Field of the Invention

The present invention relates to a process for producing diacylglycerol-enriched fat or oil. More particularly, the present invention relates to a process for producing a diacylglycerol-enriched fat or oil containing reduced amount of impurities.

Background of the Invention

A fat or oil containing a high concentration of diacylglycerol (hereinafter is referred to as a "diacylglycerol-enriched fat or oil") is known to have advantageous pharmacological properties such as a body fat burning action in the human body. Therefore diacylglycerol-enriched fat or oil has been widely used as an edible oil.

It is known that diacylglycerol can be produced by several processes, including esterification; transesterification; glycerolysis; and partial hydrolysis. These processes may be carried out with the use of a chemical alkaline catalyst, or the like; or an fatty acid-reacting enzyme catalyst such as a lipase, or the like. From the viewpoints of product purity, consumer acceptance and energy consumption, the enzymatic process is more generally used.

Some of the diacylglycerol-enriched fats or oils produced by such methods, however, contain impurities, such as free fatty acids, monoacylglycerols and other components. For the diacylglycerol-enriched fat or oil to be used as an edible oil, it is desirable to reduce such impurities to enhance the flavour.

WO2007/075079, titled "Process for the Production of Diacylglycerol" published on 5 July 2007 discloses a partial hydrolysis process for producing diacylglycerol by reacting triacyiglycerol with water, in the presence of a 1 ,3-position specific immobilized lipase. In this process, a precise control of water is required to obtain an optimal diacylglycerol yield. However, a relatively high amount of free fatty acids, in an amount of 30 to 35% by weight are also generated as by-products in the process. Removal of this high amount of by-products generated by this process is not cost effective. US Pat. No. 6,337,414, titled "Process for Producing Partial Glyceride" issued to Masakatsu Sugiura, ef a/, on 8 January 2002 describes a glycerolysis process for producing diacylglycerol from triacylglycerol and glycerol in the presence of water and a lipase. The process is carried out under such conditions that crystals are partially precipitated in the reaction system in the course of the reaction. The concentration of free fatty acids present in the oil phase amounts to 8 to 30% by weight. This concentration is relatively lower than the amount disclosed in WO2007/075079. However, the disclosed process needs to be conducted at a low temperature, in the range of 0 to 25°C, in order to allow crystallization of free fatty acids and monoacylglycerols to take place. Furthermore, the disclosed process also requires long reaction time, of about 10 to 200 hours. This leads to higher operating costs.

WO03/029392, titled "Chemical Process for the Production of 1 ,3-diglyceride Oils", published on 10 April 2003 discloses a glycerolysis process for producing diacylglycerol by reacting triacylglycerol with glycerol in the presence of potassium acetate as a catalyst. However, this process requires high reaction temperatures, in the range of 190 to 240°C. Exposure of the reaction mixture to such elevated temperatures may encourage formation of other undesirable compounds.

Summary of the Invention

The above and other problems are solved and an advance in the art is made by a process for producing a diacylglycerol-enriched fat or oil in accordance with this invention. It is an advantage of a process in accordance with this invention that the process provides a diacylglycerol-enriched fat or oil obtained in accordance with the process of the invention has reduced amount of free fatty acids and monoacylglycerols. A second advantage of this invention is that the process allows glycerolysis reaction to occur without causing any inactivation of the immobilized lipase preparation by the glycerol itself.

In accordance with an embodiment of this invention, the process produces a diacylglycerol-enriched fat or. oil by adding glycerol to a reaction mixture containing a fat or oil and an immobilized lipase preparation at a rate of not more than 40 parts by weight of glycerol per 100 parts by weight of the immobilized lipase preparation per hour. In accordance with these embodiments, the process is performed in the following manner. The process begins by charging a reaction chamber with a fat or oil and an immobilized lipase preparation to obtain a reaction mixture. Glycerol is then added to the reaction mixture at a rate of not more than 40 parts by weight of glycerol per 100 parts by weight of the immobilized lipase preparation per hour. Water in the reaction mixture is removed by a dehydration method. Unreacted materials and by-products contained in the reaction mixture after the reaction are then separated from the reaction mixture to obtain the desired diacylglycerol-enriched fat or oil.

In accordance with some embodiments of this invention, additional water is added to the reaction mixture. In particular, in one of these embodiments, the additional water is pre- mixed with glycerol to obtain a glycerol-water mixture prior to adding the glycerol-water mixture to the reaction mixture at a rate of not more than 40 parts by weight of glycerol- water mixture per 100 parts by weight of the immobilized lipase preparation per hour. In accordance with other of these embodiments, the additional water is added in an amount of at least 0.2 parts by weight to 100 parts by weight of the immobilized lipase preparation.

In accordance with some embodiments of this invention, glycerol is added to the reaction mixture is in an amount of not more than 15% by weight of the fat or oil.

In accordance with some embodiments of this invention, the by-products produced by the process include free fatty acids and monoacylglycerols. In accordance with some of these embodiments, the total concentration of the free fatty acids and the monoacylglycerols in the reaction mixture after the glycerol is added is preferably not more than 15% by weight of the fat or oil. In accordance with other of these embodiments, the concentration of the free fatty acids in the reaction mixture after the glycerol is added is more preferably not more than 10% by weight of the oil or fat.

In accordance with some embodiments of this invention, the immobilized lipase preparation is prepared by immobilizing the lipase on a hydrophilic carrier.

Brief Description of the Drawings The above and other advantages and features of this invention are described in the following detailed description and are shown in the following drawings:

Figure 1 is a graph showing time course of the diacylglycerol production from Examples 1 to 5 and Comparative Examples 1 to 5.

Description of the Invention

The present invention provides a cost effective and environmentally less detrimental process for producing a diacylglycerol-enriched fat or oil which has an improved degree of purification as an edible oil while retaining the biologically advantageous components of the fat or oil used in the production of the fat or oil product.

According to the present invention, there is provided a process for producing a diacylglycerol-enriched fat or oil whereby glycerol is added to a reaction mixture containing a fat or oil and an immobilized lipase preparation at a rate of not more than 40 parts by weight of glycerol per 100 parts by weight of the immobilized lipase preparation per hour, to obtain a mixture comprising triacylglycerol, diacylglycerol, monoacylglycerol and free fatty acids. In one preferred embodiment, the glycerol is added to the reaction mixture at a rate of from 5 to 40 parts by weight of glycerol per 100 parts by weight of the immobilised lipase preparation per hour. The glycerol is added to the reaction mixture in a continuous or semi-continuous manner.

In accordance with one embodiment of the invention, the process comprises charging a reaction chamber with a fat or oil and an immobilized lipase preparation to obtain a reaction mixture. Glycerol is then added to the reaction mixture at a rate of not more than 40 parts by weight of glycerol per 100 parts by weight of the immobilized lipase preparation per hour. Glycerol is added to the reaction mixture in a controlled manner such that glycerolysis reaction can occur without causing any inactivation of the immobilised lipase preparation by the glycerol itself.

The process in accordance with an embodiment of this invention also comprises removing water content in the reaction mixture by a dehydration method. In one embodiment of the invention, the water in the reaction mixture is removed from the reaction chamber while glycerol is added to the reaction mixture. In another embodiment of the reaction, the water in the reaction mixture is removed after the glycerolysis reaction is completed. Unreacted materials and by-products contained in the reaction mixture after the glycerolysis reaction is completed are then separated from the reaction mixture to obtain the desired diacylglycerol-enriched fat or oil.

Examples of fat or oil used in the process of the present invention include crude or refined vegetable oils and/or animal oils. The vegetable oils may include any one of or a mixture of two or more of the following oil: canola oil, coconut oil, corn oil, cottonseed oil, palm oil, palm kernel oil, peanut oil, rapeseed oil, safflower oil, soybean oil, sunflower oil, olive oil, rice bran oil, corn bran oil, borage oil, evening primrose oil, flaxseed oil, grape seed oil, linseed oil, argania oil alfalfa oil, almond seed oil, apricot kernel oil, avocado oil, babassu oil, baobab oil, blackcurrant seed oil, brazil nut oil, cocoa seed oil, camellia oil, carrot oil, cashew nut oil, hazelnut oil, hemp seed oil, kiwi seed oil, macadamia nut oil, mango seed oil, melon seed oil, niger seed oil, peach kernel oil, perilla oil, pistachio oil, poppy seed oil, pumpkin seed oil, rambutan seed oil, rosehip oil, sesame oil, shea seed oil, tall oil, walnut oil and wheat germ oil. The Animal oils may include beef tallow, lard, and/or fish oil. The fat or oil used in the process may further be fat or oil obtained from the process of hardening, hydrogenation, transesterification, or randomisation, fractionation, distillation; and mixtures thereof. The processed fat and oil in accordance with this invention comprises C ₂ -C ₂ 4 saturated or unsaturated fatty acids or mixtures thereof. Example of such fats or oils include acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, zoomaric acid, stearic acid, oleic acid, elaidic acid, petroselinic acid, linoleic acid, a- linolenic acid, γ-linolenic acid, stearidonic acid, arachidic acid, arachidonic acid, gadoleic acid, arachic acid, dihomo-y-linolenic acid, eicosapentaenoic acid, behenic acid, erucic acid, adrenic acid, docosapentaenoic acid, docosahexaenoic acid, or nervonic acid. Furthermore, an isomeric form of any of the preceding acids may be used without departing from this invention.

The immobilised lipase preparation used in the present invention preferably is a lipase having immobilised onto a hydrophilic carrier. The hydrophilic carrier preferably is a silica gel. However, one skilled in the art will recognize that other suitable hydrophilic carriers may be employed without departing from this invention. The lipase used in the present invention can be a non-position specific lipase or a I mposition specific lipase. Preferable examples of such lipase include lipases derived from microorganisms of the genera Rhizopus, Aspergillus, Mucor, Candida and Pseudomonas, more specifically, lipases derived from Rhizopus delemar, Rhizopus japonicus, Rhizopus niveus, Aspergillus niger, Mucor javanicus, Mucor miehei, Candida rugosa, Candida antarctica, Pseudomonas cepacia and the like.

The immobilised lipase preparation is obtained by immobilising a lipase in accordance with a publicly known method for immobilising an enzyme, preferably by means of adsorption and/or absorption. Commercially available immobilised lipase preparations that can be used in the present invention include "Lipozyme TL IM" (trade name, product of Novozymes).

In the present invention, the proportion of the total glycerol added to the reaction mixture is not particularly limited. In one embodiment of the invention, the glycerolysis reaction is conducted in such a manner that the proportion of the total glycerol added to the reaction mixture is not more than 15% by weight of the fat or oil used in the process, preferably from 5 to 10% by weight of the fat or oil used in the process.

The process in accordance with this invention can be conducted in the absence or presence of additional water other than the water contained in the immobilised lipase preparation and/or the raw materials. In a preferred embodiment of the invention, the glycerolysis reaction is conducted in the presence of additional water. In this embodiment, the glycerol to be added to the reaction mixture is first pre-mixed with water to obtain a glycerol-water mixture. The resultant glycerol-water mixture is then added to the reaction mixture at a rate of not more than 40 parts by weight of glycerol-water mixture per 100 parts by weight of the immobilized lipase preparation per hour, preferably at a rate of from 5 to 40 parts by weight of glycerol-water mixture per 100 parts by weight of the immobilised lipase preparation per hour, to obtain a mixture comprising triacylglycerol, diacylglycerol, monoacylglycerol and free fatty acids.

The glycerolysis reaction in the process of the present invention is conducted in such a manner that the amount of water present in the reaction mixture at any point of time of the reaction is not more than 10 parts by weight to 100 parts by weight of the immobilised lipase preparation, more preferably, in an amount from 0.2 to 2 parts by weight to 100 parts by weight of the immobilised lipase preparation. During the glycerolysis reaction, the amount of water in the reaction mixture may change. As the amount of water increases, excess water may be removed from the reaction chamber by any known dehydration methods. In one preferred embodiment of the invention, the excess water is removed from the reaction chamber by means of reducing the pressure in the reaction chamber.

The process in accordance with this invention is conducted at a temperature within the operational temperature of the immobilised lipase preparation. In one preferred embodiment of the invention, the glycerolysis reaction is carried out at a temperature in the range of 50°C to 90°C, more preferably, in the range of 60°C to 80°C.

The by-products generated by the process of the present invention include free fatty acids and monoacylglycerols. These by-products can be easily separated from triacylglycerol and diacylglycerol contained in the reaction mixture after the glycerolysis reaction is completed, using standard separation techniques. These techniques include deodorisation, steam distillation, molecular distillation, adsorption chromatography, and any combination thereof. However, one skilled in the art will recognise that other separation methods may be employed without departing from the invention.

Preferably, the content of free fatty acids and monoacylglycerols in the reaction mixture after the glycerolysis reaction is not more than 15% by weight of the fat or oil used in the process. Preferably, the content of free fatty acids in the reaction mixture after the glycerolysis reaction is not more than 10% by weight of the fat or oil used in the process.

The diacylglycerol-enriched fat or oil produced by the process of the present invention preferably contains diacylglycerol in an amount of 25 to 60% by weight of the fat or oil used in the process. A higher yield of diacylglycerol is obtained by the process of the present invention. Preferably, a yield of 45 to 55% by weight of diacylglycerol is obtained.

The process of the present invention produces a diacylglycerol-enriched fat or oil which has an improved degree of purification as an edible oil while retaining the biologically advantageous components of the fat or oil used in the process, as the amount of undesirable by-products generated by the process is reduced. This also makes the process of the present invention more cost effective as less time and steps are required to remove the undesirable by-products. The process of the present invention is also more cost effective in that the process uses relatively inexpensive fats or oils as the raw materials and relatively low-cost lipase preparation as the catalyst. The process of the present invention also allows one to control the amount of glycerol or glycerol-water mixture to be added to the reaction mixture to allow glycerolysis reaction to occur without causing any inactivation of the immobilized lipase preparation by the glycerol itself.

The following examples are provided to further illustrate and describe the particular specific embodiments of the present invention, and are in no way to be construed to limit the invention to the specific procedures, conditions or compositions described therein.

EXAMPLES

Example 1

A 500 ml flask was first charged with 100g of palm olein and 10g of Lipozyme TL IM. Thereafter, 8g of glycerol were mixed with 0.08 ml of water and the resultant glycerol- water mixture was added at a rate of 1.6g glycerol/hour. The reaction mixture was reacted at 70°C for 8 hours. During the course of the reaction, samples were taken from the reaction mixture at 1 hour interval. The samples were then analysed for free fatty acids, monoacylglycerols (MAG), diacylglycerols (DAG) and triacylglycerols (TAG) by using high performance liquid chromatography. The results are shown in Table 2.

The amounts of glycerol and water in the glycerol-water mixture and its rate of addition vary in Examples 1 to 5, as shown in Table 1.

Comparative Example 1

A 500ml flask was first charged with 100g of palm olein and 10g of Lipozyme TL IM. Thereafter, 8g of glycerol were mixed with 0.08ml of water and the resultant glycerol- water mixture was added all at once at the start of the reaction. The reaction mixture was reacted at 70°C for 8 hours. During the course of the reaction, samples were taken from the reaction mixture at 1 hour interval. The samples were then analysed for free fatty acids, monoacylglycerols (MAG), diacylglycerols (DAG) and triacylglycerols (TAG) by using high performance liquid chromatography. The results are shown in Table 2. The amounts of glycerol and water in the glycerol-water mixture vary in Comparative Examples 1 to 5, as shown in Table 1.

Table 1

Rate of

Rate of Addition of

Amount Addition Glycerol-

Type Amount Amount

Amount of Reaction of Water

Example of of of Temp.

of Oil Lipozyme Time Glycerol Mixture per

Oil Glycerol Water

TL IM -Water g of

Mixture Lipozyme

TL IM

0.16 g/g

Palm 0.08ml 1.6

Ex. 1 100 g 8g (8%) 10 g 70 °C 8 hours Lipozyme TL

Olein (1 %) g/hour

IM/h

0.22 g/g

Palm i ig 0.11 ml 2.2

Ex. 2 100 g 10 g 70 °C 8 hours Lipozyme TL

Olein (11 %) (1 %) g/hour

IM/h

0.32 g/g

Palm 16g 0.16ml 3.2

Ex. 3 100 g 10 g 70 "C 8 hours Lipozyme TL

Olein (16%) (1%) g/hour

IM/h

0.42 g/g

Palm 21g 0.21ml 4.2

Ex. 4 100 g 10 g 70 °C 8 hours Lipozyme TL

Olein (21 %) (1 %) g/hour

IM/h

0.52 g/g

Palm 26g 0.26ml 5.2

Ex. 5 100 g 10 g 70 °C 8 hours Lipozyme TL

Olein (26%) (1 %) g/hour

IM/h

Comp. Palm 0.08ml

100 g 8g (8%) 10 g 70 °C 8 hours - - Ex. 1 Olein (1 %)

Comp. Palm ng 0.11ml

100 g 10 g 70 °C 8 hours - - Ex. 2 Olein (11 %) (1 %)

Comp. Palm 16g 0:i6ml

100 g 10 g 70 °C 8 hours - - Ex. 3 Olein (16%) (1 %) Comp. Palm 21g 0.21 ml

100 g 10 g 70 °C 8 hours - Ex. 4 Olein (21 %) (1 %)

Comp. Palm 26g 0.26ml

100 g 10 g 70 °C 8 hours - Ex. 5 Olein (26%) (1 %)

Comp. Palm 8g

100g 10 g 70 °C 8 hours - Ex. 6 Olein (8%)

Table 2

Example % FFA and MAG % DAG % TAG % FFA (by titration)

Ex. 1 8.90 46.06 45.04 7.00

Ex. 2 6.07 35.24 59.69 4.43

Ex. 3 4.45 25.80 72.19 3.85

Ex. 4 4.01 21.83 76.07 3.28

Ex. 5 2.73 11.26 95.47 2.09

Comp. Ex. 1 0.14 6.75 93.11 -

Comp. Ex. 2 0.06 4.09 95.85 -

Comp. Ex. 3 0.08 4.04 95.88 -

Comp. Ex. 4 0.05 3.77 96.18 -

Comp. Ex. 5 0.04 4.17 95.6 -

Comp. Ex. 6 1.73 16.59 82.57 - The above is a description of the subject matter the inventor regards as the invention and is believed that others can and will design alternative systems that include this invention based on the above disclosure.