FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
[See section 10, Rule 13]
NOVEL METHODS OF ISOLATION OF POLYUNSATURATED FATTY ACIDS;
PHARMED MEDICARE PVT. LTD., A COMPANY INCOPORATED UNDER THE COMPANIES ACT, 1956, WHOSE ADDRESS IS 141 WALCHAND HIRACHAND MARG, MUMBAI - 400 001, MAHARASHTRA, INDIA.
THE FOLLOWING SPECIFICATION DESCRIBES THE INVENTION.
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TECHNICAL FIELD
Invention relates to methods of isolation of fats, more particularly, polyunsaturated fatty acids from compositions containing the same including a biomass.
DESCRIPTION
Essential fatty acids are those which are essential in the health of an organism but can not be synthesized in animal / human body and must be supplied through food sources. Omega-6 fatty acids and Omega-3-fatty acids are the fatty acids amongst this group that play a crucial role in brain function as well as normal growth and development. Both these classes of essential fatty acids belong to polyunsaturated fatty acids (PUFAs), generally necessary for stimulating skin and hair growth, maintaining bone health, regulating metabolism, and maintaining reproductive capability.
Omega-6 fatty acids have the first double bond in the carbon backbone of the fatty acid that occurs in the sixth carbon from the methyl end of the fatty acid. The biological effects of the Omega-6 fatty acids, particularly linoleic acid which is also an essential fatty acid, are largely mediated by their interactions with omega-3 fatty acids. Arachidonc acid, another physiologically important n-6 fatty acid and is the precursor of prostaglandins and other physiologically active molecules.
A family of polyunsaturated fatty acids that have a carbon-carbon double bond in the w-3 position are known as Omega 3 fatty acids. ct-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid, that have either 3, 5 or 6 double bonds in c/s-configuration in a carbon chain of 18, 20 or 22 carbon atoms respectively, are considered as Omega 3 fatty acids important in human nutrition. . 2

aonongst Omega-3 fatty acids, Long Chain-PUFAs, also abbreviated as LC-PUFAs .by definition, are fatty acids that have chain lengths greater than 18 carbon atoms and contain two or more double bonds. . Two such fatty acids that are important in infant nutrition are arachidonic acid (ARA or AA) and docosahexonoic acid (DHA). These are present in small concentrations in human milk. Attention to these two fatty acids was attracted when it was found out that infants fed on infant formulas did not develop as well as infants fed on breast milk.
Biosynthesis of ARA is possible within the body from the essential fatty acid alpha-linoleic acid, and that of DHA from the essential fatty acid alpha-linolenic acid. Both term and pre-term infants can synthesize the LC-PUFAs from the respective essential fatty acids, but controversy has centered around the fact that breastfed infants have higher plasma concentrations of these DHA than formula-fed infants. This information could be interpreted to imply that formula-fed infants cannot synthesize enough DHA to meet ongoing needs.
ARA is known to be important in brain development in third trimester of pregnancy and DHA is found in high concentration in brain and also is an important component of the photoreceptor of the retina. These fatty acids are supplied to the fetus from maternal plasma during pregnancy, and it is believed that the pre-term infant born during the third trimester is at much greater risk for deficiency of ARA as well as DHA than the term infants and term infants in at least DHA in absence of mother's milk. It is hypothesized that the addition of DHA to infant formula will improve infant visual function as well as brain development, and direct supplementation of ARA is important, in addition to DHA, for pre-term infants. 3

In the context for adults as well as infants, the balance of Omega 6 to Omega 3 is considered as most critical, which is recommended to be between 4:1 or 3:1. Lesser ratio is not considered to be of additional utility, although there is no harm in lesser ratios too, if not uneconomical. In developed countries, particularly in case of wester diets, due to dominance of seeds and nuts, and the oils extracted from them forming almost 20% of calories consumed through food, are known to have led to a highly undesirable ratio of about 11:1 to 30:1 and at time 50:1 also, which can be corrected only by appropriate supplementation. Balance of Omega 6 and Omega 3 fatty acids is considered very critical because the body also produces hormones from these essential fatty acids that have opposite effects. Those from omega-6 fatty acids are pro-inflammatory (an important component of the immune response), blood clotting, and cell proliferation, and those from omega-3 fatty acids are anti-inflammatory. Imbalance of these esential fatty acids is also known to lead to long-term diseases such as heart disease, cancer, asthma, arthritis, and depression.
It is generally acknowledged, although conclusive work is yet to be done, that omega-6 fatty acids may be useful for treating Anorexia Nervosa, Attention deficit/Hyperactivity Disorder (ADHD), Diabetes, Eye Disease, Osteoporosis, Menopausal Symptoms, Premenstrual Syndrome (PMS), Acne and Psoriasis, Eczema, Alcoholism, Allergies, Rheumatoid Arthritis, Cancer, Weight Loss, High Blood Pressure and Heart Disease, Tuberculosis, and Ulcers.
For the purpose of direct supplementation, the only feasible source at present is to use natural raw material sources for production, extraction and isolation of the essential fatty acids, whether for adult foods or infant foods, because the biomass can not be used unprocessed for it contains many other constituents
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that make consumption of effective quantities unpracticable or are also associated with undesirable constituents.
Presently known feasible natural sources for the omega 3 fatty acids are fish or marine protists. Various methods have been reported in literature and patents about the extraction, purification and isolation of the Polyunsaturated fatty acids.
US no. 6,528,669 broadly provides a method for recovering polyunsaturated fatty acids from an urea adduct containing saturated and/or monounsaturated fatty acids and said urea adduct to extraction treatment with a subcritical or supercritical fluid at a temperature not above 70.degree. C.
In US patent no. 3,950,365 the claimed invention is the development of a method for separation of small amounts of polyunsaturated components from mixtures of fatty acids or fatty acid esters, consisting essentially of heating a mixture of higher fatty acid compounds selected from the group consisting of higher fatty acids and esters thereof with alkanols having 1 to 4 carbon atoms and glycerol, said mixture containing a major amount of mono-unsaturated higher fatty acid compounds and from 3% to 25% by weight of polyunsaturated higher fatty acid compounds, to a temperature of between 90.degree.C and 150.degree.C in the presence of an organic macroporous, acid ion exchange resin having a specific surface area of at least 35 m.sup.2 /gm and devoid of gel characteristics, for a time sufficient to lower to the desired value the amount of polyunsaturated higher fatty acid compounds in said mixture, distilling and recovering said mixture of fatty acid compounds substantially free of polyunsaturated components.
In U.S. Pat. No. 4,377,526, a mixture of fatty acids containing EPA is treated with urea in order to remove saturated fatty acids and fatty acids of lower unsaturation. The resultant solution is then subjected to fractional distillation in order to obtain higher yields of EPA. The fractional distillation, however, requires
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a temperature of at least 180.degree. C. over a period of at least 40 minutes. The best purity which can be obtained by this method set forth in any of the examples of this patent is 92.9%. Furthermore, it has been discovered that a substantial amount of the EPA produced by this method, in some cases as high as 20%, has some degree of cis-trans conversion. Any amount of the trans- form of EPA is strictly undesirable for food or pharmaceutical use.
Abu-Nasr, A. N. et al, J. Am. Oil Chemists Soc. 31, 41-45 (1954) discloses isolation of methyl eicosapentaenoate and ethyl docosahexaenoate starting with cod liver oil acids, using preliminary concentration by precipitation of the pure complexes followed by chromatographic separations. This technique does not give high enough purity.
Teshima, S. et al, Bulletin of the Japanese Society of Scientific Fisheries, 44 (8) 927 (1978) describe a method for isolation of EPA and DHA from squid liver oil by saponifying with ethanolic potassium hydroxide, extracting the fatty acids with ether and methylating. The crude fatty acid methyl ester is purified by column chromatography on Silica Gel 60 and then the EPA is separated from the DHA by column chromatography on a mixture of silver nitrate and silica gel. The problem with this technique is that there are often traces of silver left in the final product, which is extremely undesirable in a food or pharmaceutical for human consumption. Furthermore, very high amounts of solvent are necessary in order to conduct the column chromatography. Other disclosures of the use of column chromatography to separate and purify, to some extent, EPA are described in Japanese Kokai No. 56-115736 and Russian 973,128.
Another prior art method of obtaining high purity EPA is disclosed in British patent publication No. 2,148,713. This publication describes a process in which the double bonds of the unsaturated fatty acids, in a mixture of fatty acids, are
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iodinated, followed by saponification of the iodinated oil, extraction of the fatty acids from the saponification mixture, methylation of the iodinated fatty acids, separation of the fatty acids by column chromatography, and then deiodination of the desired fractions. This process permits excellent resolution of the fatty acids upon eventual column chromatography, and protects the fatty acids from oxidation during processing. When used to separate EPA from a natural source of EPA, such as cod liver oil, a yield of over 90% and a purity of 96-100% may be obtained. It has been found, however, that a substantial amount of cis-trans conversion occurs in the course of this process, so that the product obtained of a purity of 96-100% is not pure all cis- EPA. Furthermore, iodine is not on the list of GRAS materials.
Privett, O. S. et al, J.Am. Oil Chemist. Soc, 36, 443-449 (1959), describe a technique involving a combination of low temperature crystallization and urea complexes. In the analysis of pork liver lipids, low temperature fractionation was first used to obtain two fractions and the filtrate was subjected to fractionation from methanol via the urea inclusion compounds. Each fraction and the filtrate was esterified and distilled, and the various distillates subjected to analysis. Swern, D. et al, J. Am. Oil Chemist. Soc, 29, 614-615 (1952) disclose precipitating urea complexes from olive oil to remove saturated and mono-unsaturated compounds and then subjecting the acids or esters isolated from the urea complexes to low temperature crystallization and to fractional distillation in order to produce oleic acid at 97-99% purity. Swern, D. et al, J.Am. Oil Chemist. Soc, 29, 431-434 (1952) and U.S. Pat. No. 2,838,480 isolate oleic acid from tallow, grease or red oil in 80-95% purity by first separating saturated acids by crystallization from 90% methanol at 0.degree. C, followed by addition of urea to the filrate to precipitate the adduct of oleic acid at room temperature. 7

Rubin, in British Patent No. 2,148,713, describes a process in which the double bonds of the unsaturated fatty acids, in a mixture of fatty acids, are iodinated, followed by saponification of the iodinated oil, extraction of the fatty acids from the saponification mixture, methylation of the iodinated fatty acids, separation of the fatty acids by column chromatography, and then deiodination of the desired fractions. This process permits excellent resolution of the fatty acids upon eventual column chromatography, and protects the fatty acids from oxidation during processing. When used to separate EPA from a natural source of EPA, such as cod liver oil, a yield of over 90% and a purity of 96-100% may be obtained. It has been found, however, that a substantial amount of cis-trans conversion occurs in the course of this process, so that the product obtained is not pure all-cis EPA. Furthermore, iodine is not on the list of GRAS materials. Rubin, in U.S. Pat. No. 4,792,418, describes a process for obtaining pure polyunsaturated fatty acids such as EPA and DHA and their esters, without degradation thereof. This process involves first hydrolyzing the triglycerides of the oil source under mild conditions, as with lipase, removing non-saponifiable material by washing with organic solvent, treating with urea in order to remove saturated and monounsaturated fatty acids to form a urea complex with saturated and mono-saturated fatty acids, dissolving the remainder in an organic solvent, preferably acetone, slowly cooling and fractionally removing solidified material as it forms.
Barclay, in U.S. Pat. Nos. 5,518,918, and 6,451,567, discloses production of .omega.-3 fatty acids by growing Thraustochytrium, Schizochytrium, and mixtures thereof with high omega- 3 fatty acid content in a fermentation medium.
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However, this process produces a mixture of .omega.-3 fatty acids, rather than individual omega.-3 fatty acids.
Best et al., in U.S. Pat. No. 5,928,696, extract oils from native substances using centrifugation. Again, this method produces mixtures of unsaturated fatty acids rather than pure individual fatty acids.
Kyle et al., in U.S. Pat. No. 5,397,591, disclose a method for obtaining DHA from cultivation of Dinoflagellates in a fermenter, induction of the dinoflagellates to produce single cell oil having a high proportion of DHA, and recovery of that oil. Preferably, the oil recovered contains at least about 20% by weight of DHA, and more preferably, more than about 35% by weight DHA. The product recovered is not pure DHA, but a mixture of DHA in other oils.
Corley et al in US 6,399,803 relates generally to adsorptive separation of triglycerides. This invention relates to a process for separating a first triglyceride comprising a docosahexaenoic acid residue from at least one other triglyceride using a chromatographic separation zone having a stationary phase which comprises metal ions that are capable of coordinating with a double bond of a fatty acid residue of the first triglyceride to form a metal complex with the fatty acid residue.
US 5,897,994 describes an enzymatic process for the fractionation of polyunsaturated fatty acids, in which esterification of a mixture of fatty acids is carried out by enzymatic catalysis, the esters formed are then separated from the fatty acids and is enriched in the desired polyunsaturated fatty acids, characterized in that the esters of the fatty acids are separated by controlled saponification of the fatty acids, extraction of the soaps formed with water, acidification of the aqueous phase and extraction of the acids formed with a non-polar solvent
Cornieri et al., in U.S. Pat. No. 5,130,061, disclose a process for extracting polyunsaturated fatty acid esters from fish oils. However, the product obtained is a mixture of EPA and DHA esters.
Rubin, in U.S. Pat. No. 4,792,418,describes a process for Pure DHA and pure EPA can be obtained from a mixture of EPA and DHA in a solution by forming salts of DHA and EPA which have different solubility in the solvent, cooling the solution until the salt of EPA is formed, filtering the solution to recover the salt of EPA, and acidifying the EPA salt and the DHA salt to obtain pure EPA and pure DHA.
Hoeksema in US6166231 provides a novel method for extraction of lipids, specifically edible oil, from microbial biomass. The invention uses an appropriate solvent to extract oil from relatively fine particles in an aqueous slurry without the need to dry the slurry or reform the material to create larger-sized particles.
Reuker et al in US6750048 describes a process for obtaining lipid from microorganisms by lysing cells of the microorganisms, treating said lysed cell mixture using an extraction process conducted in a medium that containing less than about 5% of an organic solvent wherein two layers are obtained one being light and one heavy and a step of treating said lipid to obtain non-emulsified lipid in light layer.
US6441208 describes a process for the isolation of Polyunsaturated fatty acids from microbial biomass, by culturing microorganisms in a fermentation broth under conditions, pasteurising either the fermentation broth or a microbial biomass derived there from; and extracting, isolation or recovering the compound from the microbial biomass.
A process in US6727373 is explained wherein for obtaining an oil comprising at least one polyunsaturated fatty acid from a microbial biomass, by providing
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biomass with a dry matter content of from 25 to 80%; granulating and drying the biomass into granules and then extracting or isolating the oil from the granules.
However, present methods are not user friendly enough in terms of operations and on account of the loss of Poly unsaturated fatty acids by oxidation. We hereby report a novel route of purification which involves absolutely ideal conditions for recovery of the said fatty acids by a most economical route. Also the process involves almost no heat treatment during the process and avoids any product loss.
Object of this invention is to develop more efficient methods for isolation of essential fatty acids from compositions containing them and from which their extraction and isolation is necessary and shall also cover one or more of a product prepared by the process of invention.
The results given below are an account of interim results on one of the preferred embodiments, which shall be improved upon by further work and fine tuned until complete specification is submitted. The details of work done so far disclosed below serve as illustrations and do not limit the scope of actual techniques used or scope of or range of reaction conditions or process conditions claimed. The techniques and reaction conditions or process conditions disclosed below are subject matter of ongoing trials and fine tuning or trials in alternative or better conditions. Several other adaptations of the embodiments will be easily anticipated by those skilled in this art and they are also included within the scope of this work.
Further, a singular shall also cover within its scope, unless context does not permit, pleural of the same and also includes one or more of an equivalent of the same that shall perform the same function, if substituted. Thus a mention of "an essential fatty acid" includes one or more of a linoleic acid and alpha linolenoic
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acid and the like and " a long chain polyunsaturated acid" includes EPA, DHA and the like.
In the present invention, we report a very economical and industrially viable purification process for isolation and purification of fatty acids, more particularly polyunsaturated fatty acids, with affinity chromatography and molecular separation techniques wherein the process involves minimal loss of the fatty acids by oxidation, less harsh treatment during the hydrolysis or esterification of the fatty acids.
In the present embodiment, the omega 3 fatty acids contained in phospholipids or membrane lipids of any natural source either from microorganisms, plants or fish oil is passed through a column containing a polystyrene based resin. This resin is non ionic adsorbent such as Tulsion ADS600 from Thermax. The lipids with the fatty acids get adsorbed on to the resin. The resin is washed to remove any unbound impurities. Then the resin is desorbed with alkaline methanol solution in water at alkaline pH. The fatty acids in lipids then start eluting out of the resin during which the insitu hydrolysis of fatty acids take place. Then the free fatty acids and lipids in the methanol layer were filtered and taken for methanol removal by distillation.
The removal of methanol is done in a falling film evaporator under reduced pressure. The temperature of the mixture during distillation was well controlled. Addition of suitable antioxidants such as ascorbic acid and butylated hydroxy toluene prevents the fatty acid to get oxidized. The aqueous solution was then neutralized.
The neutralized fatty acid solution was then passed through a nanofiltration membrane with a molecular weight cut off between 250 to 280 daltons. The lower molecular weight fatty acids such as Butyric acid, myristic acid, lauricacid,
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etc pass out of the membrane as permeate. The higher molecular weight fatty acids such as Docosahexaenoic acid, Docosapentaenoic acid and Eicosapentaenoic acid were retained.
The retained concentrated poly unsaturated fatty acids were then again diluted with water and reconcentrated in the nanofiltration membrane to remove any further removal of short chain fatty acids. This was repeated 3 -5 times till complete removal of short chain fatty acids.
Then the concentrated polyunsaturated fatty acids were mixed and diluted with water and then passed through a polystyrene based resin wherein all the fatty acids were adsorbed and water was eluted out. The resin was washed and then eluted with 100% acidified ethanol and the eluent was heated to 60 -65°C with stirring. The ethyl esters obtained were then isolated by removal of excess ethanol. The fatty acid ethyl ester mixture was analysed by GC.
Similarly this process is also extended and covers purification of other unsaturated fatty acids covered under omega 6 fatty acids such as arachidonic acid, linolenic acid, etc as well as omega 9 fatty acids such as erucic acid, nervonic acid, etc.
The starting material for isolation of these fatty acids can be from any of the sources such as from microbial biomass generated from various micoroganisms which produce these poly unsaturated fatty acids, plant resources, fish oil, etc.
The above said chromatographic method can be applied to one or more of a varient of a chromatographic method, including but not limited to following :
1. Fixed bed Chromatographic separation carried out in pulse, continuous-pulse, or continuous mode:
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a. fixed bed adsorbent is contained within a column, the feed and
desorbent being injected at one end and the separated or enriched
fractions, following an axial traverse, being collected at the other
b. fixed bed adsorbent is contained within a column, the feed and
desorbent being injected at the circumference and the separated or
enriched fractions, following a radial traverse, being collected
through an inner channel at the center
c. fixed bed adsorbent is contained within a column, the feed and
desorbent being injected through an inner channel at the center
and the separated or enriched fractions, following a radial traverse,
being collected at the circumference
d. fixed bed of solid adsorbent is contained within a vertically
mounted, rotating annulus, the feed and desorbent being injected at
the top and the separated or enriched fractions being collected at
the bottom.
e. fixed bed of solid adsorbent is contained within several serial
sections or columns in a closed loop, each individually capable of
receiving and relieving fluid, and equipped with a fixed arrangement
of feed, desorbent and take-off ports, that ratchet forward at fixed
intervals in a direction concurrent with the liquid flow, simulating
countercurrent movement of the fixed-bed adsorbent
2. Expanded bed chromatography: The adsorbent media is expanded by an upward liquid flow to increase the distance between the chromatographic beads. Given the created distance, particulate material is allowed to pass through the column without clogging the system. The unwanted material is
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washed away and then desorption can be carried out in either fluidized bed or packed bed.
3. 3. Moving bed chromatography is one of the ways by which the fixed-bed system can be made continuous. The desorbent is continuously fed into one end of the column, the adsorbent is made to move in the direction opposite to that of the desorbent, while the feed mixture is also supplied at the middle of the adsorbend bed.
4. A continuous Liquid-Solids Circulating Fluidized Bed (LSCFB) that consists of two fluidized bed columns, a fluidized bed adsorber (downer) operating in conventional fluidized bed mode for adsorption of molecules of interest and a fluidized bed riser for desorption of molecules (operating as a riser fluidized bed) to provide regenerated particles. Resin particles circulate continuously between the riser and the downer i.e. the particles that have adsorbed molecules in the absorber pass from the adsorber (downer) to the desorber where they are regenerated and the so regenerated particles are return to the adsorber near the top of the adsorber column. The LSCFB can be used in processes for continuous recovery of the molecules of interest.
5. Simulated moving bed chromatography: SMB is a chromatographic technique based on a flow of liquid (mobile phase) moving countercurrent to a constant flow of solid (stationary phase). Countercurrent flow enhances the potential for the separation and, hence, makes the process more efficient. It also allows a continuous flow of feed material to be separated, which improves the throughput of the equipment compared to traditional batch chromatography.
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Further purification of the fatty acid mixture to obtain pure fatty acids can be carried out by subjecting the fatty acid mixture to polystyrene based resin wherein the fatty acids are sequentially eluted by varying different strength of methanol water mixture.
Thus, in one such experiments, one liter of crude fish oil was mixed in a high speed shear mixer with 1 liter of water and the suspension in water was as such loaded in a 1 Liter expanded bed column containing polystyrene based hydrophobic resin like Tulsion ADS-600 from Thermax. A flow rate of ~1.5BV/hr was maintained. The lipids were adsorbed on the resin and the cell debris was separated.
1BV (bed volume - equivalent to the quantity of resin taken) of water wash was given at the same flow rate. Elution was done in counter current direction using 1BV of methanolic NaOH. 1gm of ascorbic acid was added to the eluent to prevent oxidation. It was then neutralized using 0.1 N HCI. The methanol was then removed under reduced pressure in a falling film evaporator at room temperature. The neutralized fatty acid solution was then passed through a nanofiltration membrane with a molecular weight cut off between 250 daltons. The lower molecular weight fatty acids such as oleic acid, myristic acid, linoic acid, etc were passed out of the membrane as permeate. The higher molecular weight fatty acids such as Docosahexaenoic acid, Docosapentaenoic acid and Eicosapentaenoic acid were retained.
The retained concentrated poly unsaturated fatty acids were then again diluted with water and reconcentrated in the nanofiltration membrane to achieve any further removal of short chain fatty acids. This was repeated untill complete removal of short chain fatty acids, usually up to four times. 16

Then the concentrated polyunsaturated fatty acids were diluted with water to a volume of 1 Liter and were further purified in a packed bed chromatographic column. The resin used was Tulsion ADS-600 from Thermax. A flow rate of 1BV/hr was maintained during loading and washing. Elution was done using Acidified Ethanol at 0.5BV/hr. The eluent was heated to 65°C with stirring for 2hours.The ethyl esters obtained were then isolated by removal of excess ethanol. The fatty acid ethyl ester mixture was analysed by GC.
The analysis showed 50% DHA-ethyl ester, 17% of EPA-ethyl ester and 8% of DPA- ethyl ester.
Dated this 19th day of June, 2007.