WO2009063500A2

WO2009063500A2 - Novel methods of isolation of poly unsaturated fatty acids

Info

Publication number: WO2009063500A2
Application number: PCT/IN2008/000595
Authority: WO
Inventors: Rakesh Ratnam; Sundeep Aurora; Shreeram Joshi
Original assignee: V.B.Medicare Pvt. Ltd.
Priority date: 2007-09-19
Filing date: 2008-09-17
Publication date: 2009-05-22
Also published as: WO2009063500A3

Abstract

A process is described comprising one or more of a step of separation, isolation, purification, concentration, hydrolysis of one or more of a lipid in general or its/their derivative, from a process stream; wherein the said lipid comprises any fat soluble natural molecule and the said process comprising selective capture of lipids or other derivatives on an adsorbent and washing followed by elution using either a solvent for lipids or an alkaline alkanol. The said process stream may be an emulsion of crude oil or sonicated suspension of microbial biomass. Solution of isolated fatty acids obtained during the process may be subjected to nano-filtration to achieve enrichment of polyunsaturated fatty acids.

Description

NOVEL METHODS OF ISOLATION OF POLY UNSATURATED FATTY ACIDS. TECHNICAL FIELD

Invention relates to methods of isolation of fats, more particularly, polyunsaturated fatty acids from compositions containing the same including a biomass.

BACKGROUND OF INVENTION

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 fo prostaglandins and other physiologically active molecules.

A family of polyunsaturated fatty acids that have a carbon-carbon double bond in the ω-3 position are known as Omega 3 fatty acids, α-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid, that have 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. .

Amongst 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.

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, particualrly in case of western 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 essential 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-3 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 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 containing lipids from the natural source including marine protists.

PRIOR ART

Prior art on various ways in which lipids are isolated or further fractionated in the context of lipids containing polyunsaturated fatty acids comprises the following.

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 7O.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 90⁰C and 150⁰C 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 a temperature of at least 180⁰C. 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.

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 that accompanies moderate heating. 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.The adsorbent is mixture of silver nitrate and silica gel based on a distinctive property of unsaturated organic compounds, that has the ability to complex with transition metals, in this instance with silver. Separation mainly occurs due to the complexation reaction with silver ions and double bonds. Silica gel 60 adsorbs unsaturated fatty acids and elution is carried out sequentially by organic solvents such as methanol, Acetonitrile. etc. based on the number of double bonds of each of the fatty acids. The lower number of double bonds elute out first followed by the higher ones. 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 and DHA are described in Japanese Kokai No. 56-115736 by subjecting the fatty acid mixture to the inverted-phase partition column chromatography using silica chemically bonded with 8W28C alkyl groups. A mixture of tetrahydrofuran, alkanol or acetonitrile and water or aqueous acetic acid solution is used as an eluting solvent. After elution, each objective compound is obtained by treatment as a usual manner.

Another prior art method of obtaining high purity EPA is disclosed in British patent publication No. GB 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 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 The adsorbent is conventional silica gel and separation occurs due to iodination of the fatty acids 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, in J.Am. Oil Chemist. Soσ, 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, in 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, in 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° C₁ followed by addition of urea to the filtrate to precipitate the adduct of oleic acid at room temperature.

Rubin, in U.S. Patent 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, and fractionally crystallized by slowly cooling removing solidified material as it forms to separate substantially pure EPA and DHA.

Best et al., in U.S. Patent No. 5,928,696, extract oils from native substances using centrifugation. Hoeksema in US Patent no. 6166231 provides a novel method for extraction of lipids, specifically edible oil, from microbial biomass. In one aspect, the process comprises: (a) contacting a solvent with an aqueous suspension of microbial material containing lipids in a counter-current manner, wherein the solvent is essentially immiscible in water; (b) collecting the solvent, wherein the solvent contains lipids extracted from the aqueous suspension of microbial material; and (c) separating the lipids from the solvent. In another aspect of the invention, the process comprises: (a) adding an alkali to an aqueous suspension of microbial material containing lipids, wherein the pH of the aqueous suspension is greater than 5; (b) contacting a solvent with the aqueous suspension of microbial material, wherein the solvent is essentially immiscible in water; and (c) collecting the solvent, wherein the solvent contains lipids extracted from the aqueous suspension of microbial material. In yet another embodiment, the process comprises: (a) disrupting cells in an aqueous suspension of a microbial material containing lipids; (b) increasing the pH of the aqueous suspension to be greater than 5 after disrupting cells in the aqueous suspension; (c) contacting a solvent with the aqueous suspension of microbial material; (d) collecting the solvent, wherein the solvent contains lipids extracted from the aqueous suspension of microbial material and further wherein the solvent is essentially immiscible in water; and (e) separating the lipids from the solvent..

Reuker et al in U.S. patent no. 6750048 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 obtaining lipid from the light layer. U.S. patent no. 6441208 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 US patent no.6727373 is explained wherein for obtaining an oil comprising at least one polyunsaturated fatty acid from a microbial biomass, by providing a 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, or due to high requirement of solvents or / and on account of the loss of Poly unsaturated fatty acids by oxidation.

SUMMARY OF INVENTION

We hereby report a novel route of recovery of lipids based on their selective capture on an adsorbent, isolation and purification. The process of invention also involves substantially low heat treatment and low quantity of solvents during the process when compared to other processes.

For the purpose of this specification, lipids are defined as any fat-soluble, lipophilic, naturally-occurring molecule, Such molecules comprise, without limitation, fats, fatty acids, oils, waxes, cholesterol, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, carotenoids, chlorophylls and others.

In one embodiment, the invention comprises obaining an aqueous process stream. In an embodiment comprising preparation of this process stream from a natural source of lipids that comprises a microbial biomass, the same is achieved by sonication of a suspension of microbial biomass followed by filtration.

In an embodiment where the raw material is a fat, preferably an oil, the same is subjected to high shear mixing or a suitable treatment that transforms into a stable emulsion / suspension of oil globules in water.

In another embodiment of the invention, lipids in an aqueous solution are separated and isolated from other dissolved constituents on the solution by adsorbing on an adsorbent that is capable of selectievly capturing lipids, washing off other dissolved constituents from the bed of the adsorbent/s and desorbing the adsorbed lipids in an appropriate eluent. The eluents are selected such that they result in the desired affinity and/or interaction ability of the lipids or its derivatives with the adsorbent matrix as required. In one embidiment, this eluent may be a lipid dissolving eluent so that the product is solution of lipids desorbed from the adsorbent.. In another embodiement, the eluent may be an alkaline alkanol, preferably an alkaline methanol, which, simultanous to desorption, shall also lead to saponification and hydrolysis of the adsorbed fats, which after elution followed by neutralization shall lead to solution containing mixture of fatty acids. Methanol is removed by distillation under reduced pressure and an antioxident is added during distillation to avoid oxidation. Optionally, in a further embodiement of this invention, mixtures of fatty acids obtained may be subjected to nanofiltration using a membrane having cut off for smaller molecular weight fatty acids and capability to efficiently filter out them, so that proportion of polyunsaturated fatty acids improves in the resulting product.

The isolated fatty acids may be converted into their esters, the most preferred product in pharmeceutical applications, by heating them with an alkanol, preferably ethanol. Thus, an integrated inventive process to produce esters of fatty acids from a cell free aqueous solution/emulsion/suspension of fats and oils comprises subjecting the fats and oils to capture on the above-said adsorbent, preferably by way of column chromatography, washing the adsorbent after loading to wash off the contaminants, eluting by alkaline methanol to get saponified and hydrolysed mixture of alkaline metal salts of fatty acids, neutralizing by acid to get a mixture of fatty acids, removing methanol under reduced pressure and in presence of anti-oxidants, recovering fatty acids by capture on the said adsorbent by column chromatography, eluting the adsorbed fattya acids by acidified ethanol and heating the eluted solution of fatty acids in acidified ethanol to about 65⁰C with stirring for 2 hours to get esters of fatty acids. In this process, the mixture of fatty acids obtained by neutralizing eluate of alkaline methanol may optionally be subjected to nanofiltration for a few number of times, usually for fourt times, with a membrane capable of efficient filtering off of lower molecular weight fatty acids so that in the final product, proportion of polyunsaturated fatty acids improves.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this specification, 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, alpha linolenoic acid, Arachidonic acid and the like and " a long chain polyunsaturated acid" includes one or more of EPA, DHA and the like. Similarly, mention of "a lipid" includes two or more types of lipids or a mixture thereof, and mention of "an adsorbent capable of selective capture of a lipid" includes of two or more than two adsorbents, or a mixture of two or more adsorbents" having capability of the said capture of two or more lipids or their combination too with each of such adsorbents.

In the present invention, we report an economical and industrially viable purification process for isolation and purification of fatty acids from an aqueous liquid containing fatty acids including 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. The compounds which are covered within the scope of the invented method also includes all lipids; where the term "lipids" is defined as as any fat-soluble, lipophilic, naturally-occurring molecules. Such said molecules shall at least include fats, fatty acids, oils, waxes, cholesterol, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, carotenoids and chlorophylls.

In the embodiment illustrated here, the omega 3 fatty acids contained in phospholipids or membrane lipids of any natural source either from microorganisms, plants or fish oil are extracted in an aqueous liquid that is passed through and selectively captured on a column containing an absorbent which is a non-ionic adsorbent derived from cross linked polystyrene-divinyl benzene or polymethylacrylate based mixtures or derivaive thereof made by surface modification such that they will selectively adsorb/capture the lipids including fats or fatty acids or their derivatives on them under the conditions of contacting/loading. It may be mntioned here that these adsorbents would adsorb any non-polar molecule, however, in the embdiements of this invention, the non- polar constituents are lipds only as defined above. An example of such an adsorbent is the commercially available non-ionic adsorbent polystyrene based resin Tulsion AD 600 available from Thermax™. . 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 in-situ 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.

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 and inorganic salts/impurities were 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 prepared 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: 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 washed away and then desorption can be carried out in either fluidized bed or packed bed.

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.

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.

The examples and their results given 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 or the scope of the claims. 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 specification.

Examplei : Purification of crude fish oil containing Omega 3 fatty acids.

One liter of crude fish oil containing 10% Docosahexaenoic acid 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 ~1.5 BV/hr (bed volume per hour) was maintained. The lipids were adsorbed on the resin and the cell debris was separated.

1 BV (bed volume - equivalent to the quantity of resin taken in terms of volume of the settled resin bed) of water wash was given at the same flow rate. Elution was done in counter current direction using 1 BV of 10% methanolic NaOH. 1 gm 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 - 300 daltons. Some part of the lower molecular weight fatty acids such as oleic acid, myristic acid etc passed out of the membrane as permeate. The proportion of higher molecular weight fatty acids including Docosahexaenoic acid, Docosapentaenoic acid and Eicosapentaenoic acid increased in the final product as a result.

The retained concentrated lipids including poly unsaturated fatty acids were then again diluted with water and reconcentrated in the nanofiltration membrane to achieve as much further removal of short chain fatty acids as much as possible. This was repeated , usually up to four times, for achieving further removal of short chain fatty acids. Extent of removal of short chain of fatty acids was limited by the efficiency of available mebranes and present status of nano-filtration tehcnology. There exists a scope for improving this efficiency to achieve better proporotions of polyunsaturated fatty acds in the final product.

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 1

BV/hr was maintained during loading and washing. Elution was done using

Acidified Ethanol at 0.5 BV/hr. The eluent was heated to 65⁰C with stirring for 2 hours.The ethyl esters obtained were then isolated by removal of excess ethanol. The fatty acid ethyl ester mixture was analysed by GC (Gas Chromatography).

The analysis of the final product showed 50% DHA-ethyl ester, 17% of EPA-ethyl ester, 8% of DPA- ethyl ester amongst polyunsaturated fatty acids the complete profile is as follows: Table 1 : Fatty acid profile comparison between the crude oil and oil obtained after purification in Example 1

Thus, on account of nano-filtration, the content of polyunsaturated fatty acids has increased from 10 % to 77.8 %

The reaction time and the consumption of solvents are substantially lower.

Example 2: Purification of Omega 3 fatty acids from microbial biomass (A) Start of seed culture of Schizochythum aggregatum ATCC 28209

100 ml of seed medium was prepared in 250 ml conical flask containing 100 ml water, 2 g dextrose, 0.25 g yeast extract, 4 g soy flour and 3.2 g sea salt. The pH was adjusted to 7.5 using 1 N sodium hydroxide and was autoclaved. One loop full of Schizochythum aggregatum ATCC 28209 was transferred to the flask and was incubated at 22°C at 220 RPM for 48 hours.

750 ml of seed medium was prepared in 1000 ml conical flask containing 750 ml water, 15 g of dextrose, 7 g starch, 1.125 g yeast extract, 3Og soy flour 1.125 g ammonium phosphate and 24 g sea salt. The pH was adjusted to 7.5 using 1 N sodium hydroxide and was autoclaved. 200 mg of bacterial amylase enzyme was added to the flask and 7.5 ml of seed inoculum generated from 100 ml seed flask was transferred to it. The flask was kept in a shaker for 58 hours at 24°C.

(B) Submerged Batch fermentation for DHA

Water, 5000 ml, was taken in a 5 L fermenter. Dextrose, 100g, , 47 g of starch, 7.5 g of yeast extract, 23.5 g glycerol, 200 g soy flour, 12 g of ammonium phosphate and 160 g of sea salt was added to the fermenter and all the contents were dissolved. The pH of the contents in the fermenter was adjusted to 3.5 using dilute HCI. The sterilization of the media components was carried out and after that the pH was adjusted to 7.5 using 1 N sodium hydroxide. 2.0 g of bacterial amylase enzyme was added followed by 500 ml of inoculum from example 1 was transferred via peristaltic pump.

The temperature was maintained at 22°C for 12 hours. The temperature was allowed to rise till 26°C and was not allowed to proceed further. The dissolved oxygen was not allowed to drop beyond 20% and the stirrer RPM was increased till 800 RPM. The pH of the medium was not allowed to drop below 5.0. Samples were taken intermittently and analyzed for biomass and the DHA content. The fermentation was stopped after 120 hours and the following results were obtained. The total biomass accumulated was found to be 750 g wet weight and the moisture was found to be 50%. The total fat content was found to be 300 g and DHA was found to be 6Og.

One Liter of fermented broth of schizochytrium containing 12 g of DHA was taken for extraction and purification. The broth was first flocculated with 0.6% of Ammonium sulphate. The supernatant without the microbial cells was decanted off and the concentrated slurry containing the biomass was subjected to sonication for 60 minutes at a temperature below 20⁰C to carry out cell disruption. The slurry is then filtered and 450 ml of filtrate obtained was passed from the bottom through 1 Liter expanded bed column containing polystyrene based hydrophobic resin Tulsion ADS-600 from Thermax preferably of particle size o.3 - 1 mm, specific area 375 - 750 m² / g. This resulted in selective capture of lipids on the adsorbent. The flow through liquid was collected from the top of the column. The resin bed was then washed with 1 BV of water (bed volume - equivalent to the volume of resin bed settled in the column) passed from bottom to top of the column.

Elution was done by introducing 1 BV of 30% methanolic NaOH from bottom and collecting the overflow from the top. The eluted fraction was then neutralized using dilute HCI. The methanol was then removed under reduced pressure in a falling film evaporator at room temperature.

Then the concentrated fatty acids containing polyunsaturated fatty acids were , diluted with water to 1 Liter, mixed to an emulsion using high shear mixer and were further purified in a packed bed chromatographic column. The resin used was Tulsion ADS-600 from Thermax. The aqueous solution containing the polyunsaturated fatty acids were passed through the packed bed chromatographic column from bottom to top direction. The washing was done from top to bottom. A flow rate of 1 BV/hr was maintained during loading and washing. Elution was carried out using 10% acidified Ethanol at 0.5 BV/hr passed from bottom to top. The eluate I.e. the fraction collected after elution was heated to 65°C with stirring for 2 hours. The ethyl esters obtained were then extracted into hexane and isolated by distilling off hexane. The fatty acid ethyl ester mixture was analysed by GC.

The analysis showed 26% DHA-ethyl ester, 7% of EPA-ethyl ester and 2% of DPA- ethyl ester. The complete fatty acid profile is as follows

Table :2

In this process, the reaction time and the consumption of solvents are lower.

Example 3 : Adsorptive isolation and purification of lipids from fermentation broth

One Liter of fermented broth of schizochytrium containing 12g of DHA from Example 2 was taken for extraction and purification. The broth was first flocculated with 0.6% of Ammonium sulphate. The supernatant without the microbial cells was decanted off and the concentrated slurry containing the biomass was subjected to sonication for 60 minutes at a temperature below 20⁰C to carry out cell disruption. The slurry was then filtered and 450 ml of filtrate obtained was passed from the bottom through 1 Liter expanded bed column containing polystyrene based hydrophobic resin Tulsion ADS-600 from Thermax. This resulted in selective capture of lipids on the adsorbent. The flow through liquid was collected from the top of the column. The resin bed was then washed with 1 BV of water passed from bottom to top of the column.

Desorption was carried out by introducing 1BV of Cyclohexane from bottom and collecting the overflow from the top. The eluted fraction was then concentrated to remove Cyclohexane and the lipid profile was analyzed. The analysis is as follows.

Table: 3

Example 4 : lnsitu hydrolysis and saponification of lipids from fermentation broth

One Liter of fermented broth of schizochytrium containing 12g of DHA from Example 2 was taken for extraction and purification. The broth was first flocculated with 0.6% of Ammonium sulphate. The supernatant without the microbial cells was decanted off and the concentrated slurry containing the biomass was subjected to sonication for 60 minutes at a temperature below 20⁰C to carry out cell disruption. The slurry is then filtered and 450 ml of filtrate obtained was passed from the bottom through 1 Liter expanded bed column containing polystyrene based hydrophobic resin Tulsion ADS-600 from Thermax. This resulted in selective capture of lipids on the adsorbent. The flow through liquid was collected from the top of the column. The resin bed was then washed with 1 BV of water passed from bottom to top of the column.

Elution was carried out by introducing 1 BV of 10% ethanolic NaOH from bottom and collecting the overflow from the top. The eluted fraction was then neutralized and ethanol was evaporated off and the free fatty acids were obtained. The fatty acid profile is as shown in table 4 as follows.

The neutralized fatty acid was diluted 5 times of its original volume with water and the solution was then passed through a nanofiltration membrane with a molecular weight cut off between 250 - 300 daltons. Some part of the lower molecular weight fatty acids such as oleic acid, myristic acid etc passed out of the membrane as permeate. The proportion of higher molecular weight fatty acids including Docosahexaenoic acid, Docosapentaenoic acid and Eicosapentaenoic acid increased in the final product as a result.

The retained concentrated lipids including poly unsaturated fatty acids were then again diluted with water and reconcentrated in the nanofiltration membrane to achieve as much further removal of short chain fatty acids as much as possible. This was repeated , usually up to four times, for achieving further removal of short chain fatty acids. Table 4 : Comparison between fatty oil proile of the fermented broth and the oil obtained after purification in Example 3

Thus, content of DHA increased from 26.7 % to 32.2 % in the total fatty acids and total content of poly-unsaturated fatty acids increased from 37.9% to 49.2% as a result of nano-filtraion.

Claims

1. A process comprising one or more of a step of separation, isolation, purification, concentration, hydrolysis of one or more of a lipid in general or its/their derivative, from a process stream; wherein the said lipid comprises any fat soluble natural molecule and the said process comprising at least one or more of a following steps:

a. selective capture of the most of the lipids or lipid derivative/s on an adsorbent and the exclusion of non-lipid components of the said process stream by bringing the said process stream in contact with the said adsorbent, where the said adsorbent is not a silica gel or a porous or gel cation exchange resin,

b. selective elution of one or more of an adsorbed lipid from the said adsorbents, in an unchanged chemical form or a changed chemical form including in a hydrolysed form, individually or elution of a group of related lipid compounds, and where the said adsorbent is not a silica gel or a porous or gel cation exchange resin,

c. subjecting the eluant of step (b.) to one or more of a next process step for producing a product including a process of isolation and purification of one or more of a lipid or its derivative compound.

2. A process of claim 1 comprising one or wherever required more than one of a following:

a. the said process stream or a reaction mixture further comprises an aqueous liquid composition produced during the course of a process step of isolation of or purification of a lipid or its derivative, from a natural source containing the same, b. the said lipids include fats and oils containing residues of fatty acids,

c. the said adsorbent matrix is capable of interacting with a lipid or its derivative, and comprising one or more of following: (i) a non sulfonic resin (ii) non ionic resin (iv) having a surface, which has interacting ability with a lipid, (v) which is rigid and porous, (vi), (vii) has synthetic polymeric base matrix, (viii) has a synthetic base matrix of polystyrene, divinylbenzene (PSDVB), polymethacrylates, polyacrylamide and the like, (x) is crosslinked,

d. the mobile phase used for equilibration, washing, elution and regeneration in both purification and polishing contains one or more of following: (i) water, preferably at neutral pH of 7, (ii) a lipid dissolving polar solvent, preferably an acidified alkanol, preferably ethanol, (iii) alkaline alkanol preferably alkaline methanol, chosen to achieve the desired affinity and/or interaction ability of the lipids or its derivatives with the adsorbent matrix as required.

3. A process of claim 1 or claim 2 comprising one or more of a step of:

a. using an adsorbent having on their matrix one or more of an interacting chemical group or a ligand selective for saturated as well as unsaturated fats and/or fatty acids, comprising a benzyl or a phenyl group and the like to adsorb lipids/fatty acids on the adsorbent,

b. washing away unadsorbed components of the feed, if any, comprising one or more of non-lipid compenents, inorganic salts, organic salts, organic solvents and the like by a mobile phase comprising one or more of following: (i) water, (ii) alkanol aicidifed or un acidified, preferably acidified ethanol, (iii) alkaline alkanol, preferably alkaline methanol and any suitable combination of one or more of (i) to (iii) mentioned above; chosen to achieve the desired affinity and/or interaction ability of the lipds/fatty acids with the adsorbent matrix as required, the mobile phase constituted in a manner suitable for washing of unadsorbed components of the feed, if any, comprising one or more of non-lipids, inorganic salts, organic salts, organic solvents and dictated by the adsorbent matrix used for purification,

c. washing or eluting with another suitably constituted mobile phase selected from the group mentioned in claim 2(d),

d. isolating the lipids including fatty acids by selective elution using one or more of a suitably constituted mobile phase from the group mentioned in claim 2(d).

A process of claim 3 comprising obtaining an aqueous liquid process stream for isolation of lipids by one or more of a following steps:

a. obaining an aqueous liquid process stream as a cell free extract containing lipids starting from a natural source of lipids as a preparatory step for isolation and purification of lipids; achieved in case of microbiological raw material preferably by sonication of a suspension followed by filtration, or

b. obtaining, from a starting material that is an extracted oil, an aqueous liquid process stream wherein the said oil is subjected to high shear mixing or a suitable treatment that transforms into a stable emulsion / suspension of oil globules in water.

5. A process of claim 4 comprising following steps: a. isolating lipids contained in the said aqueous liquid process stream by selective capture / adsorption on a Tulsion ADS-600™ adsorbent obtained from Thermax™by contacting the said aqueous liquid with the adsorbent,

b. washing off non-lipid constituents from the bed of the adsorbents by water,

c. desorbing the adsorbed lipids in alkaline methanol and neutralizling the eluted fraction by dilute HCI,

d. optionally passing the solution of fatty acids at least once through nano-fltration membrane with a molecular weight cut off between

250 - 300 daltons to remove a part of lower molecular weight fatty acids, further optionally repeating the nano-filtration on the retentate accompanied by dilution with water if required,

e. optionally furher purifying the neutralized mixture of fatty acids of step (c.) or nano-filtered mixture of fatty acids by adsorbing the mixture of fatty acids of step (d.) on Tulsion ADS-600 fromThermax™ , and desorbing the adsorbed lipids in acidified ethanol to get a mixture of lipids.

6. A process of claim 5 comprising converting the isolated fatty acids to their esters by heating with an alkanol, preferably an acidified ethanol at a temperature and for a perid, sufficient to convert them to esters; the preferred temperature being around 60° C and preferred period being around 3 hours.

7. A process of enrichment of polyunsaturated fatty acids or their derivatives in a mixture of fatty acids or their derivatives comprising subjecting the said mixture of fatty acids or their derivatives to nano-fitration at least once to retain polyunsaturated fatty acids and filter out at least some quantity of low molecular weight fatty acids.

8. A process of claim 7 where the said nanofiltration membrane has a molecular weight cut off between 250 - 300 daltons.