WO2007097523A2

WO2007097523A2 - Fat composition and preparation methods thereof

Info

Publication number: WO2007097523A2
Application number: PCT/KR2007/000097
Authority: WO
Inventors: Dong-Hun Yoon; Kwang-Hoon Yoon; Gi-Wang Han; Moon-Won Lee
Original assignee: Ilshin Wells Co., Ltd.
Priority date: 2006-02-24
Filing date: 2007-01-08
Publication date: 2007-08-30
Also published as: WO2007097523A3; KR100598607B1

Abstract

Disclosed are a fat composition and methods for preparing the fat composition. The fat composition comprises (a) 10 to 95% by weight of a triglyceride in which palmitic acid or stearic acid is bonded in the 2-position and is present in an amount of 25 to 95% by weight with respect to the weight of constituent fatty acids, (b) 4.5 to 80% by weight of a triglyceride in which medium-chain fatty acids are bonded in the 1,3-positions and are present in an amount of 1 to 50% by weight with respect to the weight of constituent fatty acids, and (c) 0.1 to 85% by weight of a diglyceride in which unsaturated fatty acids are bonded in the 1,2-positions or 1,3-positions and are present in an amount of 30 to 95% by weight with respect to the weight of constituent fatty acids.

Description

FAT COMPOSITION AND PREPARATION METHODS

THEREOF

Technical Field

[1] The present invention relates to a fat composition and methods for preparing the same. More specifically, the present invention relates to a fat composition comprising a diglyceride and a triglyceride containing a large amount of palmitic acid or stearic acid as a constituent fatty acid in the 2-position of the triglyceride, and methods for preparing the fat composition by interesterification of fats. According to the fat composition of the present invention, palmitic acid or stearic acid contained in the triglyceride is not degraded into its free fatty acid form, which is bonded to calcium in the body, particularly in infants, by digestive enzymes so that insoluble salts causing constipation are not formed. In addition, the diglyceride functions to lower the diameter of membrane-enclosed milk fat globules, which are formed from the fat composition and other nutrients, resulting in an improvement in the digestibility and absorbability of the fat composition in the body.

[2]

Background Art

[3] Fats are contained in powdered milk and infant formulas as human milk substitutes supplied to infants. More than half of the energy available from human milk and infant formulas supplied to infants is in the form of lipids, particularly triglycerides. Human milk lipids are characterized by their inherent structures, which are different from those of ordinary fats, based on the steric positions of constituent fatty acids of the lipids (see, Diagram 1). Palmitic acid accounts for more than a half (-58%) of constituent fatty acids in the sn-2 position (or β-position) of human milk lipids. Palmitic acid in the sn-2 position of human milk lipids is not hydrolyzed by digestive enzymes present in the body and is absorbed in the form of a 2-monoglyceride of mixed micelles along with bile into the body (see, Diagram 2). The structural characteristics of human milk lipids have important meanings in the energy supply to infants.

[4] Diagram 1

[5]

[6] Diagram 2 [7]

[8] In contrast, most vegetable oils are responsible for less than 20% of energy supply to infants. Palmitic acid or stearic acid in the sn-1 or sn-3 position is degraded in the form of its free fatty acid form by digestive enzymes and is bonded to adjacent calcium, which shows a strong tendency to form insoluble salts (such as calcium soaps) causing constipation in infants, after being absorbed. This salt formation lowers the absorption rate of palmitic acid as an energy source in infants as well as deteriorates the absorption of essential elements, such as calcium, magnesium and phosphorus, for the growth of infants.

[9]

Disclosure of Invention Technical Problem

[10] PCT Publication Nos. WO 94/26855 and WO 94/26854 introduce fat compositions in which at least 35% of fatty acids in the 2-position of a fat is occupied by palmitic acid or stearic acid by transesterification of the fat, and methods for preparing the fat compositions. However, no patent publication discloses about a fat composition as a human milk substitute comprising a diglyceride bonded by unsaturated fatty acids or a method for preparing a fat composition as a human milk substitute using an immobilized enzyme wherein the fat composition comprises a triglyceride containing a large amount of palmitic acid or stearic acid as a saturated fatty acid in the 2-position of the triglyceride.

[H]

Technical Solution

[12] Therefore, the present invention has been made in view of the above problems, and it is one object of the present invention to provide a fat composition that can lower the diameter of membrane-enclosed milk fat globules to improve the digestibility and absorbability of the fat composition in the body without the formation of insoluble salts causing constipation in infants.

[13] It is another object of the present invention to provide methods for preparing the fat composition by interesterification of fats.

[14] In accordance with one aspect of the present invention for achieving the above objects, there is provided a fat composition, comprising:

[15] (a) 10 to 95% by weight of a triglyceride in which palmitic acid or stearic acid is bonded in the 2-position of the triglyceride and is present in an amount of 25 to 95% by weight with respect to the weight of constituent fatty acids;

[16] (b) 4.5 to 80% by weight of a triglyceride in which medium-chain fatty acids are bonded in the 1,3-positions of the triglyceride and are present in an amount of 1 to 50% by weight with respect to the weight of constituent fatty acids; and

[17] (c) 0.1 to 85% by weight of a diglyceride in which unsaturated fatty acids are bonded in the 1,2-positions or 1,3-positions of the diglyceride and are present in an amount of 30 to 95% by weight with respect to the weight of constituent fatty acids.

[18] In accordance with another aspect of the present invention, there is provided a method for preparing a fat composition, the method comprising the steps of:

[19] (a) subjecting a fat to 1,3-position specific hydrolysis using an immobilized

1,3-position specific enzyme;

[20] (b) seperating the hydrolysate into a fatty acid fraction and a glyceride fraction ;

[21] (c) mixing the glyceride fraction, a fatty acid and a fat in a weight ratio of 0.1-90 :

0.1-90 : 10-95, and subjecting the mixture to a transesterification reaction using an im- mobilized 1,3-position specific enzyme at a stirring speed of 10 to 300 rpm and a temperature of 25 to 80°C under a reduced pressure of 0.001 to 10 torr for 1 to 48 hours to obtain a fat mixture;

[22] (d) mixing the fat mixture with medium-chain fatty acids in a weight ratio of 20-99

: 1-80, and subjecting the resulting mixture to a transesterification reaction using an immobilized 1,3-position specific enzyme at a stirring speed of 10 to 300 rpm and a temperature of 25 to 80°C under a reduced pressure of 0.001 to 10 torr for 1 to 48 hours; and

[23] (e) removing unreacted residues by distillation and purification.

[24] In accordance with yet another aspect of the present invention, there is provided a method for preparing a fat composition, the method comprising the steps of:

[25] (a) subjecting a fat to positionally non-specific hydrolysis using an immobilized po- sitionally non-specific enzyme;

[26] (b) seperating the hydrolysate into a fatty acid fraction and a glyceride fraction ;

[27] (c) mixing the glyceride fraction and a fat in a weight ratio of 0.1-90: 10-99, and subjecting the mixture to a transesterification reaction at a stirring speed of 10 to 300 rpm and a temperature of 25 to 80°C under ambient pressure for 1 to 48 hours using an immobilized 1,3-position specific enzyme to obtain a fat mixture;

[28] (d) mixing the fat mixture with medium-chain fatty acids in a weight ratio of 20-99

[29] (e) removing unreacted residues by distillation and purification.

[30]

Advantageous Effects

[31] The fat composition of the present invention comprises a diglyceride and a triglyceride containing a large amount of palmitic acid or stearic acid as a constituent fatty acid in the 2-position of the triglyceride. The diglyceride functions to lower the diameter of membrane-enclosed milk fat globules, which are formed from the fat composition and other nutrients, resulting in an improvement in the digestibility and absorbability of the fat composition in the body. Since the palmitic acid or stearic acid present in the 2-position of the triglyceride is absorbed in the form of a 2-monoglyceride into the body without being hydrolyzed, it is not degraded into its free fatty acid form by digestive enzymes so that insoluble salts causing constipation are not formed. Therefore, the fat composition of the present invention has marked preventive effects on constipation. [32] Furthermore, the methods of the present invention are relatively simplified, provide high productivity and enable the preparation of highly functional fats at reduced costs. Brief Description of the Drawings

[33] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[34] FIG. 1 is a graph showing the average diameter of membrane-enclosed milk fat globules which are formed from a fat composition prepared in Example 1 of the present invention and other nutrients;

[35] FIG. 2 is a graph showing the average diameter of membrane-enclosed milk fat globules which are formed from a fat composition prepared in Example 2 of the present invention and other nutrients; and

[36] FIG. 3 is a graph showing the average diameter of membrane-enclosed milk fat globules which are formed from a fat composition prepared in Comparative Example 1 and other nutrients.

[37]

Best Mode for Carrying Out the Invention

[38] The present invention will now be described in greater detail.

[39] The present inventors to develop a human milk fat substitute that can achieve improved digestibility and absorbability in the body. As a result, the present inventors have found that when a diglyceride bonded by unsaturated fatty acids in the 1,2-positio ns or 1,3-positions of the diglyceride was added to a triglyceride bonded by palmitic acid or stearic acid as a constituent fatty acid in the 2-position of the triglyceride and a triglyceride bonded by medium-chain fatty acids as constituent fatty acids in the 1,3-positions of the triglyceride to prepare a fat composition, the average diameter of fat globules, which are formed from the fat composition and other nutrients, was lowered as compared to that of conventional human milk fat substitutes, resulting in an improvement in the digestibility and absorbability of the fat composition in the body. The present invention has been accomplished based on this finding.

[40] The present invention provides a fat composition which comprises (a) a triglyceride bonded by palmitic acid or stearic acid as a constituent fatty acid in the 2-position of the triglyceride, (b) a triglyceride bonded by medium-chain fatty acids as constituent fatty acids in the 1,3-positions of the triglyceride, and (c) a diglyceride bonded by unsaturated fatty acids in the 1,2-positions or 1,3-positions of the diglyceride.

[41] In view of the oxidative stability of a fat and the digestibility and absorbability of fatty acids, the fat composition of the present invention preferably comprises 10 to 95% by weight of a triglyceride in which palmitic acid or stearic acid is bonded in the 2-position of the triglyceride and is present in an amount of 25 to 95% by weight with respect to the weight of constituent fatty acids. When the palmitic acid or stearic acid and the triglyceride are present below the respective lower limits defined above, the absorption rate of the palmitic acid or stearic acid is lowered and the oxidative stability of a fat may be deteriorated. Meanwhile, when the palmitic acid or stearic acid and the triglyceride are present above the respective upper limits defined above, the preparation of the fat composition is substantially difficult and the physical properties of the fat composition are greatly varied, causing a significant reduction in yield, which results in an increase in the price of the fat composition.

[42] The content of the medium-chain fatty acids in the triglyceride is preferably in the range of 1 to 50% by weight and particularly about 10% by weight, based on the weight of constituent fatty acids. The triglyceride containing the medium-chain fatty acids is preferably present in an amount of 4.5 to 80% by weight, based on the total weight of the fat composition. When the content of the medium-chain fatty acids or the content of the triglyceride containing the medium-chain fatty acids is below the corresponding lower limit, the medium-chain fatty acids are not rapidly absorbed into the body through blood and thus cannot be used as energy sources in the body. Meanwhile, when the content of the medium-chain fatty acids or the content of the triglyceride containing the medium-chain fatty acids is above the corresponding upper limit, a balance of the fatty acids supplied to the body is not maintained, causing undesirable problems in terms of dietetics, and a smell peculiar to the medium-chain fatty acids is produced.

[43] Examples of suitable medium-chain fatty acids include fatty acids having up to 14 carbon atoms, such as caprylic acid (C ,octanoic acid), pelargonic acid (C ), capric acid

8 9

(C , decanoic acid), undecanoic acid (C ), lauric acid (C , dodecanoic acid) and myristic acid (C 14 ). The medium-chain fatty acids are preferably selected from the group consisting of capric acid, lauric acid, and myristic acid.

[44] The content of the diglyceride in the fat composition of the present invention is preferably between 0.1 and 85% by weight and particularly between 5 and 25% by weight. If the content of the diglyceride is less than 0.1% by weight, the fat composition of the present invention is substantially composed of the triglycerides only, and as a result, the diameter of fat globules is increased, causing a drop in absorption rate. Meanwhile, if the content of the diglyceride is more than 85% by weight, the preparation of the fat composition is substantially difficult. Taking into consideration the diameter of fat globules provided by the human milk fat and the preparation of the fat composition, the content of the diglyceride is preferably limited to the range of 0.1 to 85% by weight.

[45] The diglyceride contains 30 to 95% by weight of unsaturated fatty acids as constituent fatty acids in the 1,2-positions or 1,3-positions. When the unsaturated fatty acids are present in an amount of less than 30% by weight, they are predominantly distributed in the 1,3-positions of the diglyceride rather than in the 2-position of the diglyceride, and as a result, they are bonded to calcium to form insoluble salts causing constipation in infants and cause an increase in the melting point of the fat composition. Meanwhile, the presence of the unsaturated fatty acids in an amount exceeding 95% by weight is undesirable in terms of the supply of raw materials and the preparation costs.

[46] Examples of suitable unsaturated fatty acids include fatty acids having at least 16 carbon atoms and at least one double bond. The unsaturated fatty acids are preferably selected from the group consisting of palmitoleic acid, oleic acid, linoleic acid, and linolenic acid.

[47] As explained above, the fat composition of the present invention meets the requirements for the content (-58% by weight) of palmitic acid in the 2-position and the content (-27% by weight) of saturated fatty acids in the 1,3-positions, which are characteristics of human milk fats having high digestibility and absorbability in the body. Since the fat composition of the present invention comprises the diglyceride composed of unsaturated fatty acids, it provides fat globules having a size smaller than that of fat globules provided by conventional human milk fat substitutes, thereby achieving an improvement in the digestibility and absorbability of the fat composition in the body. The size of fat globules increases in the order of clostrum, transitional milk and mature milk. The fat composition of the present invention can provide a human body human milk fat substitute which forms fat globules having a size similar to that of clostrum, which is the most advantageous for digestibility and absorbability, by controlling the content of the diglyceride in the fat composition. Fat globules provided by the fat composition of the present invention have an average diameter of 0.1 D to 1 D. The particulate fat globules having a size smaller than that of fat globules provided by conventional human milk fat substitutes can greatly contribute to an improvement in the digestibility and absorbability of the fat composition in the body.

[48] The palmitic acid or stearic acid contained in a large amount in the 2-position of the triglyceride is absorbed in the form of a 2-monoglyceride into the body without being hydrolyzed, but is not bonded to calcium so that insoluble salts known as causes of constipation are not formed, thereby preventing constipation. Therefore, the fat composition of the present invention can be advantageously used to produce powdered milk formulas for premature babies, powdered milk formulas for infants, functional foods, nutritional supplements, foods for infants, foods for pregnant women, and foods for the elderly.

[49] The fat composition of the present invention can be prepared by the following two methods.

[50] The first method subjecting a fat to 1,3-position specific hydrolysis, subjecting mixture of the glyceride fraction, a fatty acid and a fat to a first transesterification reaction using an immobilized 1,3-position specific enzyme to obtain a fat mixture, and subjecting the fat mixture to a second transesterification reaction using medium-chain fatty acids and an immobilized 1,3-position specific enzyme.

[51] The glyceride fraction, a fatty acid and a fat are mixed together in a weight ratio of

0.1-90 : 0.1-90 : 10-95. Out of this range, the weight ratio of residual fatty acids or monoglycerides increases, causing low yield of the subsequent purification processing and considerable drop of the transesterification reaction rates.

[52] The fat mixture obtained after the first transesterification reaction and the medium- chain fatty acids are subjected to a second transesterification reaction using an immobilized 1,3-position specific enzyme. At this time, the fat mixture and the medium- chain fatty acids are preferably mixed in a weight ratio of 20-99 : 1-80. When the medium-chain fatty acids are used in a larger amount than is necessary, the taste and fragrance of the final product are changed as well as the physical properties of the final product during processing are considerably varied. Meanwhile, when the medium- chain fatty acids are used in a smaller amount than is necessary, the fat composition of the present invention fails to provide balanced nutrition because the medium-chain fatty acids are quickly used as energy sources in the body as compared to long-chain fatty acids.

[53] The first and second transesterification reactions are performed using an immobilized 1,3-position specific enzyme at a temperature of 25 to 80°C and at a stirring speed of 10 to 300 rpm under a reduced pressure of 0.001 to 10 torr for 1 to 48 hours. If the transesterification reactions are performed under a pressure lower than 0.001 torr, the transesterification reaction rates are not increased and additional vacuum equipment is required to provide a degree of vacuum necessary for the reactions, which is disadvantageous from the economiacl view point. Meanwhile, if the transesterification reactions are performed under a pressure higher than 10 torr, moisture generated during the reactons is not readily removed, resulting in a decrease in the yield of the reactions. The reactions are not effectively performed at a temperature lower than 25°C, while the enzyme is inactivated at a temperature higher than 80°C, resulting in a considerable decrease in reaction rates. The mixing is insufficient at a stirring speed lower than 10 rpm. Emulsification occurs due to strong stirring at a speed higher than 300 rpm. This emulsification becomes serious with increasing reaction volume. If the reaction time is shorter than one hour, the transterificiation reactions are insufficiently completed. Meanwhile, if the reaction time is longer than 48 hours, the transesterification reactions no longer proceed or the palmitic acid or stearic acid in the 2-position migrates toward the 1- or 3-position, making it impossible to prepare the desired fat composition.

[54] The second method is carried out by subjecting a fat to positionally non-specific hydrolysis, subjecting mixture of the glyceride fraction and a fat to a first transester- ification reaction using an immobilized 1, 3-position specific enzyme to obtain a fat mixture, and subjecting the fat mixture to a second transesterification reaction using medium-chain fatty acids and an immobilized 1, 3-position specific enzyme.

[55] The glyceride fraction and a fat are mixed together in a weight ratio of 0.1-90.0 :

10-99.9. Out of this range, the weight ratio of residual fatty acids or monoglycerides increases, causing low yield of the subsequent purification processing and considerable drop of the transesterification reaction rates.

[56] The first transesterification reaction is performed using an immobilized 1, 3-position specific enzyme at a temperature of 25 to 80°C and at a stirring speed of 10 to 300 rpm under ambient pressure for 1 to 48 hours. When the transesterification reaction is performed at a temperature lower than 25°C, which is close to the melting point of saturated fatty acids, a crystal is formed, making the reaction solution turbid. Meanwhile, when the transesterification reaction is performed at a temperature exceeding 80°C, the activation of the enzyme is limited, resulting in a decrease in the yield of the reactions (an increase in the preparation costs) and emulsification occurs depending on the stirring conditions. The mixing is insufficient at a stirring speed lower than 10 rpm. This emulsification becomes serious with increasing reaction volume. If the reaction time is shorter than one hour, the transesterification reaction is insufficiently completed. Meanwhile, if the reaction time is longer than 48 hours, the transesterificiation reaction no longer proceeds.

[57] The second transesterification reaction between the fat mixture obtained after the first transesterification reaction and the medium-chain fatty acids is performed using an immobilized 1, 3-position specific enzyme. At this time, the fat mixture and the medium-chain fatty acids are preferably mixed in a weight ratio of 20-99 : 1-80.

[58] Enzymes that can be used in the enzymic reactions are known 1, 3-position specific

Upases. Examples of preferred 1, 3-position specific Upases include, but are not limited to, Upases derived from microorganisms, such as Rhizopus sp. Aspergillus sp. and Mucor sp. Examples of preferred positionally non-specific Upases include, but are not limited to, Candida cylindracea Upases and pancreatic Upases. In embodiments of the present invention, Lipozyme RM IM commercially available from Novo Nordisk is used as a 1, 3-position specific lipase.

[59] The enzyme is preferably used in an amount of 0.1 to 20 parts by weight, based on

100 parts by weight of the reactants for the enzymic reactions. The raw materials used for the enzymic reactions are glyceride (particularly, 2-monoglyceride), a fatty acid and a fat in the first method. The raw materials used for the enzymic reactions are glyceride and a fat in the second method. When the enzyme is used in an amount of less than 0.1 parts by weight, the conversion efficiency is considerably lowered. Meanwhile, when the enzyme is used in an amount exceeding 20 parts by weight, the economic efficiency is low.

[60] Examples of preferable fats include safflower oil, soybean oil, corn oil, canola oil, rice bran oil, olive oil, palm oil, palm olein oil, palm stearine oil, palm kernel oil, tallow, lard, mixed edible oil, shortening, margarine, sesame oil, perilla oil, sunflower oil, cottonseed oil, and peanut oil. Particularly preferred are animal and vegetable fats abundantly containing USU or USS type fats (U represents an unsaturated fatty acid and S represents a saturated fatty acid).

[61] A more detailed explanation of the respective steps of the methods according to the present invention will be given below.

[62] (1) 1,3-Position specific hydrolysis reaction

[63] The reaction conditions will be described in more detail. First, a fat is mixed with water. The mixture is completely or partially hydrolyzed using an immobilized 1,3-position specific enzyme. In this step, the water is preferably added in an amount of 40 to 80 parts by weight with respect to 100 parts by weight of the fat. This hydrolysis is performed to separate 10 to 90% by weight of fatty acids present in the glyceride, based on the total weight of the fatty acids.

[64]

[65] (2) Positionally non-specific hydrolysis reaction

[66] This hydrolysis step replaces step (1). A fat and water are used in the same amounts as used in step (1). This hydrolysis is performed to separate a portion {i.e. 0.1 to 90% by weight) of fatty acids present in the fat, based on the total weight of the fatty acids.

[67] The hydrolysis is preferably performed by stirring the mixture of the fat and the water at a speed of 10 to 300 rpm. Since the stirring speed is lower than 10 rpm, i.e. the stirring power is weak, the fat layer is separated from the water layer, causing the problem of low hydrolysis rate. Meanwhile, when the stirring speed exceeds 200 rpm, the fat and the water are emulsified, making it difficult to separate the water and the fat in the subsequent step.

[68]

[69] (3) Removal of fatty acids and separation of glyceride fraction

[70] After the reaction end point of the fat hydrolysate obtained in step (1) or (2) is identified, the stirring is stopped and the hydrolysate is allowed to stand to separate a fat fraction from the water. The fat fraction contains fatty acids, monoglycerides and triglycerides. In this step, the fatty acids, particularly, saturated fatty acids, are removed. Various separation processes, such as distillation, crystallization, low- temperature crystallization, urea-addition and chromatography, may be employed to remove the fatty acids. Particularly preferred are a urea-addition process by which the saturated fatty acids are selectively crystallized and deposited, a distillation process at ambient or reduced pressure to remove the fatty acids, and a crystallization process by which different melting points inherent to the fatty acids are utilized under low- temperature conditions. These separation processes may be employed alone or in combination to increase the removal efficiency of the fatty acids. Particularly, the distillation process under reduced pressure may be employed for final separation. When the distillation process is carried out at a pressure lower than 0.001 torr, the fatty acids, monoglycerides and diglycerides are concurrently distilled. Meanwhile, when the distillation process is carried out at a pressure higher than 10 torr, there is a difficulty in distilling the fatty acids. Therefore, it is very important to control the degree of vacuum in the application of the distillation process under reduced pressure.

[71]

[72] (4) First and second transesterification reactions

[73] Two transesterification reactions are performed in the respective methods of the present invention. The glyceride fraction, a fatty acid and a fat are subjected to a first transesterification reaction in the first method, while the glyceride fraction and a fat are subjected to a first transesterification reaction in the second method. The first transesterification reactions are performed using an immobilized 1,3-position specific enzyme to obtain a fat mixture. The fat mixture and medium-chain fatty acids are subjected to a second transesterification reaction using an immobilized 1,3-position specific enzyme to prepare the final fat composition of the present invention.

[74] According to the first method, the glyceride fraction, a fatty acid and a fat are mixed together in a suitable ratio, and then the mixture is subjected to a first transesterification reaction using an immobilized 1,3-position specific enzyme at a stirring rate of 10 to 300 rpm and a temperature of 25 to 80°C under a reduced pressure of 0.001 to 10 torr for 1 to 48 hours to obtain a fat mixture. On the other hand, according to the second method, the glyceride fraction and a fat are mixed together in a suitable ratio, and then the mixture is subjected to a first transesterification reaction using an immobilized 1,3-position specific enzyme at a stirring rate of 10 to 300 rpm and a temperature of 25 to 80°C under ambient pressure for 1 to 48 hours to obtain a fat mixture.

[75] The fat mixture obtained after each of the first transesterification reactions is mixed with medium-chain fatty acids in a suitable ratio, and then the mixture is subjected to a second transesterification reaction using an immobilized 1,3-position specific enzyme at a stirring rate of 10 to 300 rpm and a temperature of 25 to 80°C under a reduced pressure of 0.001 to 10 torr for 1 to 48 hours to prepare the final fat composition of the present invention.

[76] The selection of a reactor for the immobilized enzyme is of particular importance in order to ensure economical advantages and efficiency of the transesterification reactions. As the reactor, there can be used, for example, batch stirred tank reactor (BSTR), packed-bed reactor (PBR) or membrane reactor (MR). The use of the reactor can minimize the loss of the immobilized enzyme. In addition, it is important to determine techniques associated with the operation of the reactor by which the optimization of the transesterification reactions can be induced. Therefore, various factors, such as reaction modes, solvents, matrices, reactor arrangements, kind of Upases, immobilization methods, use and kind of immobilization assistants, and support materials, must be taken into consideration. The transesterification reactions are preferably performed at a stirring rate of 10 to 300 rpm, particularly at 150 rpm, and at a temperature of 25 to 80°C, particularly 45°C. The transfer speed must be suitably determined by varying the operating conditions while checking the quality of the product. Too slow stirring or transfer leads to a low conversion rate, and too rapid stirring or transfer leads to damage to the immobilized enzyme and a deterioration in the quality of the final product. Therefore, it is important to control the stirring and transfer speeds. When the reactions are performed at a temperature lower than 25°C, the conversion rate by the enzyme is retarded. Meanwhile, when the reactions are performed at a temperature higher than 80°C, the initial reaction rates are high but the enzyme may be affected by heat. When the reaction time is shorter than one hour, the conversion rate is poor.

[77] In conclusion, the methods of the present invention are relatively simplified, provide high productivity and enable the preparation of highly functional fats at reduced costs. The fat composition of the present invention prepared by the methods satisfies the requirements of human milk fats with digestibility and absorbability in the body. In addition, the fat composition of the present invention provides fat globules having a size smaller than that of fat globules provided by conventional human milk fat substitutes, thereby achieving an improvement in the digestibility and absorbability of the fat composition in the body.

[78]

Mode for the Invention

[79] The following examples and experimental examples are provided to assist in a further understanding of the invention. However, these examples are given for the purpose of illustration and are not intended to limit the present invention.

[80] EXAMPLES

[81] (Example 1) [82] Step 1. 1,3-Position specific hydrolysis

[83] 1,00Og of palm stearine oil, 70Og of water and 4g of a 1,3-position specific lipase

(Lipozyme RM IM, Novo Nordisk) were mixed in a 3 liter-reactor equipped with a stirrer. The mixture was allowed to react with stirring at 150 rpm and 45°C for 10 hours to obtain a fat mixture, which was a product hydrolyzed by the 1,3-position specific lipase.

[84]

[85] Step 2. Separation of glyceride and fatty acid fractions

[86] To separate a glyceride fraction, which was composed of monoglycerides, diglycerides and triglycerides, and a fatty acid fraction from the hydrolyzed fat mixture, the fat mixture was distilled under a reduced pressure of 1 torr to obtain a glyceride fraction free of fatty acids. After the glyceride fraction was sufficiently dissolved at 70°C, the solution was slowly cooled to 30°C with stirring at 15 rpm (cooling crystallization) to remove a slight amount of saturated fatty acids in the form of crystal nuclei. The cooling crystallization gave 31% by weight of a crystal portion containing a large amount of saturated fatty acids and 69% by weight of a solution portion containing a large amount of unsaturated fatty acids.

[87]

[88] Step 3. First transesterification reaction

[89] The crystal portion as a glyceride fraction, a high-oleic canola oil, and oleic acid were mixed in a weight ratio of 1 : 1 : 0.65. The mixture was subjected to a transesterification reaction using 4.255g of lipozyme (Novozyme RM IM) at 250 rpm and 45°C under a reduced pressure of 2 torr for 8 hours. Thereafter, the transesterification product was distilled to remove unreacted residues, followed by deodorization as a general purification process to obtain a fat mixture.

[90]

[91] Step 4. Second transesterification reaction

[92] 12Og of the fat mixture was mixed with 500g of a mixture of medium-chain fatty acids (capric acid (C :0)/lauric acid (C :0)/myristic acid (C :0) = 5/40/55 (w/w/w)). The resulting mixture was subjected to a transesterification reaction using 4.255g of lipozyme (Novozyme RM IM) at 250 rpm and 45°C under a reduced pressure of 2 torr for 8 hours. Thereafter, the enzyme was filtered off to obtain about 60Og of a process oil. Unreacted residues were removed from the process oil by distillation, yielding a fat composition of the present invention.

[93]

[94] (Example 2)

[95] Step 1. Positionally non-specific hydrolysis

[96] l,000g of palm stearine oil, 70Og of water and 4g of a positionally non-specific lipase (Lipozyme RM IM, Novo Nordisk) were mixed in a 3 liter-reactor equipped with a stirrer. The mixture was allowed to react with stirring at 150 rpm and 45°C for 10 hours to obtain a fat mixture, which was a product hydrolyzed by the positionally non-specific lipase.

[97]

[98] Step 2. Separation of glyceride and fatty acid fractions

[99] To separate a glyceride fraction, which was composed of monoglycerides, diglycerides and triglycerides, and a fatty acid fraction from the hydrolyzed fat mixture, the fat mixture was distilled under a reduced pressure of 1 torr to obtain a glyceride fraction free of fatty acids. After the glyceride fraction was sufficiently dissolved at 70°C, the solution was slowly cooled to 30°C with stirring at 15 rpm (cooling crystallization) to remove a slight amount of saturated fatty acids in the form of crystal nuclei. The cooling crystallization gave 60% by weight of a crystal portion containing a large amount of saturated fatty acids and 40% by weight of a solution portion containing a large amount of unsaturated fatty acids.

[100]

[101] Step 3. First transesterification reaction

[102] The crystal portion as a glyceride fraction and a high-oleic canola oil were mixed in a weight ratio of 1 : 1.1. The mixture was subjected to a transesterification reaction using 4.255g of lipozyme (Novozyme RM IM) at 250 rpm and 45°C under ambient pressure for 10 hours, followed by deodorization as a general purification process to obtain a fat mixture.

[103]

[104] Step 4. Second transesterification reaction

[105] 12Og of the fat mixture was mixed with 500g of a mixture of medium-chain fatty acids (capric acid (C 10 :0)/lauric acid (C 12 :0)/myristic acid (C 14 :0) = 5/40/55 (w/w/w)).

The resulting mixture was subjected to a transesterification reaction using 4.255g of lipozyme (Novozyme RM IM) at 250 rpm and 40°C under a reduced pressure of 5 torr for 10 hours. Thereafter, the enzyme was filtered off to obtain about 60Og of a process oil. Unreacted residues were removed from the process oil by distillation, yielding a fat composition of the present invention.

[106]

[107] (Comparative Example 1)

[108] The diglyceride and residual fatty acids were distilled off from the fat composition, which prepared through 1,3-position specific hydrolysis, separation of glyceride and fatty acid fractions and a first transesterification reaction in Example 1 and whose ingredients and amounts thereof are shown in Table 2, to prepare a fat composition. The fat composition was comprised of a triglyceride containing 87.7 '% by weight of palmitic acid or stearic acid as a constituent fatty acid in the 2-position of the triglyceride. [109]

[110] (Analysis Example)

[111] 1. Gas chromatography for analysis of fatty acid composition

[112] A sample was injected at a concentration of 25 g/1 under the following analytical conditions.

[113] Column: HP-INNOWAX (Agilent, USA)

[114] Carrier Gas: Helium (2.1 ml/min.)

[115] Temperature of oven: 150-260°C

[116] Temperature of flame ionization detector (FED): 275°C

[117]

[118] 2. Liquid chromatography for analysis of glyceride composition

[119] A sample was injected at a concentration of 1 mg/ml (solvent: chloroform) under the following analytical conditions.

[120] Column: Supelcosil LC-Si (5 D, 25 cm, Aupelco, USA)

[121] Mobile phase solvent: Solvent A (benzene/chloroform/acetic acid = 70/30/2) and

Solvent B (ethyl acetate)

[122] Detector: Evaporative light scattering detector (ELSD)

[123] Row rate: 2.3 ml/min.

[124]

[125] 3. Liquid chromatography for analysis of glyceride position isomers

[126] A sample was injected at a concentration of 1 mg/ml (solvent: chloroform) under the following analytical conditions.

[127] Column: ChromSpher Lipids (5 D, 25 cm, Varian, USA)

[128] Mobile phase solvent: n-Hexane plus 0.5% acetonitrile

[129] Detector: evaporative light scattering detector (ELSD)

[130] Row rate: 2.3 ml/min.

[131] [132] Analysis of fatty acids and glycerides contained in the fat mixtures (Example 1-

Step 1, Example 2- Step 1) were conducted by the corresponding analytical method described above. The results are shown in Table 1. [133] [Table 1]

[134]

[135] The glyceride positional isomers contained in the fat mixtures (Example 1- Step 3, Example 2- Step 3) were analyzed and the results are shown in Tables 2 and 3.

[136] [Table 2] [137]

[138] NOTE: P, O and S represent palmitic acid, oleic acid and stearic acid, respectively, which are constituent fatty acids of the triglycerides. Specifically, PPP represents a triglyceride in which palmitic acid as a constituent fatty acid is bonded in the 1,2,3-positions, and POP represents a triglyceride in which palmitic acid as a constituent fatty acid is bonded in the 1,3-positions and oleic acid as a constituent fatty acid is bonded in the 2-position.

[139] [Table 3] [140]

[141] Analysis of fatty acids contained in the fat compositions (Example 1, Example 2) were conducted by the corresponding analytical method described below. The results are shown in Table 4.

[142] [Table 4] [143]

[144] The results of Table 4 lead to the conclusion that each of the fat compositions was comprised of a triglyceride containing 87.7% by weight of palmitic acid or 78.7% by weight of stearic acid as a constituent fatty acid in the 2-position of the triglyceride, a triglyceride containing capric acid, lauric acid and myristic acid as medium-chain fatty acids in the 1,3-positions of the triglyceride, and a diglyceride containing unsaturated fatty acids.

[145] [146] (Experimental Examples) [147] (Experimental Examples 1 and 2) [148] Each of the fat compositions prepared in Examples 1 and 2 and Comparative Example 1 was mixed with the ingredients present in the amounts shown in Table 5. The mixture was processed with stirring at 800 rpm for 10 minutes. The processed mixture and other nutrients were used to form fat globules. The average diameter of the fat globules was measured. The results are shown in FIGs. 1 to 3.

[149] [Table 5] [150]

[151] From FIGs. 1 to 3, it could be confirmed that the fat globules formed from the fat composition of Comparative Example 1, which was composed of a triglyceride containing palmitic acid or stearic acid as a constituent fatty acid bonded in the 2-position of the triglyceride, had an average diameter of 10 D, and the fat globules formed from each of the fat compositions of Examples 1 and 2, which was comprised of a diglyceride containing unsaturated fatty acids, had an average diameter of 0.1 to 1 D.

[152] Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

[153]

Industrial Applicability [154] The fat composition of the present invention comprises a diglyceride and a triglyceride containing a large amount of palmitic acid or stearic acid as a constituent fatty acid in the 2-position of the triglyceride. The diglyceride functions to lower the diameter of membrane-enclosed milk fat globules, which are formed from the fat composition and other nutrients, resulting in an improvement in the digestibility and absorbability of the fat composition in the body. Since the palmitic acid or stearic acid present in the 2-position of the triglyceride is absorbed in the form of a 2-monoglyceride into the body without being hydrolyzed, it is not degraded into its free fatty acid form by digestive enzymes so that insoluble salts causing constipation are not formed. Therefore, the fat composition of the present invention has marked preventive effects on constipation.

[155] Furthermore, the methods of the present invention are relatively simplified, provide high productivity and enable the preparation of highly functional fats at reduced costs. [156]

Claims

[1] A fat composition, comprising:

(a) 10 to 95% by weight of a triglyceride in which palmitic acid or stearic acid is bonded in the 2-position of the triglyceride and is present in an amount of 25 to 95% by weight with respect to the weight of constituent fatty acids;

(b) 4.5 to 80% by weight of a triglyceride in which medium-chain fatty acids are bonded in the 1,3-positions of the triglyceride and are present in an amount of 1 to 50% by weight with respect to the weight of constituent fatty acids; and

(c) 0.1 to 85% by weight of a diglyceride in which unsaturated fatty acids are bonded in the 1,2-positions or 1,3-positions of the diglyceride and are present in an amount of 30 to 95% by weight with respect to the weight of constituent fatty acids.

[2] The fat composition according to claim 1, wherein the medium-chain fatty acids are selected from the group consisting of capric acid, lauric acid, and myristic acid.

[3] The fat composition according to claim 1, wherein the unsaturated fatty acids are selected from the group consisting of palmitoleic acid, oleic acid, linoleic acid, and linolenic acid.

[4] A food containing the fat composition according to claim 1.

[5] The food according to claim 4, wherein the food is selected from the group consisting of functional foods, nutritional supplements, powdered milk formulas for premature babies, powdered milk formulas for infants, foods for infants, foods for pregnant women, and foods for the elderly.

[6] A method for preparing a fat composition, the method comprising the steps of:

(a) subjecting a fat to 1,3-position specific hydrolysis using an immobilized 1,3-position specific enzyme;

(b) seperating the hydrolysate into a fatty acid fraction and a glyceride fraction;

(c) mixing the glyceride fraction, a fatty acid and a fat in a weight ratio of 0.1-90 : 0.1-90 : 10-95, and subjecting the mixture to a transesterification reaction using an immobilized 1,3-position specific enzyme at a stirring speed of 10 to 300 rpm and a temperature of 25 to 80°C under a reduced pressure of 0.001 to 10 torr for 1 to 48 hours to obtain a fat mixture;

(d) mixing the fat mixture with medium-chain fatty acids in a weight ratio of 20-99 : 1-80, and subjecting the resulting mixture to a transesterification reaction using an immobilized 1,3-position specific enzyme at a stirring speed of 10 to 300 rpm and a temperature of 25 to 80°C under a reduced pressure of 0.001 to 10 torr for 1 to 48 hours; and (e) removing unreacted residues by distillation and purification.

[7] The method according to claim 6, wherein the medium-chain fatty acids are selected from the group consisting of caprylic acid, capric acid, lauric acid, myristic acid, and mixtures thereof.

[8] The method according to claim 6, wherein the fat is selected from the group consisting of safflower oil, soybean oil, corn oil, canola oil, rice bran oil, olive oil, palm oil, palm olein oil, palm stearine oil, palm kernel oil, tallow, lard, mixed edible oil, shortening, margarine, sesame oil, perilla oil, sunflower oil, cottonseed oil, and peanut oil.

[9] The method according to claim 6, wherein the 1,3-position specific lipase is a lipase derived from a microorganism selected from the group consisting of

Rhizopus sp. Aspergillus sp. and Mucor sp.

[10] The method according to claim 6, wherein the enzyme is used in an amount of

0.1 to 20 parts by weight, based on 100 parts by weight of the reactants for the enzymic reactions.

[11] A method for preparing a fat composition, the method comprising the steps of:

(a) subjecting a fat to positionally non-specific hydrolysis using an immobilized positionally non-specific enzyme;

(c) mixing the glyceride fraction and a fat in a weight ratio of 0.1-90 : 10-99, and subjecting the mixture to a transesterification reaction at a stirring speed of 10 to 300 rpm and a temperature of 25 to 80°C under ambient pressure for 1 to 48 hours using an immobilized 1,3-position specific enzyme to obtain a fat mixture;

(d) mixing the fat mixture with medium-chain fatty acids in a weight ratio of 20-99 : 1-80, and subjecting the resulting mixture to a transesterification reaction using an immobilized 1,3-position specific enzyme at a stirring speed of 10 to 300 rpm and a temperature of 25 to 80°C under a reduced pressure of 0.001 to 10 torr for 1 to 48 hours; and

(e) removing unreacted residues by distillation and purification.

[12] The method according to claim 11, wherein the medium-chain fatty acids are selected from the group consisting of caprylic acid, capric acid, lauric acid, myristic acid, and mixtures thereof.

[13] The method according to claim 11, wherein the fat is selected from the group consisting of safflower oil, soybean oil, corn oil, canola oil, rice bran oil, olive oil, palm oil, palm olein oil, palm stearine oil, palm kernel oil, tallow, lard, mixed edible oil, shortening, margarine, sesame oil, perilla oil, sunflower oil, cottonseed oil, and peanut oil.

[14] The method according to claim 11, wherein the 1,3-position specific lipase is a lipase derived from a microorganism selected from the group consisting of

Rhizopus sp. Aspergillus sp. and Mucor sp. [15] The method according to claim 11, wherein the positionally non-specific lipase is a Candida cylindracea lipase or a pancreatic lipase. [16] The method according to claim 11, wherein the enzyme is used in an amount of