KR20190140517A - Method for improving efficiency of enzymatic esterification by addition of ethanol - Google Patents

Abstract

The present invention relates to a method for improving efficiency of enzymatic esterification, and more specifically, to a method capable of improving overall process efficiency and economic feasibility. To this end, ethanol is added to a mixture of reaction starting materials in performing a reaction in which an acyl group of triacylglyceride is exchanged with other acyl groups using an enzyme. Therefore, compared to a case where no ethanol is added, reduction of enzyme activity due to progress of the reaction can be prevented.

Description

Method for improving efficiency of enzymatic esterification by addition of ethanol

The present invention relates to a method for improving the efficiency of enzymatic esterification, and more particularly, in the reaction of exchanging acyl groups of triacylglycerides with other acyl groups using enzymes, ethanol is added to a mixture of reaction starting materials. The present invention relates to a method of preventing the reduction of enzyme activity caused by the progress of the reaction by further adding ethanol, thereby further improving the overall process efficiency and economic efficiency.

Triacylglycerides fats and oils are very widely used in the food industry, among which 1,3-dioleoyl-2-palmitoyl glycerides (OPO) are breast milk fats. As an important component of nutritional research has been conducted a lot.

Fat compositions containing the major fatty acids that predominantly make up breastmilk fats are found a lot in oils and fats of plant origin, but almost all plant origin glycerides do not have saturated fatty acids in the 2-position. In contrast, palmitic acid, a significant amount of saturated fatty acids in breast milk, occupies the 2-position of glycerides.

Recently, as people's interest in infant health increases, the demand for foods similar to breast milk is increasing.

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

However, conventional milk powder is a mixture of powdered milk and vegetable oil, and contains little palmitic acid at the 2-position when analyzing the oil component of the milk powder.

Accordingly, the application of a high quality breast milk substitute for the addition of 1,3-dioleoyl-2-palmitoyl glyceride (OPO) containing palmitic acid at 2-position, which is similar in composition to breast milk fat, in milk powder. This is increasing.

Mohamad, et al (Lipase-catalyzed interesterification reactions for human milk fat substitutes production, 2013, 115, 270-285), substituted 1,3-positions of the triacylglycerides with lipase. The study of synthesizing dioleoyl-2-palmitoyl glycerides has been mainly conducted.

Due to the 1,3-position specific reaction properties of the triacylglycerides of lipases, the provision of a glyceride backbone containing saturated fatty acids at the 2-position of the triacylglyceride fats is possible with 1,3-dioleoyl-2-palmi. Important in synthesizing soil glycerides (OPO).

While these prior arts utilize enzymatic esterification reactions, the enzyme activity decreases with the progress of the reaction, resulting in lower overall process efficiency. Therefore, in the enzymatic esterification reaction, a method for preventing the decrease of the enzyme activity according to the progress of the reaction and producing the OPO with higher efficiency than the existing 1,3-dioleoyl-2-palmitoyl glyceride (OPO) production method Development is being requested.

It is a technical object of the present invention to provide a method capable of improving the overall process efficiency and economic efficiency by preventing a decrease in the enzyme activity due to the progress of the reaction in the enzymatic esterification reaction.

In order to achieve the above technical problem, the present invention, in carrying out the reaction for the exchange of acyl groups of triacylglycerides with other acyl groups using enzymes, by reducing the enzyme activity according to the progress of the reaction by adding ethanol to the mixture of the reaction starting materials An enzymatic esterification method is provided, which is characterized in that it is prevented.

According to another aspect of the present invention, there is provided a reaction mixture comprising (1) a triacylglyceride as a reaction starting material and a fatty acid having an acyl group different from an acyl group in the triacylglyceride, to which ethanol is added, And (2) exchanging the acyl group of the triacylglyceride with an acyl group of the fatty acid by performing an enzymatic esterification reaction on the reaction mixture to which ethanol is added, obtained in step (1). A method is provided.

According to another aspect of the present invention, an enzymatic esterification reaction comprising a fatty component comprising triacylglycerides and a fatty acid having an acyl group different from an acyl group in the triacylglycerides, to which ethanol is added As a result, a fat composition is provided, wherein the acyl group of the triacylglyceride comprises triglyceride in which the acyl group of the fatty acid is exchanged.

According to another aspect of the present invention, an enzymatic esterification reaction comprising a fatty component comprising triacylglycerides and a fatty acid having an acyl group different from an acyl group in the triacylglycerides, to which ethanol is added A milk powder composition is prepared, comprising triglycerides, wherein the acyl groups of the triacylglycerides are exchanged with acyl groups of the fatty acids.

According to the present invention, in carrying out a reaction for exchanging acyl groups of triacylglycerides with other acyl groups by using an enzyme, it is possible to excellently prevent the decrease of the enzyme activity with the progress of the reaction, and as a result, overall process efficiency and economy Can be improved. Thus, according to an embodiment of the present invention, it is possible to produce dioleoyl monopalmitoyl glycerides from tripalmitoylglycerides with high efficiency and economically through enzymatic esterification reaction.

1 is a graph showing the C52 concentration change according to the reaction time according to the amount of ethanol in the enzymatic esterification reaction (Set 1: Novozym 40086 used, 55 ℃) carried out in the embodiment of the present invention.
2 is a graph showing the change in C52 concentration according to the reaction time according to the amount of ethanol in the enzymatic esterification reaction (Set 2: using Lipozyme TL IM, 60 ° C.) performed in the embodiment of the present invention.
Figure 3, in the enzymatic esterification reaction (Set 1: Novozym 40086, 55 ℃) carried out in the embodiment of the present invention, after the reaction for 4 hours, sn-2 palmitic acid (PA) ratio according to the amount of ethanol added A graph showing the change in.
Figure 4 in the enzymatic esterification reaction (Set 2: using Lipozyme TL IM, 60 ℃) carried out in the embodiment of the present invention, after the reaction for 4 hours, sn-2 palmitic acid (PA) according to the amount of ethanol added It is a graph showing the change of ratio.
5 is a graph showing the change in the USU structure ratio according to the amount of ethanol added after the reaction for 4 hours in the enzymatic esterification reaction (Set 1: Novozym 40086 used, 55 ℃) carried out in the embodiment of the present invention.
FIG. 6 is a graph showing the change in the USU structure ratio according to the amount of ethanol added after 4 hours in the enzymatic esterification reaction (Set 2: using Lipozyme TL IM, 60 ° C.) carried out in the Examples of the present invention. .

Hereinafter, the present invention will be described in detail.

Triacylglycerides used as starting materials for enzymatic esterification in the present invention may be edible oils and fats.

The edible fats and oils can be used as long as it is suitable for edible oils such as vegetable oils or animal fats of liquids or solids, cured oils or transesterified oils of the fats and oils, liquid oils obtained by fractionating the fats and oils and solid fats.

Specific examples of the vegetable oil and fat include corn oil, rapeseed oil, soybean oil, cottonseed oil, palm oil, palm oil, rice bran oil, sesame oil, cacao fat, olive oil and combinations thereof. Specific examples of the animal oil and fat include fish oil, And pork fat, beef fat (beef oil), fat of chicken, and a combination thereof. These edible fats and oils can be used individually or in combination of 2 or more.

In one embodiment, the triacylglycerides may be palm fractionation oil that is fractionated palm oil in vegetable oils and fats.

In one embodiment, the acyl groups present in the triacylglycerides are independently saturated or unsaturated having 4 to 24 carbon atoms, more specifically 12 to 22 carbon atoms, even more specifically 14 to 20 carbon atoms. It may be selected from linear aliphatic acyl groups of. For example, the acyl group present in the triacylglyceride may be independently selected from palmitoyl group, stearoyl group, oleoyl group and linoleoyl group, preferably palmitoyl group, but is not limited thereto.

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

The fatty acids used as other starting materials of the enzymatic esterification reaction in the present invention have an acyl group different from the acyl group in the triacylglyceride.

In one embodiment, the fatty acid is selected from saturated or unsaturated straight chain fatty acids having 4 to 24 carbon atoms, more specifically 12 to 22 carbon atoms, even more specifically 14 to 20 carbon atoms. You can use more than one. More specifically, the fatty acid may be selected from the group consisting of palmitic acid, stearic acid, oleic acid, linoleic acid, and combinations thereof, preferably oleic acid, but is not limited thereto.

The mixing ratio of the starting material triacylglycerides to fatty acids may be 1: 1 to 1: 7 by weight, more specifically 1: 1 to 1: 5, and even more specifically 1: 1 to 1: May be three.

In the present invention, ethanol is added to the mixture containing the triacylglyceride and the fatty acid as starting materials of the enzymatic esterification reaction in order to prevent the decrease in the enzyme activity as the reaction proceeds.

The amount of ethanol added to the reaction mixture may be specifically determined according to the type and ratio of the starting materials, the reaction conditions such as the reaction temperature, the type of the enzyme used, and the like within the range capable of preventing the reduction of enzyme activity. .

For example, the amount of ethanol added to the reaction mixture may be 5 wt% or less, or 4 wt% or less, or 3 wt% or less, or 2 wt% or less, based on the total weight of the reaction mixture. More specifically, the ethanol in 100% by weight of the reaction mixture is at least 0.01%, or at least 0.05%, or at least 0.1%, or at least 0.2%, or at least 0.3%, or at least 0.4% or or 0.5 May be included in an amount of at least 5% by weight, and may also be included in an amount of 5% by weight, 4.5% by weight, 4% by weight, 3.5% by weight, 3% by weight, 2.5% by weight, or 2% by weight or less. It is not limited to this. In a preferred embodiment, the amount of ethanol added to the reaction mixture may be 0.1 to 4.5% by weight, more preferably 0.5 to 4% by weight, based on the total weight of the reaction mixture.

In the present invention, the reaction temperature of the enzymatic esterification reaction may be determined according to 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. 85 ° C. or less, 80 ° C. or less, 75 ° C. or less, 70 ° C. or less, or 65 ° C. or less, but is not limited thereto. In one embodiment, the reaction temperature of the enzymatic esterification reaction may preferably be from 40 ° C. to 80 ° C., more preferably from 55 ° C. to 75 ° C.

As the enzyme of the enzymatic esterification reaction in the present invention, known enzymes can be used without particular limitation. Specifically, Candida Antarctica B , Thermomyces ranuginosus ( Thermomyces) lanuginose , Rhizomucour miehei , Rhizopus delemar , Mucormiehei , Alcaligenes sp . , Aspergillus niger , Candida cilindrases cylindraces ), Burkholderia cepacia , Geotricum candidum ) and esterification enzymes derived from microorganisms selected from the group consisting of, but are not limited thereto.

According to the present invention, it is possible to effectively prevent the decrease of the enzyme activity during the enzymatic esterification reaction, so that a large amount of starting material can be continuously processed without changing the enzyme. In one embodiment, 100 parts by weight to 3000 parts by weight, or 100 parts by weight to 2000 parts by weight, or 100 parts by weight to 1000 parts by weight of the reaction mixture per 1 part by weight of the enzyme may be continuously processed without replacing the enzyme, but the activity of the enzyme As long as it is retained, further amounts may continue to be processed. For example, if the enzyme activity is appropriate, the reaction mixture can be continuously treated per 500 parts by weight of enzyme, at least 500 parts by weight, at least 1000 parts by weight, at least 2000 parts by weight or at least 3000 parts by weight, without replacing the enzyme, and the upper limit has no special limitation. And even 10000 parts by weight or more can be processed. According to one preferred embodiment, the reaction mixture throughput per 1000 parts by weight of enzyme may be 1000 parts by weight to 3000 parts by weight.

According to another aspect of the present invention, there is provided a reaction mixture comprising (1) a triacylglyceride as a reaction starting material and a fatty acid having an acyl group different from an acyl group in the triacylglyceride, to which ethanol is added, And (2) exchanging the acyl group of the triacylglyceride with an acyl group of the fatty acid by performing an enzymatic esterification reaction on the reaction mixture to which ethanol is added, obtained in step (1). A method is provided.

In the above production method, as the reaction starting materials, triacylglycerides and fatty acids, the amount of ethanol added to the reaction mixture, and the enzymatic esterification reaction are as described above.

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

Also in one embodiment, in the ratio of neutral lipid (USU) in the form of unsaturated fatty acid (U) in the sn-1,3 position and saturated fatty acid (S) in the sn-2 position, prepared without the addition of ethanol The relative ratio of USU ratio of triglycerides to USU ratio of triglycerides prepared according to the process of the present invention may be at least 1.05, more preferably at least 1.1, even more preferably at least 1.2 (eg, 1.2 to 4).

The acid value of the triglyceride prepared according to the production method of the present invention is 0.6 or less (for example, 0.1 to 0.6), and the peroxide value is characterized in that 3.0 or less (for example, 0.1 to 3.0).

Here, the number of mg of KOH required to neutralize the free fatty acid contained in 1 g of fats and oils is referred to as an 'acid value', and the milli equivalents of peroxide oxygen converted from milli equivalents of iodine free to 1 kg of fats and oils is referred to as a 'peroxide value'.

The acid value of the triglyceride prepared according to the production method of the present invention may be 0.1 to 0.6, more preferably 0.2 to 0.6, even more preferably 0.38 to 0.58. The peroxide value of the triglyceride prepared according to the production method may be 0.1 to 3.0, more preferably 0.15 to 3.0, even more preferably may be 0.2 to 3.0. The production method according to the present invention effectively reduces the acid value and peroxide value in the fats and oils to ensure the food safety of the fats for edible oil, by using the fats and oils as a food additive or a supplement additive of cooking to increase the taste and flavor of food Can increase nutritional value.

In one embodiment, the ratio of the neutral lipid (PPP, PPO, OPO, etc.) having palmitic acid in the sn-2 position in the triglyceride prepared according to the production method of the present invention may be 50 to 90% And more preferably 50 to 80%.

According to another aspect of the present invention, an enzymatic esterification reaction comprising a fatty component comprising triacylglycerides and a fatty acid having an acyl group different from an acyl group in the triacylglycerides, to which ethanol is added As a result, a fat composition is provided, wherein the acyl group of the triacylglyceride comprises triglyceride in which the acyl group of the fatty acid is exchanged.

Further, according to another aspect of the present invention, a fatty acid component comprising triacylglycerides and a fatty acid having an acyl group different from an acyl group in the triacylglyceride, wherein the reaction mixture is added to the enzymatic ester There is provided a milk powder composition comprising triglycerides, wherein the acyl groups of the triacylglycerides produced by the reaction are exchanged with acyl groups of the fatty acids.

In the fat composition and the milk powder composition, the triacylglycerides and fatty acids, the ethanol addition amount in the reaction mixture, the enzymatic esterification reaction, and the triglyceride are as described above.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, this is only provided to help the understanding of the present invention, the scope of the present invention is not limited thereto.

[ Example ]

As a substrate, 25 g of a 1: 2 weight ratio mixture of tripalmitoylglycerides (PPP, superstearin) to oleic acid (OA) was added to a 100 mL Erlenmeyer flask, where ethanol was respectively 0 (comparative), 0.5 to the total weight of the substrate. , 1, 1.5, 2, 3 and 4% by weight.

Enzyme (Set 1: Novozym 40086, Set 2: Lipozyme TL IM) was separately weighed in an amount of 2.5 g (10% by weight of the substrate), and then added to the mixture of the substrate and ethanol immediately before the reaction.

The enzymatic esterification reaction was carried out at a stirring speed of 150 rpm in a shaking incubator in which the temperature (Set 1: 55 ° C., Set 2: 70 ° C.) was set for each experimental set. 2, 3 and 4 hours) elapsed time was analyzed by taking 1 mL of the upper portion of the reaction mixture. The analysis method is as follows.

1) Neutral lipid Carbon number  analysis

Neutral lipids are separated by carbon number using gas chromatography, and neutral lipids containing 52 carbon atoms (e.g., C18-C16-C18, C16-C18-C18, etc.) such as SPS, SSP, OPO, OOP , LPL, LLP, OPL, etc.) were analyzed. The results are shown in Table 1 below.

In addition, as an indicator showing the effect of preventing the reduction of enzyme activity, the trend line slope (R ^2r value) of the C52 concentration change compared to the reaction time was calculated and shown in Table 2 for each ethanol addition amount. The larger the value of the trend line slope, the better the effect of preventing a decrease in enzyme activity.

2) sn Fatty acid composition analysis at -2 position

Four hours after the start of the reaction, the fatty acid at the sn-2 position was analyzed by gas chromatography, and the ratio of neutral lipids (PPP, PPO, OPO, etc.) having palmitic acid at the sn-2 position was analyzed. It was confirmed. The results are shown in Table 3 below.

3) USU  Ratio analysis

4 hours after the start of the reaction, the structure of the neutral lipid was analyzed using HPLC (detector: ELSD, column: silver ion column) and saturated at unsaturated fatty acid (U) and sn-2 at sn-1,3 position. The proportion of neutral lipids (USU) in which the fatty acid (S) is located (usually expected to be OPO) was identified. The results are shown in Table 4 below.

In addition, the relative ratio of the ratio of neutral lipid (USU) at the time of ethanol addition to the ratio of neutral lipid (USU) at the time of no ethanol addition was calculated, and the ethanol addition amount is shown in Table 5 below.

In addition, from the following results, the change in C52 concentration according to the reaction time for each ethanol addition amount is shown in Figure 1 (Set 1) and Figure 2 (Set 2), after 4 hours of reaction, sn-2 palmitic acid ( 3 (Set 1) and 4 (Set 2), the change in the ratio of PA, and after 4 hours of reaction, changes in the ratio of USU structure according to the amount of ethanol added in Figure 5 (Set 1) and Figure 6 (Set 2) ).

Meanwhile, the C52 concentration of the reaction mixture was analyzed for each time period while performing the same enzymatic esterification reaction (Novozym 40086) by adding no alcohol component and other lower alcohols (1% and 2% of methanol and propanol, respectively). The reaction time to reach the target C52 concentration (40%) was measured. The results are shown in Table 6 below with the results obtained when ethanol was added (1% and 2%).

In addition, by adding no alcohol component and other lower alcohols (1% each of methanol and propanol), an enzymatic esterification reaction (Novozym 40086) was performed for 4 hours, and the fatty acid at the sn-2 position was analyzed by type, sn The proportion of neutral lipids (PPP, PPO, OPO, etc.) having palmitic acid at the −2 position was confirmed. The results are shown in Table 7 below with the results obtained when ethanol was added (1%).

As can be seen from Tables 1 to 5 and FIGS. 1 to 6, as the ethanol content in the reaction starting material mixture increased, the initial synthesis rate of C52 increased, and after 4 hours of reaction, the degree of USU synthesis increased, Novozym The same trend was observed for both 40086 and Lipozyme TL IM. In particular, in the case of Novozym 40086, the time taken for the C52 content to reach 41% was reduced to less than half when 0.5% was added (1.5h) compared to ethanol-free (4h), and in the case of Lipozyme TL IM, The time to reach 30% was halved with 1% addition (2h) compared to no ethanol addition (4h). That is, when ethanol was added to the starting material mixture of the enzymatic esterification reaction according to the present invention, it was possible to significantly prevent the decrease of the enzyme activity due to the progress of the reaction, as compared with the comparative example without the addition of ethanol, and as a result It can be seen that the process efficiency and economics were improved.

As can be seen from Table 6, when ethanol was added to the starting material mixture of the enzymatic esterification reaction according to the present invention, no alcohol component was added or other lower alcohols such as methanol and propanol were added in the same amount. Compared to the above, the reaction (process) time required to reach the same target C52 concentration (40%) was reduced to 1/4.

In addition, as can be seen from Table 7, the ratio of the neutral lipid having palmitic acid at the sn-2 position in the resulting fat obtained by adding ethanol to the starting material mixture of the enzymatic esterification reaction according to the present invention, It was on the same level as when not added, and was significantly higher than when other lower alcohols, methanol and propanol, were added in the same amount.

Thus, according to the present invention, it has been confirmed that dioleoyl monopalmitoyl glyceride from tripalmitoylglyceride can be produced with high efficiency and economically through enzymatic esterification reaction.

Claims (17)

In carrying out a reaction for exchanging acyl groups of triacylglycerides with other acyl groups using an enzyme, enzymatic esterification, characterized in that ethanol is added to a mixture of reaction starting materials to prevent a decrease in enzymatic activity due to the progress of the reaction. Way. The method of claim 1, wherein the reaction starting material is a triacylglyceride and a fatty acid having an acyl group different from the acyl group in the triacylglyceride. 3. The acyl group present in triacylglycerides is independently selected from saturated or unsaturated linear aliphatic acyl groups having 4 to 24 carbon atoms, and the fatty acid is saturated or having 4 to 24 carbon atoms. At least one selected from unsaturated linear fatty acids. The acyl group present in the triacylglyceride is independently selected from palmitoyl group, stearoyl group, oleoyl group and linoleoyl group, and fatty acids are palmitic acid, stearic acid, oleic acid, linoleic acid and Enzymatic esterification process, selected from the group consisting of a combination thereof. The method of claim 2, wherein the triacylglyceride is tripalmitoylglyceride and the fatty acid is oleic acid. The method of claim 1, wherein up to 5% by weight of ethanol is added based on the total weight of the mixture of reaction starting materials. The method of claim 1, wherein the throughput of the reaction starting material mixture per 1000 parts by weight of enzyme is 1000 parts by weight to 3000 parts by weight. (1) providing a reaction mixture comprising triacylglycerides and fatty acids having an acyl group different from the acyl group in the triacylglycerides, to which ethanol is added, and
(2) performing an enzymatic esterification reaction on the reaction mixture to which ethanol is added in step (1) to exchange acyl groups of the triacylglycerides with acyl groups of the fatty acids;
Method for preparing triglycerides. 9. The acyl group present in the triacylglycerides is independently selected from saturated or unsaturated linear aliphatic acyl groups having 4 to 24 carbon atoms, and the fatty acid is saturated or having 4 to 24 carbon atoms. A process for producing triglycerides, wherein the compound is at least one selected from unsaturated linear fatty acids. The acyl group present in the triacylglyceride is independently selected from palmitoyl group, stearoyl group, oleoyl group and linoleyl group, and fatty acids are palmitic acid, stearic acid, oleic acid, linoleic acid and A method for producing triglycerides, selected from the group consisting of combinations thereof. The method for producing triglyceride according to claim 8, wherein the triacylglyceride is tripalmitoylglyceride and the fatty acid is oleic acid. The process for producing triglycerides according to claim 8, wherein up to 5% by weight of ethanol is added based on the total weight of the reaction mixture. The process for producing triglycerides according to claim 8, wherein the throughput of the reaction starting material mixture per 1 part by weight of enzyme is 1000 parts by weight to 3000 parts by weight. The method for producing triglyceride according to claim 8, wherein the triglyceride prepared is dioleoyl monopalmitoyl glyceride. The method of claim 8, wherein in the ratio of neutral lipid (USU) in the form of unsaturated fatty acid (U) at the sn-1,3 position and saturated fatty acid (S) at the sn-2 position, it is prepared without adding ethanol. The relative ratio of the USU ratio of the prepared triglycerides to the USU ratio of triglycerides is 1.05 or more, a method for producing triglycerides. Fatty acid component including triacylglyceride and fatty acid having an acyl group different from acyl group in the triacylglyceride, and as a result of enzymatic esterification reaction of the reaction mixture, to which ethanol is added, of triacylglyceride Fatty composition, wherein the acyl group comprises triglycerides exchanged with the acyl group of the fatty acid. A fatty component comprising a triacylglyceride and a fatty acid having an acyl group different from the acyl group in the triacylglyceride, and prepared by enzymatic esterification of the reaction mixture, to which ethanol is added, of the triacylglyceride Milk powder composition, wherein the acyl group comprises triglycerides exchanged with the acyl group of the fatty acid.

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