN)2 has a highly electronegative anion [N(CN)2] - This anion is a strong hydrogen bond acceptor, readily forming hydrogen bonds with monoglycerides, thereby increasing monoglyceride selectivity. However, the fatty acid conversion rate of [BMIM]N(CN)2 is very low. Ionic liquids with high β values ​​are generally hydrophilic, resulting in a strong interaction between the enzyme and the solvent. This interaction can disrupt the enzyme's protein structure, reducing enzyme activity and resulting in a lower conversion rate.

[0048] (3) Effect of hydrophobicity log P on MAG selectivity

[0049] The hydrophobicity of ionic liquids is characterized by log P (octanol-water partition coefficient), which is the logarithmic value of the ratio of the concentration of ionic liquid in octanol and water phases. - , [Tf2N] - It is active in ionic liquids with hydrophobic anions, but very inactive in liquids with hydrophilic anions. The fatty acid conversion rate is positively correlated with log P. [PF6] - , [Tf2N] - The fatty acid conversion rate of ionic liquids is significantly higher than that of [BF4] - and [N(CN)2] -This is consistent with the relationship between enzyme activity and hydrophobicity in ionic liquids. In addition, there is no obvious correlation between the selectivity of MAG and log P. In comparison, the selectivity of monoglycerides in ionic liquids with high hydrophobicity is relatively low.

[0050] (4) Effect of viscosity on MAG selectivity

[0051] Overall, there is no obvious correlation between viscosity and MAG selectivity and fatty acid conversion. However, the viscosity of different types of ionic liquids shows different trends. For [Tf2N] - For example, the viscosity increases with the increase of cationic acyl chain length, the fatty acid conversion rate increases, and the monoglyceride selectivity decreases. However, [PF6] - The ionic liquids showed the opposite trend. With the increase of cationic acyl chain length, the fatty acid conversion rate decreased and the monoglyceride selectivity increased. As can be seen from Table 2, the viscosity of ionic liquids with different anions showed great differences. [PF6] - and [BF4] - The viscosity of ionic liquids (100-700 cP) is much higher than that of the same type of [Tf2N] - and [N(CN)2] - Class of ionic liquids (20-100 cP). And, [PF6] - In ionic liquids, viscosity increases significantly with increasing acyl chain length. The esterification reaction between glycerol and fatty acids is a multi-step process, initially forming monoglycerides. MAG then combines with fatty acids to form diglycerides (DAG) and TAG. High-viscosity solvents result in higher mass transfer resistance, which reduces the reaction conversion rate. Furthermore, more substrate forms DAG, which is difficult to convert to DAG and TAG, resulting in high selectivity for MAG. [PF6] - It is for this reason that ionic liquids exhibit a trend opposite to the polarity law mentioned above.

[0052] In summary, polarity and hydrogen bonding properties are the main properties that affect the selectivity of glycerides in ionic liquids. Ionic liquids with high polarity and some functional groups are conducive to the enrichment of MAG. Log P and β value mainly affect the conversion rate of fatty acids. Ionic liquids with high log P and low β value (NTf2 - PF6 - It has high FFA conversion rate. It has low polarity, high log P value and high DAG and TAG selectivity in ionic liquids with long carbon chain cations. - In the case of [Tf2N]- In the ionic liquid class, the cation substituents of the mono- or poly-substituted ionic liquids with carbon chains of 4 to 6 carbon atoms include one or more of [BMIM][Tf2N], [HMIM][Tf2N], [BMMIM][Tf2N] or [B3 MeN]NTf2; the cation substituents of the mono- or poly-substituted ionic liquids containing hydroxyl or ether functional groups include [HO(CH2)2MIM][Tf2N] or [MeO(CH2)2MIM][NTf2]. [PF6] - The cation substituents in the ionic liquids are monosubstituted ionic liquids with carbon chains of 8 to 10 carbons, including [OMIM][PF6] or [C 10 MIM][PF6]. [N(CN)2] - Ionic liquids include [BMIM][Tf2N].

[0053] 1.2 Optimization of process conditions

[0054] In addition, [HO(CH2)2MIM]NTf2 has high MAG selectivity, high conversion rate, and the product is easy to separate. Therefore, we chose [HO(CH2)2MIM]NTf2 as the solvent to simply optimize the MAG synthesis.

[0055] The optimal conditions were as follows: oleic acid and glycerol were added to the reactor in a molar ratio of 1:4, ionic liquid [HO(CH2)2MIM]NTf2 accounting for 30% of the total mass of the substrate was added, a stirrer was placed, and the mixture was preheated in a constant temperature water bath at 50°C. The magnetic stirrer was turned on and stirred at 450 rpm. Lipase Novozyme435 accounting for 4% of the total mass of the substrate and 4% 4A molecular sieve were added to start the reaction. After 8 hours of reaction, the product was taken out and centrifuged at 10,000 rpm for 10 minutes to remove the ionic liquid and lipase. Liquid phase analysis showed that the monoglyceride content in the product was 60.1%.

[0056] Example 2:

[0057] Lauric acid and glycerol were added to a reactor in a molar ratio of 1:1, and an ionic liquid [HO(CH2)2MIM]NTf2 accounting for 30% of the total mass of the substrate was added. A stirring bar was placed and preheated in a constant temperature water bath at 60°C. The magnetic stirrer was turned on and stirred at a speed of 450 rpm. The lipase Novozyme435 accounting for 4% of the total mass of the substrate and 4% 4A molecular sieve were added to start the reaction. After 8 hours of reaction, the product was taken out and centrifuged at 10000rpm for 10 minutes to remove the ionic liquid and lipase. Liquid phase analysis showed that the monoglyceride content in the product was 52.5%.

[0058] Example 3:

[0059] Caprylic acid and glycerol were added to a reactor in a molar ratio of 1:1, and an ionic liquid [MeO(CH2)2MIM]NTf2 accounting for 30% of the total mass of the substrate was added. A stirring bar was placed and preheated in a constant temperature water bath at 60°C. The magnetic stirrer was turned on and stirred at a speed of 450 rpm. The lipase Novozyme435 accounting for 4% of the total mass of the substrate and 4% 4A molecular sieve were added to start the reaction. After 8 hours of reaction, the product was taken out and centrifuged at 10000rpm for 10 minutes to remove the ionic liquid and lipase. Liquid phase analysis showed that the monoglyceride content in the product was 51.0%.

[0060] Example 4:

[0061] Lauric acid and glycerol were added to a reactor in a molar ratio of 1:1, and an ionic liquid [B3 MeN] NTf2 accounting for 30% of the total mass of the substrate was added. A stirring bar was placed and preheated in a constant temperature water bath at 60°C. The magnetic stirrer was turned on and stirred at 450 rpm. The lipase Novozyme435 accounting for 4% of the total mass of the substrate and 4% 4A molecular sieve were added to start the reaction. After 8 hours of reaction, the product was taken out and centrifuged at 10000rpm for 10 minutes to remove the ionic liquid and lipase. Liquid phase analysis showed that the monoglyceride content in the product was 59.70%.

[0062] Example 5

[0063] Linoleic acid and glycerol were added to a reactor in a molar ratio of 1:4, and an ionic liquid [HO(CH2)2MIM]NTf2 accounting for 30% of the total mass of the substrate was added. A stirring bar was placed and preheated in a constant temperature water bath at 50°C. The magnetic stirrer was turned on and stirred at a speed of 450 rpm. The lipase Novozyme435 accounting for 4% of the total mass of the substrate and 4% 4A molecular sieve were added to start the reaction. After 8 hours of reaction, the product was taken out and centrifuged at 10000rpm for 10 minutes to remove the ionic liquid and lipase. Liquid phase analysis showed that the monoglyceride content in the product was 55.3%.

[0064] Thus, the method for enzymatically synthesizing monoglycerides in ionic liquids provided by the present invention utilizes the adjustable solvent properties of ionic liquids and selects different ionic liquids as reaction media. On the one hand, the contact area between fatty acids and glycerol is increased, improving mass transfer. At the same time, the ionic liquid can retain an appropriate amount of water to maintain enzyme activity and increase the esterification reaction rate. On the other hand, under the action of polarity and hydrogen bonding properties, the ionic liquid can adjust the reaction equilibrium and selectively enrich MAG, so that its content in the reaction product reaches 60.1%. This not only increases the yield, but also effectively avoids the shortcomings of organic solvents such as easy volatility, toxic gases that can easily cause harm to the human body and pollute the environment.

[0065] 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 the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for enzymatically synthesizing monoglycerides in an ionic liquid system, characterized in that: Using ionic liquid as the reaction medium, glycerol and fatty acids are mixed, lipase is used as a catalyst, molecular sieves are used as a dehydrating agent, the mixture is stirred and reacted, and finally the monoglyceride product is obtained by centrifugation. Wherein, the ionic liquid is the ionic liquid [HO(CH2)2MIM]NTf2, and the addition amount of the ionic liquid is 30% of the substrate mass; The fatty acid is oleic acid, and the molar ratio of glycerol to fatty acid is 4:1; The lipase is immobilized enzyme Novozym435, and its addition amount is 4% of the total mass of the substrate; The dehydrating agent is 4A molecular sieve, and its addition amount is 4% of the substrate mass; The stirring reaction was carried out at a temperature of 50°C, a stirring reaction speed of 450 rpm, and a reaction time of 8 h; The centrifugal separation was performed at a rotation speed of 10,000 rpm and a time of 10 min.

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