KR20190139349A - Method for improving efficiency of enzymatic esterification by pH adjustment of reactant mixture - Google Patents

Abstract

The present invention relates to a method for improving efficiency of enzymatic esterification. More specifically, the present invention relates to a method which can further improve the overall process efficiency and economic feasibility. To this end, the pH of a mixture of reaction starting materials is adjusted to a specific range in performing a reaction in which an acyl group of triacylglyceride is exchanged with other acyl groups using an enzyme. Therefore, reduction of enzyme activity due to progress of the reaction can be prevented.

Description

Method for improving efficiency of enzymatic esterification by pH adjustment of reactant mixture

The present invention relates to a method for improving the efficiency of enzymatic esterification, and more particularly, in performing a reaction for exchanging an acyl group of triacylglyceride with another acyl group using an enzyme, the pH of a mixture of reaction starting materials The present invention relates to a method of improving the overall process efficiency and economic efficiency by preventing the decrease of the enzyme activity according to the progress of the reaction by adjusting the specific range.

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 an enzyme, the reaction proceeds by adjusting the pH of the mixture of the reaction starting materials within the range of 4.5 to 8.5 An enzymatic esterification method is provided, which is characterized by preventing a decrease in enzymatic activity.

According to another aspect of the present invention, there is provided a reaction mixture comprising (1) providing a reaction mixture comprising triacylglycerides and fatty acids having an acyl group different from an acyl group in the triacylglycerides as reaction starting materials; (2) adjusting the pH of the mixture obtained in step (1) within a range of 4.5 to 8.5; And (3) performing an enzymatic esterification reaction on the pH adjusted reaction mixture obtained in step (2) to exchange acyl groups of the triacylglycerides with acyl groups of the fatty acids. This is provided.

According to another aspect of the invention, a reaction mixture comprising a fatty component comprising triacylglycerides and a fatty acid having an acyl group different from an acyl group in the triacylglycerides, the pH of which is adjusted within the range of 4.5 to 8.5 enzymatically As a result of esterification, the fatty composition is provided which contains triglyceride in which the acyl group of the said triacylglyceride was exchanged with the acyl group of the said fatty acid.

According to another aspect of the invention, a reaction mixture comprising a fatty component comprising triacylglycerides and a fatty acid having an acyl group different from an acyl group in the triacylglycerides, the pH of which is adjusted within the range of 4.5 to 8.5 enzymatically There is provided a milk powder composition prepared by esterification, wherein the acyl group of the triacylglyceride comprises triglyceride in which the acyl group of the fatty acid is exchanged.

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 shows the change in product yield (ie, change in enzyme activity) according to the progress of the reaction (based on the amount of enzyme produced per gram of enzyme) in the enzymatic esterification reaction (55 ° C.) performed in the Examples of the present invention. It is a graph for each pH.

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, in order to prevent the enzymatic activity from decreasing with the progress of the enzymatic esterification reaction, the pH of the mixture containing the triacylglyceride and the fatty acid (also referred to as the 'reaction mixture') as a starting material of the reaction is specified. Range, that is, within the range of 4.5 to 8.5. If the pH of the reaction mixture is lower than 4.5 or higher than 8.5, there is a problem that the substrate specificity is lowered while the structure of the enzyme is changed.

More specifically, the pH of the reaction mixture may be 4.5 to 8, even more specifically 5 to 8, even more specifically 5 to 7.5, even more specifically 5 to 7 days Can be.

There is no particular limitation on the method of adjusting the pH of the reaction mixture, and within the range capable of achieving the effect of preventing the decrease of enzyme activity, depending on the type and ratio of starting materials, the type of enzyme used, and the like, which are commonly known in the art. The method can be used appropriately. In one embodiment, the pH may be adjusted by adding an aqueous solution of a suitable base (eg, NaOH, etc.) to the reaction mixture, but is not limited thereto.

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 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, more preferably 45 ° C to 70 ° 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 enzyme can 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) providing a reaction mixture comprising triacylglycerides and fatty acids having an acyl group different from an acyl group in the triacylglycerides as reaction starting materials; (2) adjusting the pH of the mixture obtained in step (1) within a range of 4.5 to 8.5; And (3) performing an enzymatic esterification reaction on the pH adjusted reaction mixture obtained in step (2) to exchange acyl groups of the triacylglycerides with acyl groups of the fatty acids. This is provided.

In the above production method, the triacylglycerides and fatty acids as the reaction starting materials, the pH of 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).

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.

According to another aspect of the invention, a reaction mixture comprising a fatty component comprising triacylglycerides and a fatty acid having an acyl group different from an acyl group in the triacylglycerides, the pH of which is adjusted within the range of 4.5 to 8.5 enzymatically As a result of esterification, the fatty composition is provided which contains triglyceride in which the acyl group of the said triacylglyceride was exchanged with the acyl group of the said fatty acid.

According to another aspect of the present invention, there is also provided a reaction mixture comprising a fatty component comprising triacylglycerides and a fatty acid having an acyl group different from an acyl group in the triacylglycerides, the pH of which is adjusted within the range of 4.5 to 8.5. There is provided a milk powder composition prepared by enzymatic esterification comprising triglycerides in which the acyl group of the triacylglyceride is exchanged with an acyl group of the fatty acid.

In the fat composition and the milk powder composition, the triacylglycerides and fatty acids, the pH of the reaction mixture, the enzymatic esterification reaction, and the triglycerides 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 ]

Prepare a 1: 2 weight ratio mixture (= pH 4) of tripalmitoylglycerides (PPP, superstearin) to oleic acid (OA) as starting materials, and adjust the pH of the mixtures using a 10N NaOH aqueous solution for each experimental set. , 7 and 8, and then homogenized with Homogenizer.

The homogenized reaction mixture was subjected to an enzymatic esterification reaction using a metering pump, continuously at a constant flow rate (SV = 1.3) in a continuous reactor in which the enzyme (Novozym 40086) was present. The reaction temperature was maintained at 55 ° C. After the reaction, the product exited the reactor and collected in the product tank.

The initial reaction mixture (substrate) input compared to the amount of enzyme was three times, and the product sample was taken every time the substrate input was increased by 200 times compared to the initial sample and the amount of enzyme thereafter, and the concentration of C52 was analyzed by gas chromatography. Calculated. The methods used at this time are as follows.

Triglyceride 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.) concentration (%) was analyzed.

The analysis results are shown in Tables 1 and 2 below, and the change in C52 concentration according to substrate throughput (= production compared to enzyme g) for each pH is shown graphically in FIG. 1.

In addition, as an indicator showing the effect of preventing the reduction of enzyme activity, the trend line slope of the C52 concentration change for each range of substrate treatment (= production compared to enzyme g) was calculated, and is shown in Table 3 for each pH. The smaller the absolute value of the trend line slope, the better the effect of preventing the decrease of enzyme activity.

Acid value measurement  And calculation

Oil samples collected for acid value were used for analysis after reaction by-products (free fatty acids, etc.) were removed through pretreatment of the thin film distillation and deodorization process.

First, about 20 g of the pretreated fat sample was dissolved in 100 to 300 mL of toluene / isopropanol mixed solvent [toluene: isopropanol = 1: 1 (V / V)], and then 2 to 3 drops of 1% phenolphthalein indicator were added thereto, and 0.1 N The end point was a time when the scarlet lasted for 30 seconds by titration with KOH solution. Acid value was calculated by the following formula, the acid value calculated for each pH is shown in Table 4 below.

Acid  = [ 0.1N  KOH titration amount (mL) X 0.1N  Of KOH solution Titer ] / [Sample Weight (g)]

Peroxide Value Measurement and Calculation

About 10 to 20 g of the oil or fat sample was placed in a 250 mL Erlenmeyer flask, 10 mL of chloroform was added thereto to completely dissolve, and 20 mL of glacial acetic acid was added thereto. After further adding 1 mL of saturated KI solution, the cap was closed and shaken for 1 minute, and then left in a cool dark place for 10 minutes. Thereafter, 75 mL of distilled water was added thereto, and 1 mL of 1% starch solution was added thereto, followed by titration with 0.01 N Na ₂ S ₂ O ₂ solution to make the colorless black to brown. The peroxide value was calculated by the following formula, and the peroxide value calculated for each pH is shown in Table 4 below.

Peroxide value =

[(Appropriate amount of this examination- Blank test  Appropriate amount) (mL) X 0.01N Na ₂ S ₂ O ₂  Solution Titer ] / [Sample Weight (g)] × 10

From Table 1 to Table 3, when the pH of the starting material mixture of the enzymatic esterification reaction was adjusted within the range according to the present invention, it was possible to significantly prevent the decrease of the enzyme activity according to the progress of the reaction, as a result of the overall process efficiency and It can be seen that the economics were improved. In addition, it can be seen from Table 4 that the oil or fat product obtained according to the present invention satisfies both an acid value of 0.6 or less and a peroxide value of 3.0 or less.

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 (15)

In carrying out the reaction for exchanging the acyl group of the triacylglyceride with another acyl group by using an enzyme, by adjusting the pH of the mixture of the reaction starting materials within the range of 4.5 to 8.5, it is characterized by preventing the decrease of the enzyme activity due to the progress of the reaction. Enzymatic esterification method. 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 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 acyl groups different from acyl groups in the triacylglycerides as reaction starting materials;
(2) adjusting the pH of the mixture obtained in step (1) within a range of 4.5 to 8.5; And
(3) performing an enzymatic esterification reaction on the pH adjusted reaction mixture obtained in step (2) to exchange acyl groups of the triacylglycerides with acyl groups of the fatty acids,
Method for preparing triglycerides. 8. The saturated or unsaturated linear aliphatic acyl group of claim 7, wherein the acyl group present in the triacylglyceride is independently 4 to 24 carbon atoms, and the fatty acid is saturated or has 4 to 24 carbon atoms. A process for producing triglycerides, wherein the compound is at least one selected from unsaturated linear fatty acids. 8. The compound according to claim 7, wherein 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 A method for producing triglycerides, selected from the group consisting of combinations thereof. The method for producing triglyceride according to claim 7, wherein the triacylglyceride is tripalmitoylglyceride and the fatty acid is oleic acid. The process for preparing triglycerides according to claim 7, 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 7, wherein the triglyceride prepared is dioleoyl monopalmitoyl glyceride. The process for producing triglycerides according to claim 7, wherein the acid value of the produced triglyceride is 0.6 or less and the peroxide value is 3.0 or less. As a result of enzymatic esterification of a reaction mixture comprising a fatty component including triacylglycerides and a fatty acid having an acyl group different from an acyl group in the triacylglycerides, and having a pH adjusted within the range of 4.5 to 8.5, the tree Fatty composition, wherein the acyl group of acylglycerides comprises triglycerides in which the acyl group of the fatty acid is replaced. The tree, prepared by enzymatic esterification of a reaction mixture comprising a fatty component comprising triacylglycerides and a fatty acid having an acyl group different from the acyl groups in the triacylglycerides, the pH of which is adjusted within the range of 4.5 to 8.5. The milk powder composition of claim 1, wherein the acyl group of acylglycerides comprises triglycerides in which the acyl group of the fatty acid is replaced.

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