KR102862613B1 - Method for preparing triglyceride with high yield through wet fractionation using solvent mixture - Google Patents

Abstract

The present invention relates to a method for producing triglycerides, and more particularly, to a method for producing triglycerides through an enzymatic esterification reaction in which an acyl group of a triacylglyceride is exchanged with another acyl group, and then purifying the reaction product through wet fractionation with a specific solvent mixture, thereby producing triglycerides with excellent purity in high yield.

Description

Method for preparing triglyceride with high yield through wet fractionation using solvent mixture

The present invention relates to a method for producing triglycerides, and more particularly, to a method for producing triglycerides through an enzymatic esterification reaction in which an acyl group of a triacylglyceride is exchanged with another acyl group, and then purifying the reaction product through wet fractionation with a specific solvent mixture, thereby producing triglycerides with excellent purity in high yield.

Triacylglyceride fats and oils are substances that are widely used in the food industry, and among them, 1,3-dioleoyl-2-palmitoyl glyceride (OPO) is an important component of breast milk fat and has been extensively studied nutritionally.

The fat composition, which contains the major fatty acids that make up breast milk fat, is found in abundance in vegetable oils and fats, but almost all vegetable glycerides do not have a saturated fatty acid in the 2-position. In contrast, in breast milk fat, a significant amount of the saturated fatty acid palmitic acid occupies the 2-position of the glycerides.

As people's interest in infant health has increased recently, the demand for foods with similar ingredients to breast milk is also increasing.

Innis (Adv. Nutr., 2:275-283, 2011) reports on the structural role of triacylglycerols in the infant diet.

However, regular powdered milk is a mixture of powdered milk and vegetable oil, and when the oil content of powdered milk is analyzed, it contains almost no palmitic acid in the 2-position.

Accordingly, the application of high-quality human milk fat substitutes for adding 1,3-dioleoyl-2-palmitoyl glycerides (OPO), which contain palmitic acid in the 2-position and have a composition similar to human milk fat, to infant formula is increasing.

OPO is a type of USU (Unsaturated-Saturated-Unsaturated) triglyceride, and Mohamad, et al (Lipase-catalyzed interesterification reactions for human milk fat substitutes production, 2013, 115, 270-285) showed that research has mainly been conducted to synthesize 1,3-dioleoyl-2-palmitoyl glyceride by substituting the 1,3-position of triacylglyceride using lipase. Due to the 1,3-position-specific reactivity of lipase on triacylglycerides, providing a glyceride backbone containing a saturated fatty acid at the 2-position of triacylglyceride fat is important in synthesizing 1,3-dioleoyl-2-palmitoyl glyceride (OPO).

However, the conventional enzymatic esterification method has a problem in that it is difficult to produce 1,3-dioleoyl-2-palmitoyl glyceride (OPO) in high yield while maintaining excellent purity.

Therefore, there is a need for the development of a new method for producing 1,3-dioleoyl-2-palmitoyl glyceride (OPO) using an enzymatic esterification reaction, which can increase the overall yield while maintaining excellent purity of the target product.

The present invention has as its technical object the provision of a method for producing triglycerides (e.g., 1,3-dioleoyl-2-palmitoyl glycerides (OPO)) through an enzymatic esterification reaction in which an acyl group of a triacylglyceride (e.g., tripalmitoyl glyceride (PPP)) is exchanged with another acyl group (e.g., an oleyl group), while maintaining excellent purity of the target product and increasing the overall yield.

According to one aspect of the present invention, a method for producing a triglyceride is provided, comprising: (1) performing an enzymatic esterification reaction on a reactant mixture comprising a triacylglyceride and a fatty acid having an acyl group different from an acyl group in the triacylglyceride to exchange an acyl group of the triacylglyceride with an acyl group of the fatty acid; and (2) performing a wet fractionation step on the resulting mixture of the enzymatic esterification reaction obtained in step (1), wherein the wet fractionation is performed using an organic solvent that is a mixture of acetone and hexane.

According to another aspect of the present invention, a method for producing a triglyceride is provided, comprising: (1) performing an enzymatic esterification reaction on a reactant mixture comprising a triacylglyceride and a fatty acid having an acyl group different from an acyl group in the triacylglyceride to exchange an acyl group of the triacylglyceride with an acyl group of the fatty acid; (2) distilling the resulting mixture of the enzymatic esterification reaction obtained in step (1); and (3) wet fractionating the distillation residue of step (2), wherein the wet fractionation is performed using an organic solvent that is a mixture of acetone and hexane.

According to the present invention, in producing a triglyceride (e.g., 1,3-dioleoyl-2-palmitoyl glyceride (OPO)) in which an acyl group of a triacylglyceride is exchanged with another acyl group through an enzymatic transesterification reaction, the purity of the target product can be excellently maintained while increasing the overall yield.

Hereinafter, the present invention will be described in detail.

The triacylglyceride used as a reactant in the enzymatic esterification reaction in the present invention may be an edible fat.

As the edible fat, any suitable edible fat may be used, including liquid or solid vegetable fat or animal fat, hardened oil or ester exchange oil of the fat, liquid oil obtained by fractionating the fat, solid fat, etc.

Specific examples of the above vegetable oils include corn oil, rapeseed oil, soybean oil, cottonseed oil, palm oil, coconut oil, rice bran oil, sesame oil, cocoa fat, olive oil, and combinations thereof, and specific examples of the above animal oils include fish oil, lard, beef tallow, chicken fat, and combinations thereof. These edible oils may be used alone or in combination of two or more.

In one specific example, the triacylglyceride may be fractionated palm oil obtained by fractionating palm oil among vegetable oils.

In one specific embodiment, the acyl group present in the triacylglyceride may be independently selected from a saturated or unsaturated straight-chain aliphatic acyl group having 4 to 24 carbon atoms, more specifically 12 to 22 carbon atoms, and even more specifically 14 to 20 carbon atoms. For example, the acyl group present in the triacylglyceride may be independently selected from a palmitoyl group, a stearoyl group, an oleoyl group, and a linoleoyl group, and is preferably, but not limited to, a palmitoyl group.

In one embodiment, at least one of the acyl groups present in the triacylglyceride is a palmitoyl group, preferably two, more preferably all three are palmitoyl groups. That is, in the most preferred embodiment, the starting triacylglyceride is tripalmitoylglyceride (PPP).

The fatty acid used as a reactant in the enzymatic esterification reaction in the present invention has an acyl group different from the acyl group in the triacylglyceride.

In one specific example, the fatty acid may be at least one selected from a saturated or unsaturated straight-chain fatty acid having 4 to 24 carbon atoms, more specifically 12 to 22 carbon atoms, and even more specifically 14 to 20 carbon atoms. More specifically, the fatty acid may be selected from the group consisting of palmitic acid, stearic acid, oleic acid, linoleic acid, and combinations thereof, and is preferably oleic acid, but is not limited thereto.

In the method for producing triglyceride of the present invention, the mixing ratio of triacylglyceride to fatty acid included in the reaction mixture of the enzymatic esterification reaction may be 1:1.1 or more, 1:1.2 or more, or 1:1.3 or more by volume, and may also be 1:2 or less, 1:1.95 or less, or 1:1.9 or less.

In the present invention, the reaction temperature of the enzymatic esterification reaction may be determined depending on the type of enzyme used, and specifically, may be 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, or 60°C or higher, and may also be 85°C or lower, 80°C or lower, 75°C or lower, 70°C or lower, or 65°C or lower, but is not limited thereto. In order to optimize the effect of the reaction and the efficiency of the process, the reaction temperature may preferably be 40°C to 80°C, and more preferably 45°C to 70°C.

In the present invention, the reaction time of the enzymatic esterification reaction may be, for example, 0.5 hours or more, 1 hour or more, 2 hours or more, 3 hours or more, or 4 hours or more, and may also be 12 hours or less, 10 hours or less, 8 hours or less, or 6 hours or less, but is not limited thereto.

In the present invention, known enzymes can be used as enzymes for the enzymatic esterification reaction without any particular limitation. Specifically, Candida Antarctica B , Thermomyces lanuginosus lanuginose ), Rhizomucour miehei , Rhizopus delemar , Mucormiehei , Alcaligenes sp . , Aspergillus niger , Candida cylindrase cylindraces ), Burkholderia cepacia , Geotricum candidum ) and combinations thereof. Esterifying enzymes derived from microorganisms selected from the group consisting of, but not limited to,

In one specific embodiment, 100 to 3000 parts by weight, or 100 to 2000 parts by weight, or 100 to 1000 parts by weight of the reactant mixture per 1 part by weight of the enzyme can be continuously treated without replacing the enzyme, and a higher amount can be continuously treated as long as the activity of the enzyme is maintained. For example, as long as the enzyme activity is appropriate, 500 parts by weight or more, 1000 parts by weight or more, 2000 parts by weight or more, or 3000 parts by weight or more of the reactant mixture per 1 part by weight of the enzyme can be continuously treated without replacing the enzyme, and there is no particular limitation on the upper limit, and even 10000 parts by weight or more can be treated. According to a preferred specific embodiment, the amount of the reactant mixture treated per 1 part by weight of the enzyme can be 1000 to 3000 parts by weight.

In one specific embodiment, the enzymatic transesterification reaction can be performed continuously. That is, the enzymatic transesterification reaction can be performed by continuously introducing a reactant mixture including a triacylglyceride as a starting material and a fatty acid having an acyl group different from the acyl group in the triacylglyceride at a constant flow rate into a continuous reactor in which an enzyme is present using a metering pump, and the product after the reaction exits the reactor and is collected in a tank. Here , the flow rate of the reaction mixture may have an SV of 0.05 to 2.0, more specifically, 0.1 to 1.5, and even more specifically, 0.2 to 1.0, but is not necessarily limited thereto.

In one specific embodiment, the triglyceride prepared according to the above method may be dioleoyl monopalmitoyl glyceride, and more specifically, 1,3-dioleoyl-2-palmitoyl glyceride (OPO).

A method for producing triglycerides according to a first aspect of the present invention comprises a step of wet fractionating a mixture resulting from the enzymatic esterification reaction, wherein the wet fractionation is characterized in that it is performed using an organic solvent that is a mixture of acetone and hexane. Using a mixture of acetone and hexane as an organic solvent for wet fractionation can increase the fractionation yield compared to using acetone or hexane alone.

In one specific example, the mixing ratio of acetone and hexane (acetone:hexane) used in the wet fractionation may be 1:99 to 99:1, 5:95 to 95:5, or 10:90 to 90:10 by weight.

More specifically, in terms of improving the wet fractionation yield, the hexane content in 100 wt% of the mixture of acetone and hexane used for wet fractionation may be 10 wt% or more, 15 wt% or more, 20 wt% or more, 25 wt% or more, or 30 wt% or more, and may also be 90 wt% or less, 85 wt% or less, or 80 wt% or less.

Alternatively, in terms of purification effect, the hexane content in 100 wt% of the mixture of acetone and hexane used for wet fractionation may be 3 wt% or more, 5 wt% or more, 8 wt% or more, or 10 wt% or more, and may also be 50 wt% or less, 40 wt% or less, or 30 wt% or less.

In one specific example, during the wet fractionation, the mixing ratio of the reaction result mixture to the organic solvent (i.e., the mixture of acetone and hexane) (reaction result mixture:organic solvent) may be 1:0.5 or more, 1:1 or more, or 1:1.5 or more by weight, and may also be 1:10 or less, 1:5 or less, or 1:3 or less.

In one specific example, during the wet fractionation, the temperature condition may be -5°C or lower, -10°C or lower, or -15°C or lower, and may also be -30°C or higher, -25°C or higher, or -20°C or higher.

In one specific example, the wet fractionation time may be 30 minutes or more, 40 minutes or more, or 50 minutes or more, and may also be 180 minutes or less, 150 minutes or less, or 120 minutes or less.

According to a second aspect of the present invention, a method for producing a triglyceride is provided, comprising: (1) performing an enzymatic esterification reaction on a reactant mixture comprising a triacylglyceride and a fatty acid having an acyl group different from an acyl group in the triacylglyceride to exchange an acyl group of the triacylglyceride with an acyl group of the fatty acid; (2) distilling the resulting mixture of the enzymatic esterification reaction obtained in step (1); and (3) wet fractionating the distillation residue of step (2), wherein the wet fractionation is performed using an organic solvent that is a mixture of acetone and hexane.

In the method for producing triglyceride according to the second aspect of the present invention, specific details regarding triacylglyceride, fatty acid having an acyl group different from the acyl group in the triacylglyceride, enzymatic esterification reaction, and wet fractionation are as described above.

In one specific example, the distillation of the enzymatic esterification reaction product may be performed in a short path distillator, more specifically, an internal condenser type short path distillator.

In one specific example, the pressure condition during the distillation may be 0.1 mbar or less, 0.09 mbar or less, 0.08 mbar or less, or 0.07 mbar or less, and may also be 0.01 mbar or more, 0.03 mbar or more, or 0.05 mbar or more.

In one specific example, the temperature conditions during the distillation may be 200°C or higher, 205°C or higher, 210°C or higher, or 215°C or higher, and may also be 245°C or lower, 240°C or lower, 235°C or lower, or 230°C or lower.

In one specific example, the feeding rate of the enzymatic esterification reaction product during the distillation may be 20 to 40 g/h, and more specifically, 20 to 35 g/h, 25 to 35 g/h, or 30 to 35 g/h.

In one specific example, the rotation speed of the distillation roller during the distillation may be 300 to 500 rpm, and more specifically, 300 to 480 rpm, 350 to 480 rpm, or 350 to 450 rpm.

In one specific example, the cooling temperature of the distillate during the distillation may be 10 to 30°C, and more specifically, 15 to 25°C, 18 to 25°C, or 18 to 22°C.

In one specific example, the result obtained according to the present invention may be a triglyceride composition having a content of neutral lipid (C52) having a total carbon number of 52 in the neutral lipid of 50 wt% or more, and more specifically, the C52 content may be 52 wt% or more, 54 wt% or more, or 56 wt% or more. There is no particular limitation on the upper limit of the C52 content, and for example, it may be 80 wt% or less, 75 wt% or less, or 70 wt% or less.

In one specific embodiment, the result obtained according to the present invention may be a triglyceride composition having a content of neutral lipids in the unsaturated-saturated-unsaturated (USU) form of 28 wt% or more, and more specifically, the USU content may be 30 wt% or more, 32 wt% or more, or 34 wt% or more. There is no particular limitation on the upper limit of the USU content, and for example, it may be 60 wt% or less, 55 wt% or less, or 50 wt% or less.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are provided solely to aid understanding of the present invention and are not intended to limit the scope of the present invention.

[ Example ]

Manufacturing example 1

A 1:2 volume ratio mixture of tripalmitoyl glyceride (PPP, super stearin (SS)) and oleic acid (OA) was prepared.

The prepared reaction mixture was continuously fed at a constant flow rate (28.8 mL/h, SV=0.2) into a continuous reactor containing an enzyme (Novozym 40086, an enzyme derived from Rhizomucour miehei) using a metering pump, thereby performing an enzymatic transesterification reaction. The reaction temperature was maintained at 60°C.

Thereafter, the above reaction product was fed into a single-path distiller (SPD) (KDL 1 Basic with option B, UIC) with an internal pressure set to 5.0~7.0Х10 ^{- 2} mbar and an internal temperature set to 225°C at a feeding rate of 32 g/h, and distilled under the conditions of a roller speed of 400 rpm and a cooling temperature of 20°C to remove by-products generated after the enzymatic reaction and residual fatty acids.

The distillation residue was wet fractionated using an organic solvent having the composition shown in Table 1 below (distillation residue:solvent ratio = 1:2 (g/g), fractionation temperature: -15°C, fractionation time: 1 hour). That is, the distillation residue was mixed with the organic solvent in a media bottle to completely dissolve it, placed in a circulator set to -15°C, taken out after 1 hour, membrane filtered, and the filtrate was concentrated under reduced pressure to remove the solvent.

The yield of the above wet fractionation is shown in Table 2 below.

[Table 1]

[Table 2]

The compositions of the above wet fractionated results #2 to #6 were analyzed using the following method, and the results are shown in Tables 3 and 4 below, along with the analysis results of the starting materials before fractionation.

1) MAG/ DAG Content and free fatty acid content

It was analyzed using gas chromatography.

2) Neutral lipids Carbon number analyze

Neutral lipids were separated by carbon number using gas chromatography, and the concentration of C52 (neutral lipids with a total carbon number of 52 (e.g., C18-C16-C18, C16-C18-C18, etc.), such as SPS, SSP, OPO, OOP, LPL, LLP, OPL, etc.) was analyzed.

3) Isomer analysis

Neutral lipids in the unsaturated-saturated-unsaturated (USU) form were separated using liquid chromatography, and their concentrations were analyzed. USU is primarily composed of OPO, and analysis was performed to distinguish it from SUU, which contains POO.

[Table 3]

[Table 4]

From the above Tables 2 to 4, it was confirmed that when the result of the enzymatic esterification reaction according to the present invention is purified by wet fractionation using an organic solvent that is a mixture of acetone and hexane, the purity of the target product can be maintained excellently while increasing the overall yield.

Claims (10)

(1) a step of performing an enzymatic esterification reaction on a reactant mixture including a triacylglyceride and a fatty acid having an acyl group different from the acyl group in the triacylglyceride to exchange the acyl group of the triacylglyceride with the acyl group of the fatty acid; and
(2) a step of wet fractionating the resulting mixture of the enzymatic esterification reaction obtained in step (1);
The above wet fractionation is performed using an organic solvent which is a mixture of acetone and hexane.
Method for producing triglycerides. A method for producing triglyceride, wherein the mixing ratio (acetone:hexane) of acetone and hexane used for wet fractionation in the first paragraph is 1:99 to 99:1 by weight. A method for producing triglyceride in claim 1, wherein, in wet fractionation, the mixing ratio of the reaction result mixture to the organic solvent (reaction result mixture: organic solvent) is 1:0.5 to 1:10 by weight, the temperature condition is -30°C to -5°C, and the fractionation time is 30 minutes to 180 minutes. (1) A step of performing an enzymatic esterification reaction on a reactant mixture including a triacylglyceride and a fatty acid having an acyl group different from the acyl group in the triacylglyceride to exchange the acyl group of the triacylglyceride with an acyl group of the fatty acid;
(2) a step of distilling the resulting mixture of the enzymatic esterification reaction obtained in step (1); and
(3) a step of wet fractionating the distillation residue of step (2);
The above wet fractionation is performed using an organic solvent which is a mixture of acetone and hexane.
Method for producing triglycerides. A method for producing triglycerides, wherein distillation is performed in a short path distillator in claim 4. A method for producing triglyceride in claim 4, wherein the pressure condition during distillation is 0.01 mbar to 0.1 mbar and the temperature condition is 200°C to 245°C. A method for producing triglyceride, wherein the mixing ratio of acetone and hexane (acetone:hexane) used for wet fractionation in the fourth paragraph is 1:99 to 99:1 by weight. A method for producing triglyceride in claim 4, wherein, in wet fractionation, the mixing ratio of the reaction result mixture to the organic solvent (reaction result mixture: organic solvent) is 1:0.5 to 1:10 by weight, the temperature condition is -30°C to -5°C, and the fractionation time is 30 minutes to 180 minutes. A method for producing a triglyceride according to any one of claims 1 to 8, wherein the triacylglyceride is tripalmitoylglyceride and the fatty acid is oleic acid. A method for producing a triglyceride according to any one of claims 1 to 8, wherein the produced triglyceride is dioleoyl monopalmitoyl glyceride.