Title of Invention	"A PROCESS FOR PREPRATION OF CHOLESTEROL LOWERING STRUCTURED LIPIDS FROM CONTAINING OMEGA 6 POLYUNSATURATED FATTY ACIDS."
Abstract	The present invention relates to a process for the preparation of cholesterol lowering structured lipids containing omega 6 polyvmsaturated fatty acids. The structured lipid, rich in MCFA and omega 6 PUFA, will provide a lipid source, primarily for use in a controlled diet for critically ill patients, which has lauric acid to provide quick energy and the changes in eicosanoid synthesis seen with omega 6 PUFA feeding will improve immunocompetence and a reduced inflammatory response to injury. Patients in need of elemental diets will benefit from having their immunocompetence improved. The structured lipid obtained as a result of enzymatic acidolysis reactions displayed an improved melting behavior compared to coconut oil or the oil blends. The structured lipid has a lower melting temperature than unmodified coconut oil thus maintaining them in the liquid state even at low temperatures... Hence the structured lipid at a temperature of 12-15°C remains a liquid without phase separation.

Title of Invention

"A PROCESS FOR PREPRATION OF CHOLESTEROL LOWERING STRUCTURED LIPIDS FROM CONTAINING OMEGA 6 POLYUNSATURATED FATTY ACIDS."

Abstract

The present invention relates to a process for the preparation of cholesterol lowering structured lipids containing omega 6 polyvmsaturated fatty acids. The structured lipid, rich in MCFA and omega 6 PUFA, will provide a lipid source, primarily for use in a controlled diet for critically ill patients, which has lauric acid to provide quick energy and the changes in eicosanoid synthesis seen with omega 6 PUFA feeding will improve immunocompetence and a reduced inflammatory response to injury. Patients in need of elemental diets will benefit from having their immunocompetence improved. The structured lipid obtained as a result of enzymatic acidolysis reactions displayed an improved melting behavior compared to coconut oil or the oil blends. The structured lipid has a lower melting temperature than unmodified coconut oil thus maintaining them in the liquid state even at low temperatures... Hence the structured lipid at a temperature of 12-15°C remains a liquid without phase separation.

Full Text	The present invention relates to a process for the preparation of cholesterol lowering structured lipids containing omega 6 polyunsaturated fatty acids. Coconut oil is a kernel oil which is a natural source of MCFA (53% of C 8:0 - C 12:0). Its lauric acid content is very high (48%). The lauric fats provide high nutritional value because either the portal or lymphatic systems can absorb them. They provide excellent nutrition for critically ill patients and do not cause any undue coronary difficulties despite their saturation. In fact, the lauric fats provide unexpected usefulness in protein catabolism, yielding positive nitrogen balance and enhanced protein formation. But coconut oil contains very low levels of polyunsaturated fatty acids (PUFA) (1.8% of linoleic acid). Long term feeding of coconut oil as the sole source of fat in the diet of experimental rats has shoAvn EFA deficiency symptoms characterized by scaly dermatitis, excessive water loss through the skin, impaired growth and reproduction and poor wound healing. In addition to this, myristic and palmitic acids that contribute to around 33% of the total fatty acids of coconut oil have been shown to be hypercholesterolemic which is a risk factor for cardiovascular disease. Medium chain fatty acids (MCFA) comprise fatty acids with 6 to 12 carbon chain length. MCFA offer numerous health benefits. They are easily absorbed, transported via the portal system and rapidly metabolized to yield quick energy and is not deposited in the body as fat. Medium chain triglycerides (MCT) have clinical applications in the treatment of fat malabsorption disorders, gall bladder disease, hyperlipidemia, obesity and deficiency of the carnitine system. But MCT alone cannot function as an ideal fat source for humans as they do not provide essential fatty acids (EFA) (EFA cannot be synthesized by the body and must be therefore ingested in the diet). Linoleic acid is an essential omega 6 PUFA as it cannot be synthesized by mammals. Linoleic acid can be foxmd in seeds of most plants except coconut, cocoa and palm nuts. It is utilized for the synthesis of complex lipids that provide the permeability barrier to the epidermis. They also help to maintain optimum levels of imsaturation in tissue lipids. It has been found to have a reducing effect on plasma cholesterol and an inhibitory effect on arterial thrombus formation. In the body, linoleic acid is metabolized to form arachidonic acid, which is a substrate for eicosanoid biosynthesis. Structured lipids are triacylglycerols containing mixtures of short-, medium-, and long-chain fatty acids attached to a glycerol backbone for specific functionality. Structured lipids are formed by (a) hydrolysis and esterification; (b) interesterification; (c) lipase-interesterification; (d) traditional chemical methods or (e) genetic manipulation. The structured lipids are particularly useful because of the way in which they are metabolized. Specific fatty acids can be attached. to specific portions of the glycerol backbone to ensure that these fatty acids are absorbed in a specific manner in the digestive process. Enzymatic acidolysis with specific lipase provides an efficient method to improve the nutritional and physical properties of lipids by incorporating the required acyl groups into specific positions of the triacylglycerols. A physical blend of MCFA rich triacylglycerols and PUFA rich triacylglycerols does not improve the absorption or metabolism of the fatty acids since each of the individual triacylglycerol maintains its original absorption rates. Safflower oil is a natural source of linoleic acid (68% of total fatty acids). Under controlled reaction conditions employing immobilized sn-1-3 specific microbial lipases, the saturated fatty acid of coconut oil can be partially replaced to incorporate the required amount of omega 6 PUFA from fi-ee fatty acids of safflower oil thus obtaining a unique structured lipid rich in MCFA and omega 6 PUFA. Because of faster absorption. Medium chain triglycerides (MCT) are useful as a calorie source in the treatment of hospitalized patients. Some hospitalized patients, particularly critically ill patients, require total parenteral nutrition and have a high risk of infection. These patients often have difficulty in obtaining the proper amount of nutrients and energy fi-om the diet; a diet that both minimizes the risk of infection and provides quick nutrition would be of vast benefit to these patients. These diets must provide the essential fatty acids, mcluding a limited amount of specific omega. 6 fatty acids It has been theorized that a structured lipid containing a medium chain fatty (C.6 -C.12) acid residue may provide improved absorption of other fatty acids attached to the structured lipid. A recent paper by Jensen (Am. J. Clin. Nutr.1992, 60: 518-524) disclosed that a structured lipid containing medium chain fatty acid residues and long chain fatty acid residues (omega 3 fatt>' acids from fish oil) are absorbed faster by the body than the physical mixture of the same fatty acids. There is no suggestion or teaching that a specific structured lipid containing MCFA and omega 6 PUFA would be useful to modify the lipid profile of the body. Reference may be made to an article, Kaimal, and Saroja, (1989, Journal of Oil Technologist's Association of India, Jan- Mar. 2-10). Wherein coconut oil was modified by enzymatic transesterification with safflower oil. The drawbacks of this method are that the products could not be expected to have specific type of fatty acid composition as two types of triacylglycerols were involved and the incorporation was poor as only preliminary studies were carried out. Reference may also be made to the article by Shimada, Y., Sugihara, A., Maruyama, K., Nagao, T., Nakayama, S., Nakano H., and Tominaga, Y., (1996) J. Am. Oil Chem. Soc.73, 1415-1420, wherein safflower oil or linseed oil were subjected to enzymatic acidolysis to produce structured lipids containing both EFA and MCFA. But the draw back of this invention is that the MCFA was a highly purified one obtained commercially which is not economical. Similarly, reference may also be made to the article by Akoh C.C.,.Jennings B.H and. Lillard. D.A, (1996) J. Am. Oil Chem. Soc. 72, 1059-1062 who used EPA ethyl esters (omega 3 PUFA) to modify evening primrose oil which is a rich source of y-linolenic acid, an omega 6 PUFA. The drawbacks of this invention were that, the structured lipid developed in this case had very low levels of MCFA and serves only as a single rich source of both omega 3 and omega 6 PUFA rather than aiming at optimal nutrition. Moreover, the EPA ethyl esters used are from commercial sources. Reference may be made to Lee, K-T and Akoh C.C, (J.Food Biochem. 1999, 23.197-208) wherein structured lipids were synthesized from synthetic tricaprylin and fish oil free fatty acids rich in omega 3 PUFA. The product when fed to mice for 21 days showed lower levels of serum, total cholesterol, LDL cholesterol and triacylglycerol in coiriparison with soybean oil. However it is not very clear from this study whether a physical mix (blending) of tricaprylin and fish oil (having same fatty acid composition as the structured lipids) would have the same effect as the structured lipids on the serum lipid profile. The soybean oil used as a control in this study does not provide a good comparison with the structured lipid, which had a different fatty acid composition. U.S. Pat. No. 5,661,180 to DeMichele, et al., describes a structured lipid, which provides substantial benefits in terms of modifying the prostanoid synthesis pathway, resulting in an improved response to endotoxic shock and other stress states. This structured lipid includes three components formed on a glycerol backbone. The first component is either alpha-linolenic acid or dihomogamma-linolenic acid. The second component is a medium chain (C 6 - C 12) fatty acid residue and the third component is a C 18 - C 22 fatty acid residue. The structured lipids in this case are prepared from a physical blend of oils subjected to a transesterification reaction to yield a reaction product that contains the structured lipids of this invention. The draw back of this is that the fatty acids will be randomized among the triglycerides of the two oils selected and there will not be any specificity in the positioning of fatty acids in the structured lipids. Canadian Patent Application 2000391 with a WPI Accession Number of 90-139962/19 discloses the triglyceride 2-(alpha-linolenoyl)/gamma-linolenoyl)-l,3-di (octanoyol/decanoyl) glycerol as useful in nutritional lipids. It is suggested that these tnglycerides are useful as components in nutritional compositions. The fatty acids are essential for control of tonus of smooth-muscle cells in the blood vessels or the tonus of the smooth muscle cells in the lungs and thus are useful in the control of respiratory distress. This reference neither suggests nor discloses the specific structured lipids of this invention nor the methods of using them. European Patent Application Number 87114297.2 discloses a triglyceride having a C.8 to C. 14 fatty acid residue at the 2-position of the triglyceride and a residue of C. 18 or higher fatty acids at the 1 and 3 positions thereof There is no suggestion or disclosure of the specific structured lipids of the invention or the benefits that can be realized by feeding the structured lipids of this invention. US Patent No. 5,962,712 deals with structured lipid containing gamma-linolenic or dihomogamma-linolenic fatty acid residue, a medium chain (C6-C12) fatty acid residue and N-3 fatty acid residue. The main object of the present invention is to provide A process for the preparation of cholesterol lowering structured lipids containing omega 6 polyunsaturated fatty acids . Yet another object of the present invention is to provide a process for the synthesis of structured lipids that are rich in MCFA and omega 6 PUFA, which is nutritionally advantageous by way of being hypocholesterolemic and hypotrigiyceridemic. Still, another object of the present invention is to provide a process for the synthesis of structured lipids containing omega 6 polyunsaturated fatty acids that could be clinically administered to patients in parenteral nutrition. Further another object of the present invention is to provide a process of Enzymatic acidolysis to produce fats (structured lipids) with a better triglyceride-distribution than known natural fats. Yet, another object of the present invention is to provide a process for structured lipids with an improved melting behaviour as they will hardly contain any trisaturated triglycerides. Yet another object of the present invention is to provide a process to develop a product, for use in a controlled diet for critically ill patients, comprising lauric acid to provide quick energy and n-6 PUFA to modulate their eicosanoid production especially in immune compromised patients and linoleic acid to take care of EFA requirement. Yet another object of the present invention is to develop a product by modifying coconut oil with the incorporation of omega 6 PUFA, which has a lower melting point than natural coconut oil. The present invention also provides a process for the manufacture cholesterol lowering structured lipids from coconut oil. Accordingly, the present invention provides a process for the preparation of cholesterol lowering structured lipids containing omega 6 polyunsaturated fatty acids comprising, (a) hydrolyzing triglycerides of vegetable oil source by known method to obtain free fatty acids rich in omega 6 PUFA; (b) interesterifying coconut oil with the free fatty acids obtained from step(a) at a preferable molar ratio of 1:3; (c) incubating with immobilized sn-1-3 lipase at 5-10% (w/w) of the substrate at a temperature range of 37-55°C for a period of 6-48 hours using a solvent such as herein described for enzymatic acidolysis thereby incorporating the required acyl groups into specific positions of the triacylglycerols; (d) interesterifying coconut oil with the free fatty acids obtained from step(a) at a preferable molar ratio of 1:3 molar ratio; (e) incubating with immobilized immobihzed sn-1-3 lipase at a temperature range of 37-55°C for a period of 6-48 hours using a solvent for enzymatic acidolysis thereby incorporating the required acyl groups into specific positions of the triacylglycerols; (f) separating the reaction products using adsorption chromatography using solvents selected from ethers, hexane and optionally with 1 part of acetic acid to obtain the structured lipids; and (g) recoverin g the structured lipids by scaling up in the range of 88 -92%. An embodiment of the present invention, wherein hydrolysis of triglycerides derived from an vegetable oil source, is done by a known method to obtain free fatty acids, rich in omega 6 PUFA. In another embodiment of the present invention, wherein interesterification of vegetable oil with free fatty acids thus obtained, is done at 1:3 molar ratio and at a temperature range of 37- 55°C, for a period of 6-48 hours, using a solvent, an hydrocarbon selected from petroleum ether, dioxane, isooctane, n- hexane, toluene etc.,. In yet another embodiment of the present invention, wherein the reaction is controlled by employing immobilized sn-1-3 lipase for enzymatic acidolysis thereby incorporating the required acyl groups into the specific positions of the triacylglycerols. In still another embodiment of the present invention, purification of reaction products is conducted using adsorption chromatography along with solvents selected from hexane and diethyl ether at a ratio ranging from 85:5 to 95:5 to obtain structured lipids. Further, in yet another embodiment of the present invention, stmctured lipids are recovered following scale up in the range of 88-92% having cholesterol-lowering capacity in the range of 10-36% Yet another embodiment of the present invention, wherein the fatty acid is derived trom a vegetable source of safflower oil. Still another embodiment of the present invention, wherein the vegetable oil is derived from coconut oil. Another embodiment of the present invention, wherein the interesterification is carried out using lipase enzyme at 5%(w/w) of the substrates. Yet another embodiment of the present invention, wherein Rhizomucor meihei an immobilized lipase is used. Still another embodiment of the present invention, wherein an immobilized lipase Rhizomucor meihei that can be utilized up to 25 cycles without loss of activity, is used, thus ensuring economic viability. Further another embodiment of the present invention, wherein the natural source of triglycerides is from coconut oil and fatty acid is derived from safflower oil for acidolysis reaction. The present invention also provides for a unique structured lipids rich in MCFA (Medium Chain Fatty Acid) and n-6 PUFA (Polyunsaturated fatty acid), which is nutritionally beneficial in being hypocholesterolemic and hypotriglyceridemic. An embodiment of the present invention, provides for structured lipids comprising lauric acid that produces quick energy for critically ill patients. Yet another embodiment of the present invention, wherein the structured lipids comprise n-6 PUFA to modulate eicosanoid production in immune compromised patients. Still another embodiment of the present invention, provides structured lipids that possess a very low melting point 12-15*^C and remains as a liquid without phase separation Yet another embodiment of the present invention, provides structured lipids comprising a cod liver oil fatty acids and triaglycerols of coconut oil for optimal nutrition. Still another embodiment of the present invention, comprises structured lipids having n-6 PUFA levels from 1.8 % in the unmodified coconut oil to 45.5% in the structured lipid. Yet another embodiment of the present invention , wherein the structured lipids comprise purity of triglycerides up to 96mg. In still another embodiment of the present invention, wherein structured lipids are recovered following scale up in the range of 88-92% having cholesterol lowering capacity in the range of 10-36%. Fig 1 is a pictorial representation of the steps involved in the synthesis of structured lipids. Fig 2 is a Graphical Diagram depicting the lower melting point of the Structured lipids enriched in Omega 6 PUFA. The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention. Example 1 Reaction conditions were optimized for the production of structured lipids from coconut oil with safflower oil free fatty acids employing a statistical design (Response surface methodology). Under optimized conditions as predicted by the model for maximum incorporation, lOOmg of coconut oil and 132 mg of free fatty acids from safflower oil were taken and 0.5 ml of hexane was added (the volume was maintained through out the reaction). The reaction mixture was taken in a 25 ml conical flask. 11.5 mg of immobiHzed lipase from Rhizomucor meihei was used and the incubation was carried out in an orbitally shaking waterbath at 160 rpm at 39°C for 48.5 hours. The modified triglycerides (structured Hpids) was separated by preparative thin layer chromatography with pefroleum ether: ethyl ether: acetic acid (80:20:1 v/v/v) as the developing solvent. The analysis of fatty acids by gas chromatography showed a maximum increase in omega 6 PUFA levels from 1.8% in the unmodified coconut oil to 45.5% in the structured lipids. The recovery of structured lipids was 96mg. Fatty Acid Composition (mol%) of Unmodified Coconut Oil and Free Fatty Acids Obtained from Safflower Oil and Structured Lipid. (Table Removed) Example 2 Enzymatic acidolysis reactions were scaled up in lab scale batch reactions of lOOg triglycerides and 132g of free fatty acids from safflower oil taken in a 2 liter Erlenmeyer flask that served as the bioreactor. 11.5g of immobilized lipase was used and the reaction was carried out in 500 ml of hexane. Incubation was carried out for 48.5 hours at 39°C in an orbitally shaking waterbath. Triglycerides were separated from the reaction mixture by column chromatography. A mixture of 20g each of alumina and silica gel (100-200 mesh ize) were activated at 200°C for 2 hrs and cooled in a desiccator. Slurry of this was made in hexane and packed in 4cm x 35cm glass columns. 30g of the sample from the reaction mixture was loaded on the column and eluted with 350 ml of hexane: diethyl ether (95:5 v/v). The fractions containing triglycerides were pooled and solvent was removed in a vacuum rotary evaporator. The purity of triglycerides was checked by thin layer chromatography. Recovery of the scaled up product was 90g for structured lipids rich in omega 6. The reactions when carried out at optimized conditions showed 46% incorporation of omega 6 PUFA into coconut oil. Example 3 The structured lipids were fed to rats in the diet as the sole source of fat at 10% levels for a period of 60 days. The serum and liver cholesterol was decreased by 10 and 36% respectively and the triglyceride level reduced by 17 and 16% respectively in the serum and liver. Lipid Profile of Rats fed Coconut oil. Blends and Structured lipid (Table Removed) Example 4 The structured lipids were subjected to DSC studies wherein their peak melting temperature and solid fat content was determined. The peak melting temperature of structured lipids enriched in omega 6 fatty acids was i2.7°C while that of coconut oil was 20.8°C . Advantages: (1) The present invention uses fatty acids from natural sources to create novel structured lipids with potential impacts on nutrition and health (2) The process could be employed for the modification of coconut oil, which is deficient in EFA by effectively incorporating omega 6 fatty acids. (3) The production could be scaled up without loosing the efficacy of the process. (4) The use of natural sources of triglycerides and fatty acids for the acidolysis reaction and the use of immobilized lipase which could be reused up to 25 cycles without loss of activity ensures that the process is economically viable. (5) Nutritionally the structured lipid proved to be more advantageous than oil blends with similar fatty acid composition or unmodified coconut oil in being hypocholesterolemic and hypotriglyceridemic. (6) The structured lipid, rich in MCFA and omega 6 PUFA, will provide a lipid source, primarily for use in a controlled diet for critically ill patients, which has lauric acid to provide quick energy and the changes in eicosanoid synthesis seen with omega 6 PUFA feeding will improve immunocompetence and a reduced inflammatory response to injury. Patients in need of elemental diets will benefit from having their immunocompetence improved. (7) The structured lipid obtained as a result of enzymatic acidolysis reactions displayed an improved melting behavior compared to coconut oil or the oil blends. The structured lipid has a lower melting temperature than unmodified coconut oil thus maintaining them in the liquid state even at low temperatures.. Hence the structured lipid at a temperature of 12 - 15°C remains a liquid without phase separation. We Claim: 1. A process for the preparation of cholesterol lowering structured lipids containing omega 6 polyxinsaturated fatty acids comprising, a) hydrolyzing triglycerides of vegetable oil source by known method to obtain free fatty acids rich in omega 6 PUFA; b) interesterifying coconut oil with the free fatty acids obtained from step(a) at a preferable molar ratio of 1:3; c) incubating with immobilized sn-1-3 lipase at 5-10% (w/w) of the substrate at a temperature range of 37-55°C for a period of 6-48 hours using a solvent such as herein described for enzymatic acidolysis thereby incorporating the required acyl groups into specific positions of the triacylglycerols; d) separating the reaction products using adsorption chromatography using solvents selected from ethers, hexane and with 1 part of acetic acid to obtain the structured lipids; e) removing solvent by known methods such as herein described and recovering the structured lipids in the range of 88-92%. 2. A process as claimed in claim 1, wherein the triglycerides are selected from a natural sources namely coconut oil. 3. A process as claimed in claims 1&2, wherein the fatty acids are selected from a vegetable source of safflower oil. 4. A process as claimed in claims 1-3, wherein the ethers are selected from group comprising petroleum ether, diethyl ether. 5. A process as claimed in claims 1-4, the solvent is selected from petroleum ether, dioxane, isooctane, n- hexane, toluene. 6. A process as claimed in claims 1-5, wherein the ratio of ethers.hexane used is in the range of 85:5 to 95:5. 7. A process as claimed in claims 1-6, wherein the immobilized lipase is obtained using Rhizomucor meihei 8. A process as claimed in claims 1-7, wherein an immobilized lipase obtained from Rhizomucor meihei can be used up to 25 cycles without loss of activity, 9. A process as claimed in claims 1-7 wherein the unique structured lipids obtained from interesterifying coconut oil with free fatty acids obtained from hydrolysis of triglycerides of a vegetable source, said structured lipids contain up to 46mol% of omega 6 polyunsaturated fatty acids and rich in medium chain fatty acids. 10. A process as claimed in claims 1-9 wherein the structured lipids are having a safflower oil fatty acids and triaglycerols of coconut oil. 11. A process as claimed in claims 1-8, wherein the structured lipids comprise n-6 PUFA levels from 1.8% in the unmodified coconut oil to 45.5% in the structured lipids. 12. A process for the preparation of cholesterol lowering structured lipids containing omega 6 polyunsaturated fatty acids substantially as herein described with reference to examples.

Full Text

The present invention relates to a process for the preparation of cholesterol lowering structured lipids containing omega 6 polyunsaturated fatty acids.
Coconut oil is a kernel oil which is a natural source of MCFA (53% of C 8:0 - C 12:0). Its lauric acid content is very high (48%). The lauric fats provide high nutritional value because either the portal or lymphatic systems can absorb them. They provide excellent nutrition for critically ill patients and do not cause any undue coronary difficulties despite their saturation. In fact, the lauric fats provide unexpected usefulness in protein catabolism, yielding positive nitrogen balance and enhanced protein formation. But coconut oil contains very low levels of polyunsaturated fatty acids (PUFA) (1.8% of linoleic acid). Long term feeding of coconut oil as the sole source of fat in the diet of experimental rats has shoAvn EFA deficiency symptoms characterized by scaly dermatitis, excessive water loss through the skin, impaired growth and reproduction and poor wound healing. In addition to this, myristic and palmitic acids that contribute to around 33% of the total fatty acids of coconut oil have been shown to be hypercholesterolemic which is a risk factor for cardiovascular disease.
Medium chain fatty acids (MCFA) comprise fatty acids with 6 to 12 carbon chain length. MCFA offer numerous health benefits. They are easily absorbed, transported via the portal system and rapidly metabolized to yield quick energy and is not deposited in the body as fat. Medium chain triglycerides (MCT) have clinical applications in the treatment of fat malabsorption disorders, gall bladder disease, hyperlipidemia, obesity and deficiency of the carnitine system. But MCT alone cannot function as an ideal fat source for humans as they do not provide essential fatty acids (EFA) (EFA cannot be synthesized by the body and must be therefore ingested in the diet).
Linoleic acid is an essential omega 6 PUFA as it cannot be synthesized by mammals. Linoleic acid can be foxmd in seeds of most plants except coconut, cocoa and palm nuts. It is utilized for the synthesis of complex lipids that provide the permeability barrier to the epidermis. They also help to maintain optimum levels of imsaturation in tissue lipids. It has
been found to have a reducing effect on plasma cholesterol and an inhibitory effect on arterial thrombus formation. In the body, linoleic acid is metabolized to form arachidonic acid, which is a substrate for eicosanoid biosynthesis.
Structured lipids are triacylglycerols containing mixtures of short-, medium-, and long-chain fatty acids attached to a glycerol backbone for specific functionality. Structured lipids are formed by
(a) hydrolysis and esterification;
(b) interesterification;
(c) lipase-interesterification;
(d) traditional chemical methods or
(e) genetic manipulation.
The structured lipids are particularly useful because of the way in which they are metabolized. Specific fatty acids can be attached. to specific portions of the glycerol backbone to ensure that these fatty acids are absorbed in a specific manner in the digestive process.
Enzymatic acidolysis with specific lipase provides an efficient method to improve the nutritional and physical properties of lipids by incorporating the required acyl groups into specific positions of the triacylglycerols.
A physical blend of MCFA rich triacylglycerols and PUFA rich triacylglycerols does not improve the absorption or metabolism of the fatty acids since each of the individual triacylglycerol maintains its original absorption rates.
Safflower oil is a natural source of linoleic acid (68% of total fatty acids). Under controlled reaction conditions employing immobilized sn-1-3 specific microbial lipases, the saturated fatty acid of coconut oil can be partially replaced to incorporate the required amount of
omega 6 PUFA from fi-ee fatty acids of safflower oil thus obtaining a unique structured lipid rich in MCFA and omega 6 PUFA.
Because of faster absorption. Medium chain triglycerides (MCT) are useful as a calorie source in the treatment of hospitalized patients. Some hospitalized patients, particularly critically ill patients, require total parenteral nutrition and have a high risk of infection. These patients often have difficulty in obtaining the proper amount of nutrients and energy fi-om the diet; a diet that both minimizes the risk of infection and provides quick nutrition would be of vast benefit to these patients. These diets must provide the essential fatty acids, mcluding a limited amount of specific omega. 6 fatty acids
It has been theorized that a structured lipid containing a medium chain fatty (C.6 -C.12) acid residue may provide improved absorption of other fatty acids attached to the structured lipid. A recent paper by Jensen (Am. J. Clin. Nutr.1992, 60: 518-524) disclosed that a structured lipid containing medium chain fatty acid residues and long chain fatty acid residues (omega 3 fatt>' acids from fish oil) are absorbed faster by the body than the physical mixture of the same fatty acids. There is no suggestion or teaching that a specific structured lipid containing MCFA and omega 6 PUFA would be useful to modify the lipid profile of the body.
Reference may be made to an article, Kaimal, and Saroja, (1989, Journal of Oil Technologist's Association of India, Jan- Mar. 2-10). Wherein coconut oil was modified by enzymatic transesterification with safflower oil. The drawbacks of this method are that the products could not be expected to have specific type of fatty acid composition as two types of triacylglycerols were involved and the incorporation was poor as only preliminary studies were carried out.
Reference may also be made to the article by Shimada, Y., Sugihara, A., Maruyama, K., Nagao, T., Nakayama, S., Nakano H., and Tominaga, Y., (1996) J. Am. Oil Chem. Soc.73, 1415-1420, wherein safflower oil or linseed oil were subjected to enzymatic acidolysis to produce structured lipids containing both EFA and MCFA. But the draw back of this
invention is that the MCFA was a highly purified one obtained commercially which is not economical.
Similarly, reference may also be made to the article by Akoh C.C.,.Jennings B.H and. Lillard. D.A, (1996) J. Am. Oil Chem. Soc. 72, 1059-1062 who used EPA ethyl esters (omega 3 PUFA) to modify evening primrose oil which is a rich source of y-linolenic acid, an omega 6 PUFA. The drawbacks of this invention were that, the structured lipid developed in this case had very low levels of MCFA and serves only as a single rich source of both omega 3 and omega 6 PUFA rather than aiming at optimal nutrition. Moreover, the EPA ethyl esters used are from commercial sources.
Reference may be made to Lee, K-T and Akoh C.C, (J.Food Biochem. 1999, 23.197-208) wherein structured lipids were synthesized from synthetic tricaprylin and fish oil free fatty acids rich in omega 3 PUFA. The product when fed to mice for 21 days showed lower levels of serum, total cholesterol, LDL cholesterol and triacylglycerol in coiriparison with soybean oil. However it is not very clear from this study whether a physical mix (blending) of tricaprylin and fish oil (having same fatty acid composition as the structured lipids) would have the same effect as the structured lipids on the serum lipid profile. The soybean oil used as a control in this study does not provide a good comparison with the structured lipid, which had a different fatty acid composition.
U.S. Pat. No. 5,661,180 to DeMichele, et al., describes a structured lipid, which provides substantial benefits in terms of modifying the prostanoid synthesis pathway, resulting in an improved response to endotoxic shock and other stress states. This structured lipid includes three components formed on a glycerol backbone. The first component is either alpha-linolenic acid or dihomogamma-linolenic acid. The second component is a medium chain (C 6 - C 12) fatty acid residue and the third component is a C 18 - C 22 fatty acid residue. The structured lipids in this case are prepared from a physical blend of oils subjected to a transesterification reaction to yield a reaction product that contains the structured lipids of this invention. The draw back of this is that the fatty acids will be randomized among the
triglycerides of the two oils selected and there will not be any specificity in the positioning of fatty acids in the structured lipids.
Canadian Patent Application 2000391 with a WPI Accession Number of 90-139962/19
discloses the triglyceride 2-(alpha-linolenoyl)/gamma-linolenoyl)-l,3-di
(octanoyol/decanoyl) glycerol as useful in nutritional lipids. It is suggested that these tnglycerides are useful as components in nutritional compositions. The fatty acids are essential for control of tonus of smooth-muscle cells in the blood vessels or the tonus of the smooth muscle cells in the lungs and thus are useful in the control of respiratory distress. This reference neither suggests nor discloses the specific structured lipids of this invention nor the methods of using them.
European Patent Application Number 87114297.2 discloses a triglyceride having a C.8 to C. 14 fatty acid residue at the 2-position of the triglyceride and a residue of C. 18 or higher fatty acids at the 1 and 3 positions thereof There is no suggestion or disclosure of the specific structured lipids of the invention or the benefits that can be realized by feeding the structured lipids of this invention.
US Patent No. 5,962,712 deals with structured lipid containing gamma-linolenic or dihomogamma-linolenic fatty acid residue, a medium chain (C6-C12) fatty acid residue and N-3 fatty acid residue.
The main object of the present invention is to provide A process for the preparation of cholesterol lowering structured lipids containing omega 6 polyunsaturated fatty acids .
Yet another object of the present invention is to provide a process for the synthesis of structured lipids that are rich in MCFA and omega 6 PUFA, which is nutritionally advantageous by way of being hypocholesterolemic and hypotrigiyceridemic.
Still, another object of the present invention is to provide a process for the synthesis of
structured lipids containing omega 6 polyunsaturated fatty acids that could be clinically
administered to patients in parenteral nutrition.
Further another object of the present invention is to provide a process of Enzymatic
acidolysis to produce fats (structured lipids) with a better triglyceride-distribution than
known natural fats.
Yet, another object of the present invention is to provide a process for structured lipids
with an improved melting behaviour as they will hardly contain any trisaturated
triglycerides.
Yet another object of the present invention is to provide a process to develop a product,
for use in a controlled diet for critically ill patients, comprising lauric acid to provide
quick energy and n-6 PUFA to modulate their eicosanoid production especially in
immune compromised patients and linoleic acid to take care of EFA requirement.
Yet another object of the present invention is to develop a product by modifying
coconut oil with the incorporation of omega 6 PUFA, which has a lower melting point
than natural coconut oil.
The present invention also provides a process for the manufacture cholesterol lowering
structured lipids from coconut oil.
Accordingly, the present invention provides a process for the preparation of cholesterol
lowering structured lipids containing omega 6 polyunsaturated fatty acids comprising,
(a) hydrolyzing triglycerides of vegetable oil source by known method to obtain free fatty acids rich in omega 6 PUFA;
(b) interesterifying coconut oil with the free fatty acids obtained from step(a) at a preferable molar ratio of 1:3;
(c) incubating with immobilized sn-1-3 lipase at 5-10% (w/w) of the substrate at a temperature range of 37-55°C for a period of 6-48 hours using a solvent such as herein described for enzymatic acidolysis thereby incorporating the required acyl groups into specific positions of the triacylglycerols;
(d) interesterifying coconut oil with the free fatty acids obtained from step(a) at a preferable molar ratio of 1:3 molar ratio;
(e) incubating with immobilized immobihzed sn-1-3 lipase at a temperature range of 37-55°C for a period of 6-48 hours using a solvent for enzymatic acidolysis thereby incorporating the required acyl groups into specific positions of the triacylglycerols;
(f) separating the reaction products using adsorption chromatography using solvents selected from ethers, hexane and optionally with 1 part of acetic acid to obtain the structured lipids; and
(g) recoverin g the structured lipids by scaling up in the range of 88 -92%.
An embodiment of the present invention, wherein hydrolysis of triglycerides derived from an vegetable oil source, is done by a known method to obtain free fatty acids, rich in omega 6 PUFA.
In another embodiment of the present invention, wherein interesterification of vegetable oil with free fatty acids thus obtained, is done at 1:3 molar ratio and at a temperature range of 37- 55°C, for a period of 6-48 hours, using a solvent, an hydrocarbon selected from petroleum ether, dioxane, isooctane, n- hexane, toluene
etc.,.
In yet another embodiment of the present invention, wherein the reaction is controlled by employing immobilized sn-1-3 lipase for enzymatic acidolysis thereby incorporating the required acyl groups into the specific positions of the triacylglycerols.
In still another embodiment of the present invention, purification of reaction products
is conducted using adsorption chromatography along with solvents selected from hexane and diethyl ether at a ratio ranging from 85:5 to 95:5 to obtain structured lipids.
Further, in yet another embodiment of the present invention, stmctured lipids are recovered following scale up in the range of 88-92% having cholesterol-lowering capacity in the range of 10-36%
Yet another embodiment of the present invention, wherein the fatty acid is derived trom a vegetable source of safflower oil.
Still another embodiment of the present invention, wherein the vegetable oil is derived from coconut oil.
Another embodiment of the present invention, wherein the interesterification is carried out using lipase enzyme at 5%(w/w) of the substrates.
Yet another embodiment of the present invention, wherein Rhizomucor meihei an immobilized lipase is used.
Still another embodiment of the present invention, wherein an immobilized lipase Rhizomucor meihei that can be utilized up to 25 cycles without loss of activity, is used, thus ensuring economic viability.
Further another embodiment of the present invention, wherein the natural source of triglycerides is from coconut oil and fatty acid is derived from safflower oil for acidolysis reaction.
The present invention also provides for a unique structured lipids rich in MCFA (Medium Chain Fatty Acid) and n-6 PUFA (Polyunsaturated fatty acid), which is nutritionally beneficial in being hypocholesterolemic and hypotriglyceridemic.
An embodiment of the present invention, provides for structured lipids comprising lauric acid that produces quick energy for critically ill patients.

Yet another embodiment of the present invention, wherein the structured lipids comprise n-6 PUFA to modulate eicosanoid production in immune compromised patients.
Still another embodiment of the present invention, provides structured lipids that possess a very low melting point 12-15*^C and remains as a liquid without phase separation
Yet another embodiment of the present invention, provides structured lipids comprising a cod liver oil fatty acids and triaglycerols of coconut oil for optimal nutrition.
Still another embodiment of the present invention, comprises structured lipids having n-6 PUFA levels from 1.8 % in the unmodified coconut oil to 45.5% in the structured lipid.
Yet another embodiment of the present invention , wherein the structured lipids comprise purity of triglycerides up to 96mg.
In still another embodiment of the present invention, wherein structured lipids are recovered following scale up in the range of 88-92% having cholesterol lowering capacity in the range of 10-36%.
Fig 1 is a pictorial representation of the steps involved in the synthesis of structured lipids. Fig 2 is a Graphical Diagram depicting the lower melting point of the Structured lipids enriched in Omega 6 PUFA.
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.
Example 1
Reaction conditions were optimized for the production of structured lipids from coconut oil with safflower oil free fatty acids employing a statistical design (Response surface methodology). Under optimized conditions as predicted by the model for maximum incorporation, lOOmg of coconut oil and 132 mg of free fatty acids from safflower oil were taken and 0.5 ml of hexane was added (the volume was maintained through out the reaction). The reaction mixture was taken in a 25 ml conical flask. 11.5 mg of immobiHzed lipase from Rhizomucor meihei was used and the incubation was carried out in an orbitally shaking waterbath at 160 rpm at 39°C for 48.5 hours. The modified triglycerides (structured Hpids) was separated by preparative thin layer chromatography with pefroleum ether: ethyl ether: acetic acid (80:20:1 v/v/v) as the developing solvent. The analysis of fatty acids by gas chromatography showed a maximum increase in omega 6 PUFA levels from 1.8% in the unmodified coconut oil to 45.5% in the structured lipids. The recovery of structured lipids was 96mg.
Fatty Acid Composition (mol%) of Unmodified Coconut Oil and Free Fatty Acids Obtained from Safflower Oil and Structured Lipid.
(Table Removed)
Example 2

Enzymatic acidolysis reactions were scaled up in lab scale batch reactions of lOOg triglycerides and 132g of free fatty acids from safflower oil taken in a 2 liter Erlenmeyer flask that served as the bioreactor. 11.5g of immobilized lipase was used and the reaction was carried out in 500 ml of hexane. Incubation was carried out for 48.5 hours at 39°C in an orbitally shaking waterbath.
Triglycerides were separated from the reaction mixture by column chromatography. A mixture of 20g each of alumina and silica gel (100-200 mesh ize) were activated at 200°C for 2 hrs and cooled in a desiccator. Slurry of this was made in hexane and packed in 4cm x 35cm glass columns.
30g of the sample from the reaction mixture was loaded on the column and eluted with 350 ml of hexane: diethyl ether (95:5 v/v). The fractions containing triglycerides were pooled and solvent was removed in a vacuum rotary evaporator. The purity of triglycerides was checked by thin layer chromatography. Recovery of the scaled up product was 90g for structured lipids rich in omega 6.
The reactions when carried out at optimized conditions showed 46% incorporation of omega 6 PUFA into coconut oil.
Example 3
The structured lipids were fed to rats in the diet as the sole source of fat at 10% levels for a period of 60 days. The serum and liver cholesterol was decreased by 10 and 36% respectively and the triglyceride level reduced by 17 and 16% respectively in the serum and
liver.
Lipid Profile of Rats fed Coconut oil. Blends and Structured lipid

(Table Removed)

Example 4
The structured lipids were subjected to DSC studies wherein their peak melting temperature and solid fat content was determined. The peak melting temperature of structured lipids enriched in omega 6 fatty acids was i2.7°C while that of coconut oil was 20.8°C .
Advantages:
(1) The present invention uses fatty acids from natural sources to create novel structured lipids with potential impacts on nutrition and health
(2) The process could be employed for the modification of coconut oil, which is deficient in EFA by effectively incorporating omega 6 fatty acids.
(3) The production could be scaled up without loosing the efficacy of the process.
(4) The use of natural sources of triglycerides and fatty acids for the acidolysis reaction and the use of immobilized lipase which could be reused up to 25 cycles without loss of activity ensures that the process is economically viable.
(5) Nutritionally the structured lipid proved to be more advantageous than oil blends with similar fatty acid composition or unmodified coconut oil in being hypocholesterolemic and hypotriglyceridemic.
(6) The structured lipid, rich in MCFA and omega 6 PUFA, will provide a lipid source, primarily for use in a controlled diet for critically ill patients, which has lauric acid to provide quick energy and the changes in eicosanoid synthesis seen with omega 6 PUFA feeding will improve immunocompetence and a reduced inflammatory response to injury. Patients in need of elemental diets will benefit from having their immunocompetence improved.
(7) The structured lipid obtained as a result of enzymatic acidolysis reactions displayed an improved melting behavior compared to coconut oil or the oil blends. The structured lipid has a lower melting temperature than unmodified coconut oil thus maintaining them in the liquid state even at low temperatures.. Hence the structured lipid at a temperature of 12 - 15°C remains a liquid without phase separation.

We Claim:
1. A process for the preparation of cholesterol lowering structured lipids containing omega 6 polyxinsaturated fatty acids comprising,
a) hydrolyzing triglycerides of vegetable oil source by known method to obtain free fatty acids rich in omega 6 PUFA;
b) interesterifying coconut oil with the free fatty acids obtained from step(a) at a preferable molar ratio of 1:3;
c) incubating with immobilized sn-1-3 lipase at 5-10% (w/w) of the substrate at a temperature range of 37-55°C for a period of 6-48 hours using a solvent such as herein described for enzymatic acidolysis thereby incorporating the required acyl groups into specific positions of the triacylglycerols;
d) separating the reaction products using adsorption chromatography using solvents selected from ethers, hexane and with 1 part of acetic acid to obtain the structured lipids;
e) removing solvent by known methods such as herein described and recovering the structured lipids in the range of 88-92%.

2. A process as claimed in claim 1, wherein the triglycerides are selected from a natural sources namely coconut oil.
3. A process as claimed in claims 1&2, wherein the fatty acids are selected from a vegetable source of safflower oil.
4. A process as claimed in claims 1-3, wherein the ethers are selected from group comprising petroleum ether, diethyl ether.
5. A process as claimed in claims 1-4, the solvent is selected from petroleum ether, dioxane, isooctane, n- hexane, toluene.
6. A process as claimed in claims 1-5, wherein the ratio of ethers.hexane used is in the range of 85:5 to 95:5.
7. A process as claimed in claims 1-6, wherein the immobilized lipase is obtained using Rhizomucor meihei
8. A process as claimed in claims 1-7, wherein an immobilized lipase
obtained from Rhizomucor meihei can be used up to 25 cycles without
loss of activity,
9. A process as claimed in claims 1-7 wherein the unique structured lipids obtained from interesterifying coconut oil with free fatty acids obtained from hydrolysis of triglycerides of a vegetable source, said structured lipids contain up to 46mol% of omega 6 polyunsaturated fatty acids and rich in medium chain fatty acids.
10. A process as claimed in claims 1-9 wherein the structured lipids are having a safflower oil fatty acids and triaglycerols of coconut oil.
11. A process as claimed in claims 1-8, wherein the structured lipids comprise n-6 PUFA levels from 1.8% in the unmodified coconut oil to 45.5% in the structured lipids.
12. A process for the preparation of cholesterol lowering structured lipids
containing omega 6 polyunsaturated fatty acids substantially as herein
described with reference to examples.

Documents:

323-del-2002-abstract.pdf

323-del-2002-claims.pdf

323-del-2002-complete specification (granted).pdf

323-del-2002-correspondence-others.pdf

323-del-2002-correspondence-po.pdf

323-del-2002-description (complete).pdf

323-del-2002-drawings.pdf

323-del-2002-form-1.pdf

323-del-2002-form-2.pdf

323-del-2002-form-3.pdf

323-del-2002-form-4.pdf

323-del-2002-petition-137.pdf

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Patent Number

195816

Indian Patent Application Number

323/DEL/2002

PG Journal Number

31/2009

Publication Date

31-Jul-2009

Grant Date

21-Apr-2006

Date of Filing

27-Mar-2002

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	REENA RAO	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUE, MYSORE, INDIA.
2	KARI SAMBAIAH	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUE, MYSORE, INDIA
3	BELUR RAMASWAMY LOKESH	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUE, MYSORE, INDIA

PCT International Classification Number

C11C 3/10

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA