CA2247368A1 - Enteral formula or nutritional supplement containing arachidonic and docosahexaenoic acids - Google Patents

Abstract

This invention is directed to the use of triglycerides containing fatty ester moieties that include arachidonic acid (AA) and docosahexaenoic acid (DHA) in enteral nutritionals or nutritional supplements. These triglycerides are derived from lipid mixtures which have high levels of sterols and phosphorous.
A preferred embodiment of the invention comprises an infant or enteral nutritional, or nutritional supplement, that comprises a lipid source derived form egg yolk. The lipid source derived from egg yolk is prepared by transesterification or hydrolysis, subjecting the mixture to distillation, and esterification with glycerin to result in a triglyceride containing the desired fatty acids of AA and DHA and little or no sterols and phosphorus.

Description

~'0 97/26804 PCT~US97/01138 E~rTE~L FORU~UI~ OR ~nUTRITION~L SUPPLE~E2~r ~ONT~INING A~CHIDONIC ~2~D DOCOS~HE~AE3WOIC ACIDS

FI ~ 3 OF T~E IN~3NTION

This invention relates to an enteral nutritional ~ormula and nutritional supplement cont~;n;ng triglycerides prepared by the process disclosed in this invention. The enteral formula can be used as an in~ant formula or as an adult nutritional. The invention also relates to nutritional supplements which contain arachidonic acid and other long-chain polyunsaturated fatty acids, such as maternal supplements.

R~.~OuND OF THE lNv~NllON

The composition o~ human mil~ serves as a valuable re~erence ~or improving in~ant ~ormula. Much e~ort has been directed at producing a milk based inrant ,~ormula which is similar to human milk.
One component o~ human milk that is receiving more investigation is the ~at composition. Human milk ~at contains long chain polyunsaturated ~atty acids which may play a role in in~ant de~elopment. Many inrant 'ormulas do not con-tain lipids having long chain polyunsaturated ~atty acids such as arachidonic acid (C20:4w6) (also re~erred to herein as AA), ecosapentaenoic acid (also re~erred to herein as EPA), and docosahexaenoic acid (C22:6w3) (also ref~erred to herein as DEIA). Acceptable ingredient sou--ces i~or these ~atty acids are limited, thus the lacl.; o~ such acids in ir.~ant ~ormula and adult nutritionals.
Polyunsaturated acids, in par~icular he longer chain acids such as A.~, DHA, and EPA are natural cors~ituents o_ S~ TESHEET(RU~E26) W O 97/26804 PCTrUS97/01138 many ~oodstuffs. However these acids are either intimately combined with undesirable components such as cholesterol, phosphorus compounds, or are unsuitable for food applications in their functional form.
The n-6 family o~ polyunsaturated fatty acids, based on the parent linoleic acid and higher derivatives such as AA , have long been established as essential in human and ~n;m~l nutrition. More recently, evidence has accumulated for the nutritional importance of the n-3 family of polyunsaturated ~atty acids, based on the parent linolenic acid and higher derivatives such as ecosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These polyunsaturated acids are .
the precursors ~or prostaglandins and eicosanoids, a power~ul group o~ compounds which produce diverse physiological actions at low concentrations. The prostagl~n~; n-q are known to influence blood clotting, inflammatory and anti-inflammatory response, cholesterol absorption, bronchial function, hypertension, visual acuity and brain development in infants, and gastric secretions, ~mong other effects.
Egg yolk lipids contain AA (arachidonic acid) and DHA
(docosahexaenoic acid) and are widely consumed in diets of both children and adults. Lipids isolated from egg yolks could be deemed unacceptable ~or use in infant formula due to high levels o~ cholesterol which suffers from negative public opinion, and the troublesome levels of phosphorus. The AA
and DHA are present in egg yolk lipids primarily as phospholipids. Thus, in~ant ~ormulas forti~ied with egg yolk lipids exhibit levels of cholesterol and phospholipids which far exceed the level of such nutrients found in breast milk.
Typically, the amount o~ lipids in egg yolk is about 65%
by weight (wt%) of the dry matter. In such lipids, about 66 wt% o~ the lipid is triglycerides, of whicn about 30 wt% is phospholipids, and about 4 wt% is cholesterol. The phosphorus content of the lipids is about 1 wt% to 2 wt%.

S~ TESHEET(RULE26) ~ro 97/26804 PCTrUS97tO1138 Several commercial egg lipid ingredients are presently available. The first, OVOTHIN 120, is a total egg yolk lipid extract supplied by Lucas Meyer of 765 East Pythian Ave., Decatur, Ill. 62526. OVOTHIN 120 contains triglyceride, phospholipid and cholesterol. A second ingredient, supplied by Psanstiehl Laboratories, Inc. of 1219 Glen Roc~ Ave., Waukegan, Ill. 60085 is an egg yolk extract which is 90%
phospholipids. Also, purified egg phospholipid is available from Genzyme Corporation of One Kendill Square, Cambridge, Mass. 02139. Un~ortunately, all the above ingredients negatively impact the phosphorus levels of in~ant formula when used at the proper fortification level to achieve AA and DHA target levels approximating the content of AA and DHA in human milk. The proper fortification would require that about 7-9 wt% of the fat in the infant formula be composed of phospholipid. ~t-m~n milk fat contains 1-3 wt% phospholipid.
Fur~rmore, the use of OVOTHIN 120 increases cholesterol in infant ~ormula above the levels found in human milk.
There are numerous methods in the literature ~or recovering phospholipids from lipid mixtures. For example, U.S Pat. No. 4,698,185 discloses a method of separating phospholipids from crude vegetable triglyceride mixtures.
The method involves the addition o~ water in a mass ratio about equal to the mass of phospholipids present in the lipid mixture, with or without heating, and with or without co-addition of citric or phosphoric acid, to cause the phospholipids to hydrate and separate into a second phase.
Such degumming methods, however, were designed for the removal o~ 1 to 2 weight percent of phospholipids ~rom crude vegetable triglycerides and are not directly applicable to the puri~ication of other natural lipid mixtures, such as-egg yolk lipids because of the higher levels or phospholipids ~30-40 wt%~ in egg yolk lipids. Addition of a 1:1 mass ratio of water to phospholipid with large amounts of phospholipids SUBSTITUTE SHE~T(RULE26) ~70 97/26804 PCT~US97/01138 present causes the ~ormation of a stable emulsion which prevents phase separation. Moreover, sterols tend to partition between both the phospholipid and triglyceride phases.
It is desirable to provide a process by which cholesterol and other sterol compounds (many of which can be metabolized to cholesterol or its derivatives) can be extracted ~rom various foodstuf~s, thereby producing low-cholesterol versions of such foodstu~fs. However, the process must not introduce into the foodstu~f any material which is not generally recognized as safe ~or use in foodstuf~s. In addition, the process should remove ~rom the foodstu~ not only cholesterol itsel~ but also cholesterol derivatives and other sterol compounds which can be metabolized in the body to cholesterol or derivatives thereo~, and which thus affect cholesterol levels in the body. Furthermore, the process should leave the foodstuf~ in a form which is as close as possible to that of the original, high cholesterol foodstuff. Finally, the cholesterol-removal process should not remove vit~m; n~ and other important nutrients o~ the foodstu~.
Numerous attempts have previously been made to provide a cholesterol-removal process which meets these exacting criteria. U.S. Pat. NO. 4,692,280, discloses a process for the purification of fish oils in which the oil is extracted with supercritical carbon dioxide to remove cholesterol, together with odori~erous and volatile impurities. Such carbon dioxide extraction processes, however, suffer ~rom the disadvantage that they must be operated under pressure to keep the carbon dioxide in the supercritical phase, which increases the cost o~ the apparatus re~uired. In addition, such carbon dioxide extraction processes are not very selective in the removal of cholesterol, and thus remove valuable constituents of the foodstu~f. In addition, the S~S~ TESHE}~(RULE26) W 0 97/26804 PCTrUS97/01138 properties of some foodstuffs may be altered disadvantageously by contact with supercritical carbon dioxide; for example, in some cases the carbon dioxide removes flavoring and odori~erous components which af~ect the taste and smell of the treated foodstuf~.
U.S. Pat. No. 5,091,117 discloses a process for removing at least one sterol compound and at least one saturated ~atty acid from a fluid mixture by contacting the ~luid mixture with an activated charcoal. U.S. Pat. No. 5,031,117 states however, in column 12, lines 4-19, that the process should not be used for removing cholesterol from materials, such as egg yolks which contain a combination of cholesterol and proteins, since a significant adsorption of proteins and their constituent amino acids occurs on the charcoal.
British Pat. No. 1,5~9,064 discloses a process for removing cholesterol from butter triglycerides by distillation. However, Lanzani et al [J. Am. Oil Chem.
Soc. 71, ~1994) 609] det~rm;ned that only 90~ o~ the cholesterol could be removed using the process disclosed in British Pat. No. 1,559,064 without seriously affecting the ~uality of the end product. Excessive time at the high temperatures needed for more complete cholesterol removal was found to cause cis-trans isomerization of the polyunsaturated fatty acids. The trans form of polyunsaturated fatty acids are considered undesirable in ~ood products.
Egg yolk is an example of a lipid mixture rich in polyunsaturated ~atty acids including AA and (all-cis)-4,7,10,13,16,19-docosahexaenoic acid ~DHA) in which the polyunsaturated ~atty acids are predom,n~ntly bound in the phospholipids and which contain high levels o~ cholesterol.
It is desira~le to provide a process Ior the manufacture o~
egg-derived fatty acids and ~atty acid esters high in polyunsaturated fatty acids which removes cholesterol and phosphorus residues without degrading or causing cis-trans SlJtsS ~ J rE SHEET (RULE 26) CA 02247368 l998-07-24 WO 97~6804 PCTAUS97/01138 isomerization of the essential polyunsaturated ~atty acids contained therein or the taste and ~lavor o~ ~oods prepared using such ~atty acid and ester mixtures. Moreover, the process ~or the manu~acture o~ the ~atty acid and ester mixtures should use materials which are on the Generally Recognized As Sa~e (GRAS) list Gf the U.S. Food and Drug ~m; n; stration in order ~or the ~inal product to be used in ~oods.
U.S. Patent 4,670,285 to M. Cl~n~;n;n o~ June 2, 1987 discloses the use o~ lipid extracted ~rom egg yolk in in~ant ~ormula. The lipids o~ the Clandinin re~erence include polyunsaturated lipids found in human milk such as C:20 or C:22 w6 and C:20 or C22 w3 ~atty acids The lipids o~
Cl~n~;n;n contain the unacceptable levels o~ cholesterol and phosphorus of the original egg yolk material.
Abstract o~ JP 62198351 o~ Sept. 2, 1987 to Morinaga Milk discloses a substitute mothers' milk composition which contains egg yolk lipid extracted ~rom egg yolk with ethanol.
The lipid is pre~erably combine,d so that a 100 g milk composition contains 68 mg of cholesterol. However, the 68 mg o~ cholesterol translates to about 680 mg/L (liter) or greater than ~our times that ~ound in average composition human milk.
U.S. patent 5,112,956 o~ May 1., 1992 to P. Tang, et al.
discloses a method ~or the removal o~ lipids and cholesterol from protein material such as that in egg yolk by treating the protein with an extraction mixture comprising a lower alcohol, water, and an acid in concentrations selected to extract cholesterol and lipids ~rom the protein. The pre~erred lower alcohol of this reference is ethanol and a primary ob~ect is obt~;n;ng protein suitable ~or human consumption.
PTC publication WO 89/11521 o~ Nov 30, 1989 discloses a process ~or preparing EPA and DHA and their esters ~rom oils S~ TE S~EET(RULE26) W 0 97/26804 PCTrUS97/01138 of ~nim~l and/or vegetable origin by subjecting the raw oil to alkaline hydrolysis, acidifying the soap so ~ormed with a mineral acid in aqueous solution, extracting the resulting mixture with petroleum ether and a~ter washing and concentration, the combined extracts are submitted to one or more distillation steps with the pressure and temperature parameters being suitably changed in order to obtain a whole range o~ desired products.
Abstract of JP 1160989 (application) of June 23, 1989 to NIOF. Fresh fish eggs are extracted with solvent o~
distilled water, methanol/chloro~orm, acetone, ether, under oxygen-free conditions to extract lipids and eventually isolate a docosahexaenoic acid- cont~;ning phosphatidylcholine.
Abstract of Han~guk Ch'uksan ~akhoechi, 1991, 33(8), 602-6 by Han, ~.K., et al. Egg yolk was ground with trichloromethane and methanol. Lipid extract was converted to methyl esters by transesterification with boron tri~luoride and methanol. The methyl esters were analyzed for various ~atty acids. C20-22 polyunsaturated acids accounted ~or 4.3% o~ the total.
In the present invention, egg yolk derived glyceride compositions, also simply re~erred to herein as Processed Natural Ingredients, are prepared which typically contain about 4 wt% of AA and about 1.5 wt% o~ DHA based on the weight of the Processed Natural Ingredients and wherein the amount o~ phosphorus can be reduced to less than about 0.002 wt% (20 ppm) and the amount o~ cholesterol reduced to less than about 0.1 wt% of the processed Natural Ingredients.~ 30 Pre~erably at least 95% and particularly at least 98% o~ the cholesterol and other sterols, and phosp~orus compounds are removed ~rom the lipid mixture staring material, e.g. egg yolks in the process o~ this invention, and such highly puri~ied fatty acids or esters thereo~ are re~erred to herein s~ rE SHEET(RUEE26) ~0 97126804 rcTnusg7/oll38 as being "essentially free of cholesterol, sterols and phosphorus compounds~. The Processed Matural Ingredients can be that of mono-, di-, or triglycerides as well as mixtures thereof.
Unless the context indicates otherwise, the following terms shall have the following meaning:
"AA" is arachidonic acid (~20:4w6);
~alkaline metal" is an alkaline earth metal or alkali metal such as calcium, magnesium, sodium, or potassium;
~DHA~ is docosahexaenoic acid (C22:6w3);
"egg derived triglycerides" are one of the ~rocessed Natural Ingredients (as defined below) wherein a major portion, preferably at least 75% by weight of the glycerides and particularly at least 90~ of the glycerides are triglycerides derived from egg yolki "ester route" is the process which comprises the preparation o~ ~atty acid esters by transesterifying ~atty acids o~ lipids to lower alkyl esters o~ the ~atty acids;
"essentially free of cholesterol, sterols, and phosphorus compounds~ means that at least 95%, preferably at least 98%, o~ the cholesterol and other sterols, and phosphorus compounds are removed from a lipid starting material by the process of the present invention;
"FAP~ is fatty acid profile;
"FAME~' is fatty acid methyl esters;
~'free ~atty acid route" is the process which comprises the production o~ free ~atty acids and/or esters ther~o~ by hydrolysis of naturally occurring lipids to free ~atty acids;
"GC" is gas chromatography;
"lower alkane" is an alkane having from 1 to 4 carbon atoms;
"lower alkyl" is an alkyl having ~rom 1 to 4 carbon atoms;

SU~ 111 LITE SHEET (R~JLE Z6 ~'0 97/26804 PCT~US97tO1138 "lower alkanol" is a monohydric alcohol having from 1 to 4 carbon atomsi "lower alkoxide" is an alkyl oxide group having from 1 to 4 carbon atoms such as in sodium methoxide;
"mL~ means milliliter;
"N/AP~ means not applicable "N/D~' means not detectable;
"N/R" means not reported; and "Processed Natural Ingredients" are the compositions cont~l n; ng glycerides prepared by reacting glycerol with the free fatty acids or lower alkyl esters thereof in the process of this invention;
"TLC" is thin layer chromatography.

~ TF~ DESCRIPTTON OF 'l'H ~ DE~AWTI~G

FIG. ~ is a schematic flow diagram entitled "ESTER ROUTE
FOR EGG PHOSPHOLIPID TO TRIGLYCERIDE CONVERSION" and shows important steps o~ a pre~erred method for making the triglyceride composition of the Processed Natural Ingredients by use of methanol as the extraction solvent for lipids from egg yolk solids by the ester route.

FIG. 2 is a schematic ~low diagram entitled " FREE FATTY
ACID ROUTE FO~ EGG PHOSPHOLIPID TO TRIGLYCERIDE CONVERSION"
and shows important steps o~ a pre~erred method for making the triglyceride composition of the Processed Natural Ingredients by use of methanol as the extraction solvent for lipids from egg yolk solids by the free fatty acid route.
.
DISCLOSURB OF IHB INVENTION

The present invention relates to the use o~
triglycerides in nutritional products or supplements wherein sllda~ TEsHEET~RuLL26) the triglycerides are produced in accordance with a process disclosed herein. The process produces triglycerides which are high in polyunsaturated fatty acids, which are essentially free of cholesterol and other sterols, and phosphorus, and are derived from lipid mixtures such as naturally occurring lipid mixtures. The sterols and the phosphorus compounds are removed without degrading or causing cis-trans isomerization of the essential polyunsaturated fatty acids or esters thereof contained therein or the taste and flavor of foods prepared using such lipids mixtures.
Moreover, the process of the present invention uses materials which are on the Generally Recognized As Safe (GRAS) list of the U.S. Food and Drug ~m; ni stration.
In one aspect of this invention, the process broadly comprises the steps of:
(A) su~jecting a lipid mixture contA; n; ng phospholipids, triglycerides and sterols, including cholesterol, to treatment selected ~rom the group consisting of (1) hydrolysis to form a free fatty acid phase and an aqueous phase comprised of water, glycerol and phosphorus compounds (2) alkaline transesterification with a lower alkanol to produce a lower alkyl fatty acid ester phase comprised of lower alkyl fatty acid esters and sterols and an aqueous phase comprised of water, glycerol and phosphorus compounds;
(B) separating the a~ueous phase from (l)fatty acid phase or (2) lower alkyl fatty acid ester phase products formed in Step (A);
(C) distilling the fatty acids or esters thereof o~
Step (B) at a temperature o~ at lest 100~ C to separate and recover in the distillate (l)free fatty acids or (Z) lower alkyl esters o~ the ~atty acids wherein said ~atty acids or esters thereof have reduced concentrations o~ cholesterol and SIJD~ 111 ~1TE SHEET ~RULE 26) CA 02247368 l998-07-24 ~'0 97/26804 PCTrUS97/01138 other sterols, and phosphorus compounds in relation to the lipid mi~ture; and (D) subjecting the (1) purified '~ree fatty acids or (2) purified lower alkyl esters from Ste~ (C) to treatment selected from the group consisting o~: reaction of the purified free ~atty acids with a C1 to C10 monohydric or polyhydric alcohol to produce a fatty acid ester, or (2) transesterification of the purified lower alkyl ester o~tained in Step (~) with a Cl-C1~ monohydric or polyhydric alcohol to produce a fatty acid ester of said C1-C10 alcohol wherein said alcohol has a different number of carbon atoms from that used in the transesterification OL Step (A) The selection of the specific steps in the chemical synthesis method of this invention compllment each other so as to arrive at the ~rocessed Natural Ingredients in an economic and efficient manner useful in the manufacture of enteral formulas.
In another aspect of the invention, phospholipids having a high concentration o~ AA are prepared by contac~ing a natural lipid source, e.g., egg yolk and preferably egg yolk solids, with a sol~ent consisting essentially of methanol at a temperature of about 20C C to 68~ C.
In another aspect o~ the invention a lower alkanol is included with the lipid mixture to assist or cause the mixture to separate into a top phase comprising phospholipids, sterols and alcohol and a bottom phase comprising triglycerides and sterols. The top phase is then used for subse~uent processing.
In yet another aspect of the invention, which comprises the free ratty acid route, the proc2ss comprises the steps o,: :
(A~ hydrolyzing a lipid mi~ture cont~i n i ng phospholipids, triglycerides, and sterols to rorm a two-phase p-oduct containing a fatty acid phase com~rising free fatty SUBSTITUTE SHEET (RULE 26) ~'0 97126804 PCTrUS97/01138 acids and sterols, and a~ aqueous phase comprising water, slycerol, and glycerol phosphoric acid est~s;
(B) separating the aqueous phase '-rom tke ~atty acid phase o_ the two-phase product ,or~ed in S.~-p (A);
(C) reacting the ~atty acids witn the sterols in the fatty acid phase ~rom Step (B) at a tem~erature o~ 150~ C to 250~ C to ~orm a mixture comprising sterol fatty acid esters and water; and (D) distilling the sterol ~atty acid esters ~ormed in 10 Step (C) at a temperature o~ 13~~ C to 250~ C and a pressure o~ 1 x 10-3 kPa to 0.5333 kPa, to recover purified ~atty acids which are essentially ~ree o~ cholesterol, sterols, and phosphorus compoundsi and optionally;
(E) reacting the puri~ied ~atty acids prepared in Step (D) with a monohydric or polyhydric alcohol in a molar ratio o~ 1 to 2 moles o~ ~atty acid to each hydroxy equivalent o~
the alcohol to produce a ~atty acid ester.
~ n still another aspect o~ the invention the egg yolk is extracted with a lower alkyl alcohol and the subsequent processing ~ollows the same steps as ~or that described a~ove ~or the ester route or the ~atty acid route wherein the egg yolk lipid was the starting material. The use of methanol to extract lipids is advantageous, particularly at temperatures ~rom about 20~ C to the boiling point o~
methanol, i.e , 68 degrees C, since the amount o~ AA
extracted is unexpectedly greater in comparison with the use or other alkanols such as ethanol or propanol. Additionally, methanol is a solvent accepted ~or use in preparation or rood ingredients.
In a ~urther aspect of the invention puri,ied ~ree ratty acids, lower alkyl esters o~ the ~atty acids, or mixtures thereo~ are recovered rrom the distillation step without proceeding to the es~erirication step.

S~ TESHEET(RULE26) CA 02247368 l99X-07-24 ~ro 97/26804 PCTnUS97/~1138 Still further aspects of the invention include fractionation techni~ues for concentrating fatty acids such as AA and DHA.
A further aspect of the invention is directed to liquid or powdered enteral formulas such as an enteral ~ormula comprising: from about 10 to 35 grams of protein per liter of formula; carbohydrates, which may include those of dietary fiber of between 60 and 110 grams per liter of formulai and from about 20 to 45 grams of fat per liter of formula wherein the fat includes the egg derived triglycerides in su~ficient amount to provide triglycerides having ester groups cont~; n; ng ~rom about 0.1 wt~ to 2.0 wt~ and pre~erably about O.1 to 1 wt% (weight percent) of AA based on the total ~at in the enteral ~ormula and about 0.05 wt% to 0.5 wt% o~ DHA
1~ based on total ~at in the formula and wherein the said egg derived triglyceride contains less than 0.1~ and preferably less than 0.05% phosphorus and less than 1% and preferably less than 0.5% cholesterol by weight based on the weight o~
the egg derived triglycerides. The enteral ~ormula, in a ~0 simple form, contains a nutritionally adequate source of amino nitrogen, carbohydrates and edible ~ats together with the egg derived triglycerides. There is also disclosed enteral ~ormulas wherein the AA and DHA are prepared by the method disclosed herein. Methods ~or manu~acture of AA and DHA cont~;n;ng enteral formula is still another aspect of the invention.
A number of techniques were unsuccessfully tried to obtain glycerides o~ AA and DHA in an economic and practicable manner which would be suitable for use in an enteral formula such as infant ~ormula. One of the unsuccessful tech-niques was thermal cracking. When egg yolk lipids and water were mixed and heated, there was a se~ere ~oaming problem. When water was limited to one equivalent based on phospholipid, ~oaming could be controlled A~ter 5 S~ UTE SHEET (RULE 2~i) ~'0 97/26804 PCT~US97/01138 minutes at 250~ C with no solvent, TLC (thin layer chromatography) showed a mixture of triglyceride and diglyceride and starting material (phospholipid). However, the reaction mixture was very dark in color and non-homogeneous. The dark color was indicative o~ decomposition.Lowering the temperature to 200~ C for 30 minutes showed no obvious bene~its.
Still another advantage o~ this invention is the ~inding that temperatures o~ up to about 250 degrees C can be used in some o~ the method steps without decomposition or appreciable darkening o~ the AA and ~A or methyl esters thereo~. This is believed to be unexpected since a test conducted with methyl oleate began to darken at about 75~ C.

n~.~rATT.~n n~scl7TpTIoN OF T~E lNV~;N'l'l ON

Naturally occurring lipid mixtures high in polyunsaturated ~atty acids are derived ~rom ~n; ~1 and vegetable matter. Sources o~ lipid mixtures include: marine ~n;mA 1s such as blue-colored fish, e.g., the mackerel, sardine, mackerel pike and herring; salmon; cod liver oil;
~n; m~ 1 marine plankton, such as krill and the various shrimp-like copepods; eg~s; green lea~y vegetables such as spinach, broccoli, and purslane; and oilseeds such as soya, sun~lower, flax, canola, rapeseed, and cotton seeds. Any source o~
lipid mixtures high in polyunsaturated ~atty acids may be used in the process o~ the present invention.
The lipid mixture is separated ~rom the ~n; m~ 1 or vegetable ~at or oil by extraction or leaching with a solvent such as alcohol or hydrocarbon. Illustrative of solvents for leaching or extracting lipids there can be mentioned lower alkanols having ~rom 1 to 4 carbon atoms such as methanol, ethanol, isopropanol, and the like; hydrocarbons such as hexane; ethers such as petroleum ether and diethyl ether;

SUBSTITUTE SHEFT(RULE26~

W 0 97/26804 PCTrUS97101138 lower alkanes under pressure such as those having ~rom 3 to carbon atoms and halogen substituted lower alkanes such as trichloromethane and dichloromethane; ketones such as acetone; as well as mixtures of the foregoing. For example, egg yolk powder may be mixed with a lower al~anol, e.g.,methanol, which yields a lipid mixture containing phospholipids, triglycerides and sterols in li~uid form, and solid protein material. The solid protein material is easily separated ~rom the lipid mixture by methods known in the art such as filtration or centrifugation.
The pre~erred lipid source is egg yolks. The egg yolks used in this invention are generally derived from various avian species such as the hen, turkey, etc. and preferably the hen. However, eggs of other ~nim~ s can be used, e.g.
that of ~ish such as salmon eggs as well as eggs of turtles.
A typical composition of hen's egg yolks as found in Sim, J.S. et al. , Egg Uses and Processing Technologies, page 120 (1994) is as follows on a percent by weight basis:
(a) 47.5% water, 33.0~ lipids, 17.4% protein, 0.20% of carbohydrates (free), 1.1% of inorganic elements; and others of 0.8%;
(b) as to lipid composition (~rom total lipids):
triglycerides o~ 71-73%, cholesterol o~ 4-6~, phospholipids o~ 23-25%, lecithin (in phospholipids) of 70-77%, C16-C18 fatty acids 93.5%, saturated fatty acids 44%, monounsaturated fatty acids 44% and polyunsaturated fatty acids o~ 10.2%. As far as the C16 and C18 fatty acids are concerned in the preceding egg yolk analysis, it does not appear to applicants that the analysis accounted for long chain fatty acids.
Egg yolks can be in different forms such as liquid, frozen, or solid with or without conventional additives such as silica ~low agents. Egg yolk solids can be obtained from eggs by various conventional means such as by spray drying egg yolks, freeze drying, etc. Egg yolk solids ~ypically SU~IllUTE SHEET(RUIE26~

U'O 97/26804 PCTrUS97/~1138 have 5~ maximum moisture content, a pH of 6.5 +3, a 56.0 wt~
min;mum fat conte~t, protein of 30 wt% m;~imll~ A preferred form of egg yolk useful in the present invention is egg yolk solids.
The long chain unsaturated ~atty acids such as A~ and DEA in egg yolk lipids are found predominantly in the phospholipid fraction. In the methanol solution of the egg yolk lipids o~ this invention, the amount of lipids is typically about 38 wt%; the amount of AA is about 4 wt%; and the amount of DHA is about 1.5 wt% as determined by a relative fatty acid profile. However, the quantity of these lipid components can vary depending on the species o~ ~n; mA l ~ -its diet, time of year, etc.
The amount of phosphorus and cholesterol contained in the Processed Natural Ingredients is very low. Generally, the quantity of phosphorus can vary ~rom about 0.1 wt% to 0.0001 wt~ based on the Processed Natural Ingredients. It is preferred that the quantity of phosphorus be less than 0.1 wt% and particularly less than 0.01 wt% of the Processed Natural Ingredients. It is pre~erred that the quantity o~
cholesterol be less than 0.5 wt~ and particularly less than 0.1 wt% based on the weight of the Processed Natural Ingredients. The distilled free fatty acids as well as the distilled lower alkyl esters of this invention will also have the low phosphorus and low cholesterol levels give above for the Processed Natural Ingredients. It is particularly preferred that the fatty acid and ester products of this invention be essentially free of cholesterol, sterols and phosphorus compounds.
The quantity of organic solvent used ~or extracting lipids from a lipid source, can vary over a broad range sufficient to dissolve the lipids. In the case o~ egg yolk solids, such quantity can vary ~rom about 40 ml to over 800 ml of methanol based on 100 grams (g) o~ egg yolk solids.

SlJ~:~ 111 UTE SHEFT (RULE 26) W 0 97/26804 PCT~US97/01138 Larger quantities of methanol can be used but such larger quantities serve little useful purpose since it needs to be removed in later steps of the process.
As can be seen in Example 4 herein the use of methanol to extract lipids from egg yolk provides an unexpected high concentration of AA in the egg lipid extract in the temperature range of about 20~ C to 68~ C. and prefera~ly 30~
C to 65~ C.
By extracting egg yolk with ~lethanol, a phospholipid-rich egg lipid extract is obtained. It is the phospholipids which contain most of the AA and DHA of the egg yolk. When a solvent other than methanol is used for extracting the lipids, the extraction temperature can vary from about 0~ C
to the boiling point of the solvent. The quantity of such other organic solvent can be the same as in the use o~
methanol.
The addition of a lower alkanol as used in the extraction o~ lipids from a lipid source or when simply added to a lipid mixture from which the triglycerides have not been separated from the phospholipids before hydrolysis or transesterification causes the formation of two liquid phases when the temperature is maintained between 20~ C and 68~ C, preferably 30~ C to 65~ C. The top phase is comprised of phospholipids, sterols, and alcohol, the bottom phase is comprised of triglycerides and sterols. The triglyceride phase is removed by methods known in the art such as decantation. For lipid mixtures such as egg yolks in which the polyunsaturated ~atty acids such as AA, DHA and EPA are predom;n~ntly bound in the phospholipids rather than the triglycerides, the addition of the alcohol is convenient and inexpensive method of removing the triglycerides and concentrating the polyunsaturated ~atty acids in the r~m~in;ng lipid mixture. The addition of the lower alkanol does not interfere with the subsequent hydrolysis reaction SlJt~S 111 IJTE StlEET (RULE 26) ~'0 97/26804 PCTnUS97/01138 1~
nor the transesterification reaction and can provide the lower alkanol needed for transesteri~ication o~ the ~atty acid portion o~ the phospholipid. In case methanol is used as the lower alkanol for the phase separation, the methanol is pre~erably added in a mass ratio o~ about O.S to 1 to 3 to 1 alcohol to the source o~ the lipids, e.g., egg yolk solids.
The addition o~ methanol outside this range either does not result in the ~ormation o~ a two phase mixture or results in poor partitioning o~ triglycerides and phospholipids into 1~ their respective phases. Water can be used to assist in such separation and the quantity of water can vary over a wide range such as that of from about 1 wt% to about 100 wt% based on the source of the lipids, e.g., egg-yolk solids.
A brief description o~ a preferred embodiment o~ the invention involving the ester route is as ~ollows. Lipids are extracted ~rom a lipid source, e.g.,egg yolk solids, with methanol; the lipids are separated from proteins and other insoluble constituents o~ the lipid source; the methanolic solution of lipids is submitted to alkaline transesteri~ication and subsequent neutralization to convert the ~atty acids o~ lipid glycerides into ~atty acid methyl esters wherein the reaction medium also contains sterols such as cholesterol as well as glycerine, phosphorus, and other products in the lipids or resulting ~rom the transesteri~ication and subsequent neutralization; the methyl esters and sterols o~ the ~oregoing are separated, such as by precipitation or phase separation, ~rom an aqueous phase which includes phosphorus from the lipids, principally ~rom phospholipids, as well as glycerine and some o~ the methar~ol;
the methyl esters are distilled to separate sterols ~rom the methyl esters; and the methyl esters are subjected to esteri~icatiOn, speci~ically transesteri~ication, in the presence of glycerol and subsequent neutralization or quenching o~ the reaction product to produce the egg derived S~S~IlUTE SHEET(RULE26~

W O 97/26804 PCT~US97/01138 triglycerides of fatty acids from the egg yolk lipids wherein such triglycerides have a high concentration of AA and DHA
ester groups and wherein such egg derived triglycerides contain reduced quantities of cholesterol and phosphorus.
A~ter the lipids are dissolved in the methanol or other organic solvent, the insoluble egg yolk components such as protein are separated from the methanolic solution of lipids.
This can be done by various conventional techniques such as the use of a filter press, centrifuging, vacuum ~iltration, etc.
In the case of egg yolk is extracted with metLanol, the extract is preferably separated into a triglyceride phase and a phospholipid phase by the addition of water and centrifuging. Analysis of a sample with methanol as the solvent for extracting the lipids showed that the triglyceride phase had no detectable phosphorus and was low in cholesterol. A fatty acid distribution assay of such sample showed that the triglyceride phase contained only O.37% AA and 0.13% DHA. This demonstrates that the phospholipids were cleanly separated from the triglyceride fraction. With the separation and isolation of the phospholipid phase, a large percentage of triglyceride can be removed and final products such as the purified free fatty acids, lower alkyl fatty acid esters and Processed Matural Ingredients can be prepared with a higher concentration of the polyunsaturated acids such as DHA and AA.
Although separation of phospholipids from triglycerides as described above prior to hydrolysis or transesterification is advantageous, it was ~ound that the majority o~
cholesterol also separated into the phospholipid layer. Thus, an e~fective method for removing the cholesterol and other sterols from this or subse~uent reaction mixtures needs to be used.

SUBSTITUTE SHEET (RUL 26 0 97/26804 PCTrUS97/01138 In the ester route, after removal of insoluble material from the lipid source, the solution of lipids, preferably phospholipids such as those separated from egg yolk triglycerides, are then ready ~or transesterification with a lower alkanol and a catalytic quantity of an alkaline metal lower alkoxide. In case the lipid is not dissolved in a lower alkanol, such alkanoï needs to be added for the transesteri~ication. Lipid solvents other than lower alkanols should preferably be removed at this step. At this stage neutralization might be required because egg yolk lipids are typically slightly acidic. The alkaline metal portion of the alkoxide of the transesteri~ication catalyst can be that of an alkaline earth metal or alkali metal such as calcium, magnesium, sodium or potassium. Preferred alkaline metals are those of sodium or potassium and particularly that of sodium.
The lower alkyl oxide, i.e., the al~oxide, can have from 1 to 4 carbon atoms and preferably from one to 2 carbon atoms, e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, etc.
Illustrative of the alkaline metal lower alkoxides there can be mentioned those of sodium methoxide, sodium ethoxide, sodium n-propoxide, potassium methoxide, potassium ethoxide, and the like.
The quantity of the alkaline metal lower alkoxide catalyst can vary over a wide ran~e sufficient to neutralize the lower alkanol solution of lipids as well as providing a catalytic amount for effecting the transesterification o~ the lipids in the lower alkanol to the corresponding lower alkyl esters of the fatty acids in the lipids. Alternatively, the acidity in the alcoholic solution of lipids can be neutralized with other basic materials such as calcium oxide and then an alkaline metal lower alkoxide is us~d in a cata~ytic amount, e.g., about 0.4 wt% o~ sodium methoxide based on the weight of lipid.

S~ TESHEET~RULE26 CA 02247368 l998-07-24 ~'0 97/26804 PCT~US97/Q1138 The temperature ~or the transesteri~ication o~ lipid to lower alkyl esters o~ the ~atty acids such as that of AA or DHA can vary over a broad range such as that of about 20~ ~
to the boiling point of the lower alkanol, e.g., 68~ C in the case o~ methanol, and pre~erably at a temperature of about 50~ C to the boiling point o~ the lower alkanol.
After the transesteri~icatiOn of lipids to the lower alkyl esters o~ the corresponding ~atty acids, the reaction medium is pre~erably neutralized with an acid, as is conventional with transesteri~ication reactions. ~owever, such neutralization is not necessary. Illustrative o~ acids which can be employed are inorganic acids such as phosphoric, hydrochloric, sul~uric, etc. as well as organic acids such as acetic, and the like.
Transesteri~ication o~ the lipids produces an aqueous phase cont~in;ng phosphorus compounds, generally as precipitates, and lower alkanol and glycerine. There is also produced a lower alkyl ester phase which contains the ~atty acid esters and sterols such as cholesterol. The aqueous phase material including precipitates is separated ~rom the phase co~t~in;ng the lower alkyl esters of the ~atty acids and the cholesterol. The precipitate is pre~erably separated by ~iltration or by centri~uging whereas liquid materials can be separated by means such as decanting, or centri~uging.
Although much o~ the lower alkanol is removed at this stage, about 5 wt~ to 10 wt% o~ the crude lower alkyl ester ~raction ls lower alkanol. The lower alkanol can be removed by evaporative means. Thus, a~ter neutralization o~ the transesteri~ication reaction, two distinct layers will ~orm (i.e., phase separation). The upper layer is principally crude lower alkyl esters o~ the ~atty acids, however, it contains some quantity o~ lower alkanol such as about 2 to 20%. The lower alkanol is removed by evaporation or distillation prior to the distillation o~ the alkyi esters or SUBSTITUTES~EET(RULE26) CA 02247368 l998-07-24 ~'0 97126804 PCTrUS97/01138 the egg yolk fatty acids. Once the above two phases are separated, the lower layer, principally aark (brownish) in color, contains a majority o~ the alkanol Upon extended standing or removal o~ some alkanol, additional amounts o~
crude lower alkyl esters can be isolated, thus increasing the effective yield.
The crude lower alkyl esters o~ the ~atty acids are then separated from the cholesterol by distillation under reduced pressure such as with a molecular or short path still. Since unsaturated ~atty acids such as AA and DHA are sensitive to temperature in that they degrade, particularly in formation of trans isomers, the distillation is pre~erably conducted at a temperature of 100~ C to about 250~ C. The distillation equipment is preferably of the type which permits distillation at low temperature and reduced pressure such as in the use of molecular distillation or short path distillation. Preferably, the distillation is conducted at a temperature of 130~ C to 230~ C. The pressure can vary ~rom about 1 x 1~-3 kPa to 0.533 kPa to recover the puri~ied lower alkyl esters o~ the ~atty acids ~rom the distillation.
A~ter distillation o~ the lower alkyl esters o~ the fatty acids, such esters are then converted to oter esters, e.g., glycerides by transesterification, with the removal of lower alkanol, prefera~ly in the presence of catalytic quantities o~ an alkaline metal lower alkyl oxide.
The purified (distilled) lower alkyl esters of the fatty acids which are transesterified with a monohydric or polyhydric alcohol are generally in a molar ratio o~ 1 to 2 moles o~ the lower alkyl ester of the ~atty acid to each hydroxyl equivalent of the alcohol in the transeteri~ication reaction. In order to m;n;m; ze ~ormation of mono- and diglycerides in the preparation of triglycerides, it is pre~erred that the quantity o~ glycerol in the preparation o~
the egg derived triglycerides be no more than about 95% of SU~SIllUTE SHEET(~ULE263 U~O 97/26804 PCT~US97101138 the stoichiometric quantity re~uired for formation o~ the triglycerides.
The temperature used for the transesterification reaction of the lower alkyl esters of the fatty acids should be no higher than about 250~C and preferably no higher than 200~C since the double bonds in the polyunsaturated fatty acids are heat labile and can be converted ~rom cis to trans isomers. Thus the temperature of the transesterification can vary over a wide range such as that of from about 75 to 250 C and pre~erably about 150 to 200~ C. An alkaline metal lower alkoxide is again used in catalytic quantities ~or the transesterification to glycerids and other esters.
After the formation of the glycerides, or other esters in the transestrification reaction are produced, the reaction medium is neutralized with an acid as in the case of the transesteri~ication above for the ~ormation of the lower alkyl esters of the fatty acids ~rom lipid mixtures or phospholipids. The neutralized reaction medium is then treated to remove waste materials and recover a composition cont~;ning esters, e.g., glycerides, of the lipid source, e.g., egg yolk fatty acids, including that o~ AA and DHA, i.e., the Processed Natural Ingredients. Conventional techniques can be used ~or this purification, e.g., such as washing the neutralized reaction medium with water, a~ter which the lipid is dried with heat, vacuum or both. The Processed Natural Ingredients will contain at least 1 wt% of AA such as about 1 wt% to 10 wt% of AA and at least Q.1 wt%
of DHA such as about 0.1 wt% to 5 wt~ of DHA and preferably less than 0.01 wt% of phosphorus and less than 0.5 wt% of cholesterol.
A~ter removing wastes from and drying the glycerides, ~ the glycerides are optionally subjected to decolorization such as by contact with activated carbon and the solids from such process then removed, e.g., by a filter press to recover S~J~ 111 ~ITE SHEET (RULE 26) ~0 97/26804 PCT~US97/01138 the Processed Natural Ingredients which contain the glycerides of AA and DHA together with small quantities o~
cholesterol and even smaller quantities of phosphorus.
Additionally the decolorized glycerides can be deodorized to remove all volatile components such as free fatty acids, or lower alkyl ester thereof, and residual solvent. Such processing is typical ~or the production o~ edible glyceride oils.

The Free Fatty Acid Route In the ~ree ~atty acid route, ~atty acids, fatty acid esters, and mixtures thereo~ high in polyunsaturated ~atty acids are prepared. As in the ester route the lipids can be derived ~rom naturally occurring lipid mixtures and the resulting acids, esters and mixtures thereof o~ this invention ha~e low levels of cholesterol, sterols and phosphorus compounds which are pre~erably essentially ~ree of cholesterol, sterols, and phosphorus compounds as described hereinabove. The ~ree ~atty acid route o~ this invention involves up to ~ive steps. These steps are set ~orth above in the Disclosure of the Invention in the aspect of the ~ree fatty acid route. The free ~atty acid route starts with the same starting materials as the ester route.
In the first step, Step (A), the lipid mixture contAining phospholipids, triglycerides, and sterols is hydrolyzed in water to ~orm a two-phase product cont~ n; ng a ~atty acid phase comprised o~ free fatty acids and sterols, and an agueous phase comprised o~ water, glycerol, and glycerol phosphoric acid esters.
The hydrolysis of the lipid mixture in Step (A) may be catalyzed by either the addition of an acid or a base.
Pre~erably, the hydrolysis of the lipid mixture in Step (A) is accomplished by a base-catalyzed hydrolysis reaction. Such SIJ~S 111 ~JTE StiEET (RULE 26) W O 97/26804 PCTrUS97101138 bas~-catzl~zed hydrolysis reactions ar~ co~ onl~ known as saponiIication reactions. Suita~le base cz_alysts are aaueous alkali which include sodium, lithium, calc um, and potassium salt of an h~droxide, carbonate or bicarbo~ate. Combinations 5 G,- base catalysts may also be used.
The hydrolysis reaction in Step (A) is an equilibrium-limited reaction. The base-catalyzed reaction is driven to completion through the ~ormation of a metal salt o~ the corresponding ~atty acid. The base catalyst is added in at least a stoichiometric amount up to two times the stoichiometric amount based on the equivalents o~ ~atty acid groups contained in the lipid mixture. Preferably, the base catalyst is added in an amount o~ 1.1 to 1.5 times the e~uivalent o~ ~atty acid groups contained in the lipid mixture.
In a base-catalyzed hydrolysis the metzl salts o~ ~atty acids ~ormed during hydrolysis are acidi~ied to a pH of 4 or less with a mineral acid to ~orm a two-phase product contA; n; ~g a ~atty acid phase comprised o~ free ~atty acids and sterols, and an a~ueous phase comprised o~ water, glycerol, and glycerol phosphoric acid ester residues.
Mineral acids use~ul ~or the acidi~ication o~ the metal salts o~ the ~atty acids must have a pKa lower than the pKa o, the ~ree ~atty acid. Suitable mineral acids include sul~uric acid, nitric acid, hydrochloric acid, and phosphoric acid. Com~inations o~ mineral acids may also be used. The mineral acid is added in at least a stoichiometric amount based on the amount of base catalyst. The mineral acid may be added in dilute or concentrated rorm. A preferred mineral acid is aqueous hydrochloric acid.
In the absence o~ a suitable quantity o,~ lower alkanol in Step (A), unreacred phospholipids and hydrolyzed phospholipid residues act as sur~actant and mav inter~ere with the ~ormation o~ distinct ~atty acid znd zqueous phases SU~IllUTE SHEET~RULEZ6~

W 0 97/26804 PCT~US97/01138 in Step ~A). In the event that the lipid mixture o~ Step (A~
does not contain a lower alkanol in suita~le ~uantity, a lower alkanol may be added to the hydrolysis product in Step (A) to assist in two-phase formation. The alcohol solubilizes the fatty acids and helps partition the surfactant residues into the aqueous phase. The alcohol is added at a 0.5:1 to 3:1, pre~erablY a 1.5:1 mass ratio of alcohol to phospholipid present in the lipid mixture ~ed to Step (A). Examples of lower alkanols suitable to aid in two-phase ~ormation include methanol ethanol propanol,isopropanol, isobutanol, and butanol. The addition of lower alkanol outside this range either does not result in the ~ormation o~ a two phase mixture or results in poor partitioning o~ triglycerides and phospholipids into their respective phases.
In the second step, Step(B), the aqueous phase is separated from the ~atty acid phase o~ the two-phase product formed in Step (A). The ag~eous phase is removed by methods known in the art such as decantation. It is important to note that at acidic pH, the fatty acids may ~orm ~atty acid alcohol esters with any lower alkanol used optionally in Step (A). The fatty acid alcohol esters are undesira~le as they represent a yield loss of fatty acids. Therefore, it is desirable that: (1) the two-phase product ~ormed in Step (A) be maint~; n~ at a low temperature to slow the esterifica~ion reaction, but at a temperature which maintains the ~atty acids as a liquid phase, ~etween 35~ C to 55~ C, pre~erably 40~ C to 50~ C; and (2) the aqueous phase should be removed as soon as practical ~rom the two-phase product.
In the third step, Step (C), the ~atty acid phase ~rom Step (B) is heated at a temperature of 150~ C to ~0~ C, preferably 170~ C to 230~ C, to allow the ~atty acids to react with the sterols to ~orm sterol ~atty acid esters and water. Optionally water is removed ~rom the reaction to S~ TE SHEET~RULE26) W O 97/26804 PCTrUS97/01138 drive the eouilibrium toward the ~o~mation of the sterol fatty acid esters. The formation o'- ratty acid sterol esters represents a yield loss of ~atty acids, i~cluding a s.atistical distribution of polyunsaturated fatty acids ~ased on their percentage in the mixture, equal to one mole of ~atty acid ~or each mole o~ sterol ester .ormed. This yieid loss is necessary in order to convert the sterols in~o sterol esters which can be separated easily from fatty acids.
Optionally an esteri~icatiOn catalyst can be added in Step (C) to increase the rate of sterol 'atty acid ester formation. Examples of suitable esterirication catalysts include: dibutyl tin oxide, phosphoric acid, zinc oxide, hydrochloric acid, and butyl stannoic acid.
In the ~ourth step, Step (D), the ~atty acid and sterol ester mixture ~ormed in Step (C) is dis.illed at a temperatur~ o~ 130~ C to 250~ C and a pressure of 1 x 10-3 kPa to 0.5333 kPa, to recover puri~ied ~atty acids. The distillation is pre~erably conducted zt a temperature o~ 180~
C to 220~ C and a pressure o~ 1 x 10-3 kDa to 0.0667 kPa.
The ~atty acids are relatively volatil~ and distill overhead, while the sterol ~atty acid esters are not volatile and remain with the residue. The molecular welght distribution o~ the ~atty acid residues o~ subse~uertly derived glyceride proaucts can be controlled by distillation. For example, the lower molecular weight ~atty acids tend to be the lower boiling ~atty acids and concentrate in the rirst ~ractions of the distillation; and the higher molecular weight acids are rour,d in the higher boiling ~ractions. The resulting ~atty acids are essentially ~ree o~ sterol compounds and phosphorus containing residues. Successive distillation s~ages may be used to remove lighter acids and concer.rate heavier polyunsaturated acids such as ~, D~, and ~PA.
The ~ormation o~ sterol ~atty acid est2rs are critical tG the present invention in order to r2cove- r-atty acids in SUi~SIilUTE SHEET (RULE 26) ~'097/26804 PCT~US97/01138 high yield ~hich are ~ree of sterols and sterol esters. The relative volatility between the hish molecul2r weight poli~unsaturated ~atty acids such as AA, D~, and EPA, and the sterol esters is relatively large. Thus, the pol~unsaturated ,atty acids can be separated sharply from .he sterol esters with an~y single equilibrium stage, non-refluxed high vacuum distillation apparatus known in the art, including a wiped-~ilm evaporator, a ~alling ~ilm evaporator, a short path evaporator, and a centri~ugal molecular stlll.
Alternatively, the relative volatility of the ~ree sterols and the high molecular weight polyunsaturated ~atty acids such as AA, DHA, and EPA is relatively small. Thus, a sharp separation o~ free sterols from higher mol~cular weight polyunsaturated fatty acids is not practical by single equilibrium stage, non-refluxed high vacu~m distillation methods.
Multistage ~ract~onal distillation devices with re~lux which are capable o~ sharp separations between components o~
low relative volatility such as free sterols and fatty acids must operate at higher pressures and subse¢uently higher temperature in order to allow for suf~icient pressure drop across the multistage column. The requisite higher temperatures required in a multistage distillation leads to undesir~ble heat degradation and cis-trans isomerization of the unszturated ~atty acids.
Other methods of separation of sterols such as crystallization or supercritical extractio~ are more difficult and expensive. The melting points of sterols and ~atty acids overlap and a sharp separation requires comDlicated, expensive ~ractional crystalli7ation equipment and refrigeration. Supercritical extraCtiGn reauires e~Densive high pressure equipment to m~intain the extractant at supercritical conditions.

SU~SIllUTESHEET(RULE26 CA 02247368 l998-07-24 W 0 97/26804 PCT~US97/01138 Optionally, the purified ~atty acids, ;~ree of sterols and phosphorus containing residues, ~rom St~p (D) may be mixed with a Cl-C10 alkyl monohydric or polynydric alcohol and heated to produce a fatty ester or the alcohol, Step (E).
Suitable monohydric alcohols include, ~or example, methanol, ethanol, propanol, isopropanol, and butanol. Suitable polyhydric alcohols include, ~or example, glycerin, propylene glycol, ethylene glycol, sorbitol, sucrose, erythritol, pentaerythritol, mannitol, ~ructose, glucose, xylitol, and lactitol. The monohydric or polyhydric alcohol is added in a molar ratio of 1 to 2 moles of fatty acid to each hydroxyl equivalent of the alcohol, preferably, in a molar ratio o~
1.1 to 1.3 moles o~ fatty acid to each hydroxyl e~uivalent of the alcohol. optionally, water may be removed during the esteri~ication reaction to drive the e~uilibrium toward the ester product.
The Processed Natural Ingredients in the ~ree ~atty acid route are obtained a~ter separation o~ the glycerides ~rom the esteri~icatiOn reaction as in the case o~ t'e ester route. Optionally the Processed Natural Ingredients are puri~ied such as by deodorization and decoloration. The Processed Natural Ingredients can be the glyceride composition ~rom the esterification reaction with glycerol or pre~erably such glyceride composition a~ter purification in both the ~ree ~atty acid route and the ester route.
In both the ~ree ~atty acid route and the ester route, it is o~ten desirable to increase the ratio o~ the unsaturated ~atty acids or lower alkyl esters thereo~ in relation to the saturated ~atty acids or lower al~yl esters thereo~. As shown in Examples 5, 6, ~nd 7 hereor, this can be accomplished by various ~ractionatiOn t-c~niaues such as solvent Iractionation, solid rractionation such as cold pressed techniques, etc. Such ~ractionation can rely on the ~elting or solidi~ication temperatures or the egg yolk S~51ll~TESHEET(RULF26) ~'0 97/26804 PCT~US97/01138 saturate'd ~atty acids and esters thereof in relation to the unsaturated egg yolk fatty acids and esters thereof. The fractionation can be applied to the crude free fatty acids or the lower alkyl esters thereof before the distillation step or to the puri~ied free fatty acids or lower alkyl esters thereof after distillation.
The concentration of glycerides in the Processed Natural Ingredients from either the ester route or the ~ree acid route can vary from that of at least about 60%, pre~erably at least about 70~ and particularly at least 85 to 90% based on the weight of the Processed Natural Ingredients composition.
The r~m~;n~ is generally that of various reactants, intermediate products and solvents used in the method of this invention together with the small amounts of cholesterol and phosphorus. Illustratively~ such r~;n~er can contain:
alkanols and various other solvents as well as unreacted fatty acids or lower alkyl esters thereo~.
A typical ~atty acid profile of some of the more significant individual fatty -.ids o~ the triglycerides in the egg derived triglycerides is set ~orth in Table A below.

S~J~S 11~ UTE SH--ET (RULE 26) W O 97/26804 PCT~US97/01138 T~iBLE A
ANALYSIS OF EG~ YOL~ DERIVED ~RIGLYCERIDE OF THIS lNv~NllON
-Relative Amount Based on ~ Fattv Acid Pro~ile Total o~ Fattv Acids S~own C16:0 29.5 C18:0 11.0 C18:1 40.3 C18:2 15.6 C20:4w6 (AA) 2.9 C22:6w3 (DHA) 0.8 Total 100.1 Other components O~ Egg Derived Trialvcerides Amount(mg/100 g) Cholesterol less than 50 Phosphorus less than 10 The enteral formula of this invention can generally be prepared using the ~ollowing method. An appropriate quantity o~ protein is dispersed in su~icient water or oil to solubilize or suspend it, thereby ~orming a protein solution/
suspension. Typically this protein source would be intact milk or soy proteins and/or hydrolyzed milk or soy proteins.
A car~ohydrate source, such as one or more of corn syrup solids, lactose, maltodextrins and sucrose is dissolved in water, thereby ~orming a carbohYdrate solution. A source o~
dietary ~iber, such as soy polysaccharide, may also be added.
Approp~iate minerals are dissolved in water, the carbohydrate solution or oil, so as to ~orm a mineral solution Once ~ormed, the three solutions (protein, carbohydrate, and mineral) are combined in appropriate quantities with oils, especially the oils obtained by the instant process and oil soluble vit;~m;nC:~ This resulting solution is then heat processed and homogenized. Following processing, water soluble vit~m' n.~, iron, choline and other nutrients are added S~ TESHEET(RULEZ~) ~0 97126804 PCT~US97/01138 and then nucleotides mav be added. The solution is then diluted with water to ~he appropriate caloric density, approximately 670-725 kcal per liter o~ ~o~mula. The ~ormula is then dispensed into containers and reto-ted to obtain S commercial ste~ility or packaged as~ptically using commercially available techniques and equipment. As prepared, the ~or~ula contains appropriate nutrients in compliance with the In~ant Formula Act as o~ the date o~ this application. It should also be recognized that the unique rormula o~ this invention could be prepared for use in powdered ~orm or as a concentrated liauid.
The enteral ~ormula can also simply comprise a nutritional source o~ amino nitrogen, carbohydrates, edible ~ats, minerals, or vit~mi n.~, together with the egg derived triglycerides of this invention. Pre~erabl~, the egg derived triglyçerides will provide ~rom about 0.1 wt~ to about 2 wt%
o~ AA and typically about 1 wt% based on the total ~at in the in~ant ~ormula and DHA ~rom about 0.05 wt% to about 0.5 wt%
based on the total ~at in the in~ant rormula.
An enteral ~ormula o~ this invention is shown in Table B
below S~al~l~TESHEET~RULE26) U~O 97/26804 PCT~US97/01138 T~iB~E B
FOR~nLA ACCORDING TO THE IN~NTION
-NUTRIEN~ CONCENTRATION PER LIT~R OF FORMULA
Protein 13.0-20 g Protein Source Condensed Skim Milk 55-75%
7.15-15 g Whey Protein Concentrate25-45%
3.25-9 g ~ipid 13-40 g H.O. Sa~lower Oil 35-55%
Soy Oil 20-~0%
Coconut Oil 20-45%
triglycerides from egg derived triglycerides 2-20~
Carbohydrate Lactose 70-110 g Nucleotides 70-100 mg Cytidine monophosphate29-39 mg Uridine monophosphate15-21 mg Adinosine monophosphate10-16 mg Guanosine monophosphate14-20 mg Iron 8-16 mg R,R,R, ~
tocopherol 10-30 IU
~Carotene 375-575 ,ug Selenium 14-32 mcg Calcium 475-850 mg Phosphorus '240-700 mg Ca:P Ratio 1.4-2.4 Although the percentage of triglycerides ~rom the egg derived triglycerides is 2 to 20% in the above pre~erred enteral ~ormula, the quantity o~ the other lipids can be decreased so that the triglycerides ~rom tke egg derived triglycerides can make up about 2 to 95% o~ the lipids. The amount of egg derived triglycerides in the in~ant ~ormula is generally less than about 36 g per liter o~ rormula.
Also contemplated by this invention is the use o~ the egg-derived triglycerides in a nutritional supplement ~or kumans and ~n;m~l S that may be in the .~orm o~ a pill or c~psule. ~lore speci~ically, the nutri.ior-l supplement in SUa~ I I I UTESHEET(RULE26) CA 02247368 l998-07-24 W 0 97/26804 PCT~US97/01138 accordance with this invention could be used by pregnant and/or lactating females.
The following examples are illustrative of the invention. All parts and percentages in the examples, as well as elsewhere in this application, are by weight. Room or ambient temperature is 23 degrees C, unless the context indicates otherwise.

PREPARA~ION OF EGG DERrVED TRIGhYCERIDES BY ESTER ROUTE

Type Y-l Egg yolk so:ids of Henningsen Foods, Inc. of 14334 Industrial ~oad, Omaha NE were used in this example.
Such egg volk solids have the ~ollowing chemical and physical st~n~ds: moisture of 0.5% m~X;mllm; pH of 6.5+ 0.3; fat of 56% minimllm; protein of 30~ m;nim-lm; color of 40-60 ppm Beta-carotene; and granulation so that 100~ passes through U.S.S.S. # 16 screen. Egg yolk solids(455.7 g) ~nn;ngsen Foods type Y-l were placed in a beaker (2 liters [L]) with methanol (1 L), heated to 60~C and stirred with a magnetic stir bar. The yellow slurry was stirred for 1 hour and after a brief coo~ing period the solids were removed by vacuum filtration. The insoluble egg yolk components cont~; n~ in the funnel were washed with an additional amount of methanol (2 X 200 ml). The filtrate was placed in a 3-neck round bottom flask (1 L)and a portion of the methanol was removed by distillation so that all the filtrate could by accommodated by the one liter flask. The acid content of the methanol lipid mixture was determined by titremetric measurement and an equal number of moles of sodium methoxide was added so as to neutralize any acid. An additional amount of sodium methoxide ~1 g) was added to act as catalyst ~or the transesterification of the egg lipids to methyl esters.
A~ter approximately one hour the reaction was complete as S~5lll~TE SHEET~RULE263 W O 97/26804 PCTrUS97/01138 determined by TLC (thin layer chromatography) indicating that all of the egg lipids had been converted to methyl esters.
The reaction was quenched by the addition o~ phosphoric acid (0.7 g). The acid addition caused the ~ormation o~ a precipitate. A~ter cooling the precipitate was removed by vacuum filtration. The ~iltrate separated into two phases.
The upper orange layer contained mostly methyl esters and a small amount o~ methanol solvent. The lower dark layer contained some methyl esters and most o~ the methanol. The lower layer was nearly water dispersable. A~ter removal o~
the excess methanol ~rom the lower layer, an additional amount o~ the crude methyl esters could be isolated. The combined crude methyl esters (82.4 g) were distilled with a short path glass evaporator (UIC Inc., KDL-4 Unit) at vacuum o~ 0.045 mm Hg and jacket temperature o~ 100~ C. This clear and colorless distillate (60.4 g) o~ puri~ied egg derived methyl esters contained 0.46 wt% cholesterol and less than 5 ppm o~ phosphorus. The puri~ied methyl esters (45 g) were combined with glycerin (4.6 g) in a 3 neck round bottom ~lask (100 ml). The flask was purged with nitrogen and a nitrogen atmosphere was maintained throughout. The immiscible mixture was stirred vigorously with a magnetic stir bar. A~ter drying the mixture at elevated temperatures, sodium methoxide (0.5 g) was slowly added to the reaction mixture. Heating was maintained between 110-170~ ~ ~or 24 hours. TL~
indicated that the reaction was slowly proceeding. An additional amount o~ sodium methoxide (0.2 g) was added and heating continued an additional 24 hours. A~terwards the reaction mixture was cooled and neutralized by the addition o~ 85% phosphoric acid (0.5 g). The mixture was washed with water (5 X 20 ml) and dried with heat and vacuum. The oil was deodorized with a short path glass evaporator to remove all volatiles including unreacted methyl esters in order to obtain the egg yolk derived triglycerides.

SUBSTITUTE SHEET(RULE26) ~'097/26804 PCTrUS97/01138 T~iBLE 1 This table sets ~orth the composition of the egg derived triglycerides prepared in Example 1. The extracted lipids ~rom the egg powder dissolved in methanol are referred to as "Extract~; and the decolorized and deodorized triglyceride egg derived triglycerides referred to as "Puri~ied Triglyceride". This table also shows quantities o~ ~atty acids and cholesterol obtained in another experiment involving the method o~ this invention ~or a crude triglyceride be~ore deodorization and decolorization which is simply re~erred to as ~Crude Product". The quantitative results are expressed as grams in 100 grams o~ sample. The designation ~N/D~ means that the quantity was not detectable whereas ~NJR means not reported.

SUBSTITUTE SHEET~RULE26) -CA 02247368 l998-07-24 ~'0 97/26804 PCT~US97/01138 Distilled Crude Puri~ied Extract Methvl ~ters ~roau~t TriqlYceride Cholesterol2 786 0 465 N/D N/D
Fatty acids C14:0 0 156 0 347 Q.303 0.310 C14:1 0.025 0.066 ~/~ N/R
C15:0 0.037 0.080 M/~ N/R
C16:0 13.790 27.051 24.821 23.371 C16:1 1.148 2.586 2.370 2.250 C16:2 0.024 N/R N/R N/R
C16:3 0.080 0.172 0.162 0.152 C16:4 0.073 N/R N/R N/R
C18:0 5.912 9.925 9.344 8 725 C18:1 18.695 36.121 34.095 31.969 C18:2 7.875 14.203 13.213 12.361 C18:3 0.192 0.331 0.264 0.248 C18:4 0.067 0.058 0 187 0.176 C20:0 N/R 0.030 N/~ N/R
C20:1 0.123 0.223 0.210 0.206 C20:2w6 0.152 0.157 0.219 0.224 C20:3w6 0.178 0.264 0.243 0.233 C20:4w6 (AA)2.096 2.882 2.500 2.367 C20:5w3 N/R 0 032 N/R N/R
C21:5 0.036 N/R R/R N~R
C22:4w6 0.130 0.141 0.123 0.123 C22:5w6 0 493 0.~36 0.453 0.439 C22:5w3 0.070 0.083 N/~ N/R
C22:6w3 (DHA) 0.675 0.616 0.512 0.596 TOTAL 52.026 96.568 89 124 83.750 PREPARATION OF EG& DERIVED TRIG~YCERIDES BY ESTER ROUTE

Egg yolk powder (8 batches o~ 500 g, or 4,000 g total) was mixed with methanol (8 batches o~ 1,000 ml, or 8,000 ml total) and heated to 50-60 degrees C with stirring. The egg powder did not ~reely disperse in the meth2no'1, and the clumps o~ egg powder had to be broken u~ via a spatula. The extraction time averaged about 45-60 minut-s before the slurry wzs ~iltered through a Buchner _unr-l. The egg protein _iltered very quickly, and an addi-ional 200 ml of SUBSTITUTESHEET(RULE26) ~0 97/26804 PCT~US97/01138 methanol (per batch) was used to wash the insoluble egg yolk components.
By isolation o~ the extract in a separate e-~eriment, the acid value or the extract was aoout 12. In order to reduce the usage o~ sodium methoxide ror the transesterification, 21.6 g o~ calcium oxide was added. This amount o~ calcium oxide was enough to neutralize an acid value o 12, assuming that the weight o~ the extract is 50 wt% o~ the egg powder. A~terward the yield o~ the extract ~rom egg powder was estimated to be about 33 wt~ and there~or an excess o~ calcium oxide was probably used.
To initiate the transesterirication, 36 ml o~ 2S% sodium methoxide in methanol was added to the methanol solution at room temperature. Within one hour, the reaction was nearly complete, but the reaction was stirred overnight ~or convenience. There was not a glycerol layer in the bottom o~
the reaction ~lask as would be normally expected, but there were calcium salts suspended in the mixture. Acetic acid (9.45 g ) was added to neutralize the sodium methoxide be~ore the removal o~ methanol. Methanol was removed by distillation by heating the reaction mixture up to a temperature of 75 degrees C, and ~inally heating under vacuum.
The residue was poured into centri~uge bottles, and placed in a centri~uge set to run at 4,000 rpm for 15 minutes at room temperature. A~ter centriIugation, there were two phases in the bottles, and the dark orange upper layer was decanted ~rom the calcium salt resldue. The calcium salt residue weighed 382 g. The orange colored upper layer was not totally homogeneous, and it appears that cholesterol was crystallizing out.
The orange colored decantate was dilu~-d with a~out 275 g o~ triglyceride oil previously isolated rom the egg yolk phospholipids. This nonvolatile t-iglyceride oil was added SU~SIll~TE S~EET(RULE26~

~0 97126804 PCTrUS97/01138 to lubricate the rotors o:E the Pope still because of the high cholesterol concentration o~ the decantate. The decantate was added to the still. At a vacuum of 1 mm EIg, the distillation was conduc~ed at a temperature or 200 * 20 5 degrees C. This temperature is the set point of an external heating mantle on the Pope still, and this is not the temperature where the methyl esters actually distill.
From this distillation was obtained, 820 g of~ distilled methyl esters. qlhese methyl esters contained 0.3%
10 cholesterol by GC (gas chromatographY) assay, and the distillate turned into a semi-solid upon standing. The residue weighed 489 g . From these isolated yields, one obtains the :Eollowing:

Calcium salt residue382 grams distilled methyl esters820 grams distillation residue489 grams triglyceride diluent-275 grams calcium oxide added -21 grams sodium methoxide -10 grams acetic acid -10 grams Total isolated weight1375 grams This isolated weight shows that the extract weight was~5 about 30 wt% based on egg powder.
About 741 grams oi~ the distilled methyl esters was used ~or the Einal esteri~lcation. This distillate was mixed with 82 g o~ glycerol and 10 ml of~ 25% sodium methoxide. The esterif~ication reaction started at 75~ C, and was gradually 30 increased. The temperature was started this low because analogous esterifications-with methyl oleate began to darken at this temperature. No darkening oi~ the reaction mixture occurred up to 150 degrees C. The temperature was later increased to 170 degrees bei~ore the reaction was terminated.

S~ JTE SHEET(RULE 2~i~

W 0 g7/26804 PCTrUS97/~1138 After 7 days of constant heating, there was no sign of major decomposition, and the product color was very light. The reaction mixture was cooled to 75 degrees and ~.5 g of 85%
phosphoric acid was added to neutralize the sodium methoxide catalyst, and then hot water (400 ml) was added to wash away the acid salts. Two additional hot water washes were used to remove the salts. Hot water was necessary to reduce the ~ormation of emulsions. The product was then heated to 95 degrees C under vacuum to degas and dry the sample. This product, referred to herein as the egg derived triglycerides weighed 711 g, but this number is not an accurate yield because much of the reaction mixture was removed during sampling to analyze the progress of the reaction.
A small portion of the final product above was removed and heated to liquify the methyl esters. The sample was treated with activated carbon, and later filtered through a bed of Celite in order to decolorize it. There was a slight improvement in the color by carbon treatment.
About 120 grams of decolorized product was added to the molecular still to remove the unreacted methyl esters in order to deodorize the product. After deodorization, 87.6 g of triglyceride residue and 12.6 g of methyl ester was isolated. The lost 20 grams is not indicative of the process, and it is only the holdup and loss after small scale distillation. However, the lost 20 grams is mostly methyl ester. Analysis of the product obtained by the process of this Example 2 showed the presence o~ AA and DHA in a higher than expected amount. The methyl ester product was remarkably stable and there was no apparent decomposition or darkening during the glycerol esterification reaction The decolorized and deodorized triglycerides appeared to have darkened slightly during the distillation. Decoloration may not be necessary, but it appears that if performed that it be done last.

SUBSTITUTESHEET(RULE26 ~'097/26804 PCTrUS97/01138 Table 2 below shows the ~atty acid content o~ various compositions ~rom Example 2 as a percent of total fatty acid as obtained by analyzing the ~atty acid methyl esters (FAME) or the various compositions indicated in the table.

Final Product Startiny Material FAME Triglycerides Distilled Esters Egg Powder Extract 14:0 0.14 0.11 0.31 16:0 22.35 20.90 27.63 16:1 1.84 1.71 0.53 16:3 0.16 0.16 0.15 16:4 NtR N/R O.lS
17:0 0.22 0.22 0.22 18:0 11.76 11.90 11.25 18:1 40.09 40.32 36.75 18:2 15.42 15.35 15.10 18:3w60.12 0.12 N/R
18:3w30.29 0.29 0.26 18:4 0.18 0.18 0.14 20:1 0.32 0.37 0.24 20:2 0.29 0.38 0.27 20:3 0.32 0.41 0.29 20:4w63.85 4.23 3.92 (AA) 22:0 N/R 0.13 0.14 22:4 0.26 0.35 0.24 22:5w61.02 1.18 0.98 22:5w30.11 0.21 0.15 22:6w31.27 1.47 1.26 (DHA) Total100.00 100.00 100.00 -S~SIll~TE SHEET(RULE 26) - =
CA 02247368 l998-07-24 ~'0 97/26804 PCT~US97/01138 EGG POWDER EXTRACTION OF LIPID WITH VARIOUS SOhVENTS

Solvent Tem~erature vield% tfat) ~AA

2:1 CHCl,/CH30HS0-60~ C 64.2 2.0 Isopropyl alcohol50-60~ C 60.0 1.8 Methyl alcohol 50-60~ C 37.3 4.2 Ethyl alcohol 50-60~ C 57.2 2.2 Ethyl alcohol 22~ C 41.1 2.7 Ethyl alcohol 4~ C 25.2 3. 7 The above extractions were per~ormed similarl~ to the extraction described in Example 1. It can be seen rrom the above table that mixture of trichlorometh~ne and methanol gave a high yield o~ total ~at but the ~ ~,ias only 2.0% in the ~at. The methyl alcohol gave a relatively low yield o~
total ~at but a very high yield o~ AA in the lat. The isopropyl alcohol as well as the two runs or ethyl alcohol at 50-60 and 22~ C give relatively high yields o~ total fa~ but small yields o~ AA in the ~at. The ethyl alcohol at 4 degrees C gave the sm~llest yield o_ tot~l rat but a relatively high yield o~ AA in the ~at. It can be seen ~rom the above that at temperatures above abou~ 20 degrees C, the meth~nol was superior compared to the other solvents in the percentage o~ AA extracted in the lipids. At 4 degrees C the percentage yield o~ AA i~ the ~at had increased ror ethanol but the yield was lower at that tempera~ure ~or ethanol as to total ~at and ~A in comparison to the meth~nol.

SOhVENT FRACTIONATION OF DISTILLED FATTY ACIDS

A sample or distilled egg derivea attv acids (1 g) was cissolved at room temper~ture in he~~ne (4 ml). The sample ~s placed in the r~frigerator a. a t--.,pe-~ture or S~ TESHEET(RULE26) ~'0 97/26804 PCT~US97/01138 approximately 5 degrees C. A~ter CGO1 ing L Or two days a white soLid had precipitzted. ~ ~orticn c the ciear supernatant liquid was isolated and an FAP (~atty acid pro~ile) was obtained. The resul~s are sho~n below wherein S F~E means ratty acid methyl este~; S.~. means s~arting material, namely, the distilled egg de~ived fatty acids; and Prod. means the clear supernatant liauid. The acids are merely designated ~y the number Ol carbon atoms o~ the acid and the number o~ unsaturated groups (a~ter the colon) ~or the particular acid involved.

EaM~ S.M P-od.
C16:0 22.7 13.5 C18:0 11.6 ~.2 lS C18:1 40.0 47 7 C18:2 15.3 18.8 C20:4 3 8 ~.9 C22:6 1.2 1.4 From the results o~ the above Exam~le 4 it can be seen that the solvent ~ractionation of the dis.-lled ~atty acids increased the concentration o~ the unsatu-ated ~atty acids.
The solid precipitate appears to be mos.ly saturated ~atty acids. Thus, this procedure increases the concentration of unsaturated ~atty acids such as AA and D~' which in turn reduces the amount o~ egg derived triglycerides needed in an enteral rormula to achieve desired levels OL ~ and DHA.

SOLVENT FRACTIONATION OF ~ I ~J~ ESTERS

A sample o~ egg yolk methyl es.ers e~c'racted with methanol ~rom egg yolk prior to distillation to remove cholesterol was dissolved in he~cane (- ml). The sample was S~ TESHEET(RULEZ6) CA 02247368 l998-07-24 ~'0 97/26804 PCTAUS97/01138 placed in the ~reezer at a temperature o~ approximately -20 degrees C. After cooling for two days a solid precipitate ~ormed. A portion o~ the supernatant liquid was isolated and an FAP was obtained. The abbreviations in the below table are the same as those ol Example 4 above and again it can be seen that the ~ractionation increased the concentration of unsaturated ~atty acids such as AA and DHA.

E~ S.M. Prod.
C16:0 26.7 14.7 C18:0 11.6 4.2 C18:1 36.4 46.6 C18:2 13.6 17.4 C20:4 3.7 4.8 ~22:6 0.9 1.1 COLD TEMPERAq'URE FRAC~TIONATION OF MEIHYL ESTERS

A sample o~ distilled egg yolk derived methyl esters (1 g) was placed in a syringe ~5 ml.) in which the end was plugged with a small piece of cotton. The plunger o~ the syringe was inserted and all air was removed ~rom the syringe body. The syringe, containing the sample, was placed in the refrigerator at a temperature o~ approximately 5~ C. A~ter cooling ~or two days the entire syringe contents appeared to be a solid white mass. The syringe was removed ~rom the refrigerator and pressure was quickly applied to the plunger and a clear liquid i~raction was isolated. An FAP o~ the clear liquid was obtained. The results are shown below wherein the m~n;n~ o~ abbreviatio~s is the same as in Example 4 and again it can be seen that this procedure increases the concentration o~ unsaturated ~atty acids such 2s AA and DHA.

S~ UTESHEET(RULE26 , ~0 97/26804 PCTrUS97/01138 AM~ S.M. P~?d C16:0 27.8 1~.4 C18:0 10.5 ~.6 C18:1 36.1 47.3 C18:2 14.6 19.0 C20:4 3.1 ~.0 C22:6 0.8 1.0 Li~uid egg yolk (292.5 g; "Ezsy Eggs~, M.G. Waldbaum, Gaylord Minnesota) was mixed with ethanol (690 ml) in a one liter beaker and stirred with a magnetic stir bar. The mixture was heated with a hot plate until boiling. Bolling was continued ~or lC minutes. The mixture was cooled ~or several minutes and then ~iltered with a buchner apparatus.
The insoluble egg yolk components were rirst rinsed with ethanol (100 ml) and then removed rrom the runnel and stirred in an additional amount o~ ethanol (250 ml) at room temperature ror 5 minutes. The solid material was again ~iltered and washed with ethanol (100 ml). The combined ethanol solutions were placed in single se~aratory ~unnel and allowed to stand undisturbed over night. ~ phase separation occurred ~nd the lower layer, mostly triglyceride, was removed. The eth~nolic solution or egg phospholipids was placed in a 3-neck round bottom ~las~ (lL). Sodium hydroxide pellets (2.56 g) were added to the mechanically stirred solution. Heating commenced and ethanol w-s removed by simple distillation. A~ter approximately 250 ml o~ ethanol had been removed by distillation, TLC indicated that the reaction mixture contained a signiricant ~mount o~ ethyl esters. An additional amount or sodium h~~~oxide pellets (1.5 g ) were added and ethanol distillation con~inued.
ter another 1~5 ml or ethanol was romov--, TLC indicatea S~ JTE SHEET (RULE 26) W 0 97/26804 PCT~US97/01138 that the reaction mixture contained no ethyl ester and only ~atty acids o~ the original egg phospholipid extract. Heat was removed ~rom the ~lask and a~ter cooling several minutes, concentrated HCl (6 ml) was adaed to the mixture in order to neutralize the base. Water was added to the cooled mixture and then the entire solution was extracted with hexane (2 X 400 ml). The combined hexane extracts were dried with sodium sul~ate and the hexane was removed under reduced pressure. A dark orangish oil (14.65 g ) was obtained. The oil was again dissolved in hexane (50 ml) and placed in a re~rigerator at a temperature o~ 0-5~ C and allowed to stand overnight. A solid ~raction precipitated ~rom the hexane solution and was isolated ~y ~iltration. The hexane ~iltrate was placed in the freezer (-20~ C)and allowed to stand ~or lS 6 hours. Again, a solid precipitate ~ormed that was isolated by ~iltration. The ~iltrate was stripped o~ solvent under reduced pressure to yield a dark orange oil (6.68 g). GC
analysis o~ the various ~ractions indicates that the solid materials are principally saturated ~ree ~atty acids and the liquid ~ractions show increasing concentrations o~
unsaturated ~atty acids. Tables 7A and 7B below show the relative ~atty acid pro~ile of various samples o~ this example wherein:
Sample A, also re~erred to as Folch Ext. is the Folch extract o~ liquid egg yolks;
Sample B, also re~erred to as EtOH Tri~l., is the triglyceride ~raction isolated ~rom ethanol extract;
Sample C, also re~erred to as EtOH Acids, is the ~irst ~raction o~ crude ~atty acids (no cold/solvent ~ractionation) 30Sample D, also re~erred to as 0C Li~. Frac., is the liquid ~raction ~rom 0-5~ C hexane ~ractionation;
Sample E, also re~erred to a -20C Liq. Frac., is the liquid ~raction ~rom -20~ C hexane ~ractionation;

SUBSTITUTESHEET~RULE263 WO 97/26804 PCTrUS97/01138 Sample F, also referred to as 0C Solid Frac., is the solid precipitate fraction from 0-5~ C hexzne f~actionation;
and Sample G, also referred to as -20C Solid Frac., is the solid precipitate ~raction from -20~ C hexane ~ractiOnatiOn The ~ree fatty acids, as prepared above prior to extraction with hexane, can then be ~istilled to separate such acids ~rom cholesterol, pre~erably after heating to form cholesterol esters with the ~ree fatty acids. The distillate of purified ~ree ~atty acids can then be sub~ected to esteri~ication with glycerol to prepare the egg derived triglyceride o~ this invention.

Relative FAP

Sample A Sample B S~mple C Sample D
FAME Folch Ext. EtOH Trigl. EtOH Acids 0C Li~. Frac.
C1626.71 25.95 28.76 22.04 C16:12.90 3.42 1.54 1.80 C188.89 7.67 12.46 8.68 C18:141.31 45.19 29.72 34.88 ~18:214,45 14.10 16.04 18.83 C20:4w6 2.10 1.03 6.27 7 44 C20:5w6 0.52 0.22 1.73 2.06 C22:6w3 0.47 0.20 1.70 2.02 Total -97.35 97.78 99.22 97.75 AA/DHA4.47 5.15 3.69 3.68 S~ TESHEET(RULE26~

CA 02247368 l99X-07-24 097/26804 PCTnUS97/01138 T}~BLE 7B
Relati~e FAP
Sample E SamPle F Sample G
FA~OE -20C Lig. Frac. OC Solid Frac. -20C Solid Frac.
C16 6.24 54.97 58.68 C16:1 2.42 0.00 0.37 C18 1.87 32. 3g 27. 59 C18:146.62 7.13 7.09 C18:225.41 4.00 3.55 C20:4w69.96 1.5Q ~.31 20:5w62.75 0.~)0 0.35 22:6w32.68 O.OO 0.33 Total97.95 99.99 99.27 AA/DHA3. 72 N/AP 3.97 It can be seen ~rom Table 7B that ~ractional crystallization o~ ~atty acids in hexane increases the concentration o~ the unsaturated ~atty acids while dramatically reducing the amount o~ saturated ~atty acids.

PREPAR~TION OF TRIGLY~R Tn~-C BY FREE FATTY ACID ROU~E

A 500 mL three neck ~lask e~uipped with a mechanical stirrer, re~lux condenser, addition ~unnel, thermowell, heating mantle, and nitrogen atmosphere was charged with 154 grams o~ lipid mixture, obtained by the leaching of powdered egg yolk with methanol, 193 grams o~ methanol, and 28 grams of water. Sodium hydroxide (80 g o~ 50% dilution~
was added through the addition funnel. The resulting mixture was heated at 64~ C ~or 145 minutes. Hydrochloric acid ~84 mL o~ 12 N) was added over ~ive minutes. An additional 14 mL
o~ HCl was added in small portions until a pH of 2 was attained. Stirring was stopped and the phases were allowed to separate. The aqueous (bottom) phase was separated and contained 0.58~ phosphorus. The organlc phase weighed 128 g S~ UTE SHEET(RULE26~

~097/26804 P~~ 97loll38 and contained 6% monoglycerides, 2~ ~atty acid methyl esters, 5% cholesterol, and free fatty acids.
The ~ree ~atty acids, 124 g, were charged to a 300 mL 3 neck flask equipped with a mechanical stirrer, water trap, thermowell, heating mantle, and a sparge tube. The mixture was heated at 170~ C for 4 hours with a nitrogen sparge o~
100 mL/min. Residual methanol, 14 g, and water of reaction were collected, the resulting product was distilled at 245G C
and 0.5 Torr (0.0667 kPa) on a wiped ~ilm evaporator to give 83 g o~ distillate and 16 g residue. The distillate l~atty acids) contained 0.13% cholesterol and no detectable phosphorus. The residue contained prP~om;n~ntly cholesterol esters of ~a~ty acids.
A sample of the distillate fatty acids, 67 g, was charged to a 300 mL 3 neck ~lask equipped with a mechanical stirrer, water trap, thermowell, heating mantle, re~lux condenser, and sparge tube. The sample was warmed to 110~ C, and ~.6 g o~ glycerin was added under nitrogen. The temperature was increased to 160~ C. The resulting mixture was heated ~or 29 hours with a nitrogen sparge of 100 mL/min.
The resulting product was passed through a wiped ~ilm evaporator at 0.4 Torr and 220~ C to remove excess ~atty acids. The ~atty acid distillate weighed 8 g, and the triglyceride residue weighed 50 g. Analysis of the triglycerides showed 96% triglycerides and 4% diglycerides.
Total cholesterol was less than 0.13%.

EX~NP~E 9 P~EP~ TION OF TRIG~Y~RTn~ BY FREE FAT~Y ACID ROlrrE

A 22 L reaction vessel was charged with 7733 grams (g) ~ o~ the methanol containing phase o~tained ~rom leaching 5 kg o~ powdered egg yolk with 9 L of methanol at 60~ C for 3 hours. The mix~ure was heated to re~lux and 5.7 L o~

SU~SIlIUTESHEET(RULE26) CA 02247368 l998-07-24 W O 97/26804 PCT~US97/01138 methanol was distilled off. To the resulting mixture was added 2. 5 L of water, followed by 750 g of 5096 NaOH solution.
The resulting mixture was heated at reflux (65-70~ C) with stirring ~or 2.5 hours. The heat was removed, and 785 mL of concentrated HCl was slowly added while the temperature o~
the mixture was maintained above 50~ C. Agitation was discontinued, and the phases were allowed to separate. The bottom phase was separated and weighed 5764 g and cont;~; nF~l 0.31% phosphorus. The ~atty acid phase weighed 1350 g and contained 5.296 cholesterol, 0 .17% phosphorus, and 5.5% fatty acid methyl esters. The fatty acid phase was charged to a 3 L ~lask equipped with a N2 (nitrogen gas) sparge and water trap and was heated to 170Q C i~or 7 hours with a sparge rate of about 1 L/min. A total o~ 83 g of methanol/water mixture was collected during this time. The product weighed 1216 grams and contA;ne~ O.02~ cholesterol.
The product was purified by distillation through a wiped ~ilm evaporator. Distillation at 180~ C and 0.5 Torr gave 21~ g of distillate that contained 13% fatty acid methyl esters, 41% palmitic acid, and 24% oleic acid. The residue was redistilled at 280~ C to give 745 g of distillate and 151 g o~ residue. The residue contained mainly cholesterol esters. The distillate contained the larger fraction of higher molecular weight fatty acids than the crude material.
A 2 L ~1ask equipped with a N2 sparge and water trap was charged with 708 g of the distillate obtained at 280~ C and with 71 g of glycerin. The resulting mixture was heated at 160~ C for 24 hours. The resulting product was transferred to the wiped film evaporator and distilled at 280~ C and 0.5 Torr to give 155 g fatty acid distillate and 480 g of triglyceride product. The triglyceride product contained 90%
triglycerides and 9% diglycerides.

SU~IlIUTE SHEET (RULE 26 V~O 97/26804 PCTrUS97/01138 E~ PLE 10 PREPARATION OF GLYCERIDES BY FREE FATTY ACID ROUTE

A 300 ml flask equipped with a mechanical stirrer, water trap, and N2 sparge was charged with 80.5 g of fatty acid distillate recovered ~rom Example 9, and with 7.82 g of glycerin. The resulting mixture was heated at 230~ C with a N2 sparge ~or 3 hours. The mixture contained 86%
triglycerides and 12% diglycerides.

PREPARATION OF FATTY ACIDS BY FREE FATTY ACID ROUTE

The procedure descried in Example 9 was followed except the methanol was not distilled from the saponi~ication step until a~ter the NaOH was added. A 6060 g methanol solution of lipid mixture was mixed with 750 g o~ 50% NaOH. The resulting mixture was heated at reflux while 2 L o~ methanol was distilled ~rom the mixture ~or 150 minutes. Water, 200 m~ , was added back to the mixture and heating was continued an additional 30 minutes. The mixture was acidi~ied to pH or 2 and was allowed to cool to 6 oD c over a two hour period and the phases were separated. The ~atty acid phase weighed 771 g and contained 20~ ~atty acid methyl esters.

P~ASE SEPARATION OF FATTY ACIDS IN FREE FATTY ACID ROUTE

A 500 ml ~lask was charged with 83 g o~ an egg lipid - 30 mixture ~ree o~ methanol, 122 ml o~ water, and 39 g o~ 50%
NaOH solution. The resulting mixture was heated at 70~ C ~or 3 hours. Concentrated HCl (41 mL) was added over ~ive minutes, causing a slight exotherm. The addition o~ HCl caused the product to lorm as a sticky, solid phase which SUBSTITUTE SHEET(RULE26~

_ CA 02247368 l99X-07-24 W 097126804 PCT~US97/01138 could not be cleanly separated from the aqueous phase.
Methanol, 122 g, was added to the mixture at 60~ C while stirring. The resultlng mixture was trans~erred to a warm separatory ~unnel and the phases were allowed to separate.
S The aqueous phase weighed 343 g and the fatty acid phase weighed 65.6 g. The fatty acid product contained less than 2% ~atty acid methyl esters.

EXAMP~E 13 SEPARATION OF CHOLESTEROh ESTERS IN FREE FATTY A~ID ROUTE

A 1213 g sample of ~atty acids that had been treated to esteri~y cholesterol was charged to a steam ~acketed addition funnel. The material was ~ed to a Rodney-Hunt wiped ~ilm molecular stlll at a rate of 5 mL/min. The temperature o~ the still was maintained at 150~ C and the pressure was Q.5 Torr.
A total of 21S grams o~ distillate was collected. The distillate cont~;ne~ 41% palmitic acid, 24~ oleic acid, 13%
fatty acid methyl esters, and less than 0.5~ of C20 ~atty acids. The residue was charged to the addition ~unnel and ~ed to the molecular still at a rate o~ 3.5 mL/min while the temperature o~ the still was maintained at 230~ C and the pressure was 0.~ Torr.
The distillate weighed 547 g and contained 45% oleic acid, less than 1% ~atty acid methyl esters, and greater than 3% o~ C20 and heavier ~atty acids. The residue from this fraction was charged to the addition ~unnel and ~ed to the molecular still at a rate of 3.5 mL/min while the temperature was maintained at 250~ C and the pressure was 0.35 Torr. The 3Q distillate weighed 193 g and contained 47% oleic acid, no ~atty acid methyl esters,-and greater than 4% o~ C20 and heavier ~atty acids. The residue weighed 151 g and contained mainly ~atty acid sterol esters and less than 2% free ~atty acids.

SUBSnnnESHEET(RULE26 W 0 97~6804 PCTrUS97/01138 E~ PLE 14 ~TRU~CTION OF LIPIDS WITH ~IETH~iNOL A~D

PHASE SEPARATION OF TRIGLYCERIDES FROM LIPIDS

A 1,000 gal glass-lined reactor e~uipped with a mechanical agitator, condenser, nitrogen, and vacuum system was charged with ~,000 lb of egg yolk powder and 300 gal of methanol. The resulting mixture was heated to 65~ C and agitated ~or three hours. After ~iltering o~ the protein residue and washing with methanol, the methanol-lipid ~iltrate was returned to the 1,000 gal reactor and heated with agitation to 45~ C. The agitation was stopped and the mixture was allowed to settle for one hour, with the temperature maintained between 40-45~ C. Phase separation lS spontaneously occurred. The bottom phase was decanted o~~, sampled, and weighed. Analysis showed the bottom phase to weigh 96 lb and contained 94.9% triglyceride, 509 ppm phosphorus, and a ~atty acid distribution on a relative basis o~ O.6% arachidonic acid and 0% DHA. The top phase, upon 2~ stripping o~f methanol, weighed 245 lbs, and contained 4%
triglycerides, 3.63% phosphorus, and a ~atty acid distribution on a relative basis of 6.5% arachidonic acid and 2.0~ DHA.
The Processed Natural Ingredients o~ this invention have utility in enteral ~ormulas, nutritional supplements, parenteral ~ormulas, and can serve as starting materials ~or various edible emulsi~iers such as diacetyltartaric acid esters o~ mono-and diglycerides (DHTEM), succinylated mono-and diglycerides, and acylated mono- and diglycerides. The ~ree ~atty acids or lower alkyl esters o~ the fatty acids prepared ~rom the egg yolk lipids can also serve as starting materials ~or the preparation c~ various other edible lipid ingredients such as polyglycerol esters, propylene glycol esters, sorbate esters, and the like.

SU~a ~ JTE SHEET (RULE 26) ~'0 97~6804 PCT~US97/01138 Man~ variations will suggest thems~lv~s .o those skilled in this a~ light of the above detail~ description. All such obvious modi ications are within the -ull intended scope o' the appended claims.

S~ TESHEET~RULE26)

Claims (27)

WHAT IS CLAIMED IS:

1. An enteral formula, said formula comprising:
(A) protein, said protein being of a concentration of between 10 and 35 grams per liter of formula;
(B) carbohydrates, said carbohydrates including those from total dietary fiber being of a concentration of between 60 and 110 grams per liter of formula; and (C) fat, said fat being of a concentration of between 20 to 45 grams per liter of formula wherein the fat includes an ingredient derived from egg, said ingredient contains triglycerides wherein the triglycerides have ester moieties in sufficient quantity to provide, by weight based on fat in the formula, from about 0.05% to 0.5% of DHA and about 0.1%
to 2% of AA and wherein said ingredient derived from egg contains less than about 0.1% of phosphorus and less than about 5.0% of cholesterol by weight.

2. The formula of claim 1 wherein said ingredient derived from egg is a triglyceride composition prepared by reacting glycerol with purified fatty acids or with purified lower alkyl esters of fatty acids wherein the fatty acids are obtained by a process selected from the group consisting of:
(a) hydrolyzing egg phospholipids to release the fatty acid moiety of the phospholipids as free fatty acids associated with phosphorus compounds and cholesterol; and (b) transesterifying the fatty acids from egg phospholipids to obtain lower alkyl esters of the fatty acids associated with phosphorus compounds and cholesterol; and subsequently purifying the fatty acids or lower alkyl esters thereof by removing phosphorus compounds by phase separation in the presence of a lower alkanol; and subsequently removing cholesterol by distillation.

3. The formula or claim 1 wherein the AA content in the triglycerides of the ingredient derived from egg is from about 1% to 15% based on the weight of the said ingredient.

4. The formula of claim 1 wherein the DHA content in the triglycerides or the ingredient derived from egg is from 0.1%
to 5% based on the weight of the said ingredient.

5. An enteral formula which comprises a nutritionally adequate source of amino nitrogen, carbohydrates, and edible fats, the improvement comprising the inclusion in said formula of a triglyceride composition in an amount sufficient to provide from about 0.1 to 2% of AA and about 0.05% to 0.5%
or DHA, based on the total edible fats in the formula, and wherein the said triglyceride composition contains less than about 0.1% of phosphorus and less than about 0.5% of cholesterol based on the weight of the said triglyceride composition wherein said composition is obtained by the process of:
(A) subjecting a lipid mixture containing phospholipids, triglycerides and sterols to treatment selected from the group consisting of (1) hydrolysis to form a two phase product containing a fatty acid phase comprised of free fatty acids and sterols and an aqueous phase comprised of water, glycerol, and phosphorus compounds, or (2) alkaline transesterification with 2 lower alkanol to form a two phase product containing a lower alkyl fatty acid ester phase comprised of lower alkyl fatty acid esters and sterols and an aqueous phase comprised of water, glycerol and phosphorus compounds;
(B) separating the aqueous phase from (1) the fatty acid phase containing sterols or (2) the lower alkyl fatty acid ester phase formed in Step (A);

(C) reacting the fatty acids with the sterols in the fatty acid phase from Step (B) to form a mixture comprising sterol fatty acid esters and water; and (D) distilling the (1) fatty acids and sterol fatty acid esters from the fatty acid phase or (2) lower alkyl fatty acid esters and sterols from the lower alkyl fatty acid ester phase of Step (B) at a temperature of at least about 100° C to separate and recover in the distillate (1) free fatty acids or (2) lower alkyl esters of the fatty acids; and (E) subjecting the said free fatty acids or esters from Step (D) to treatment selected from the group consisting of:
(1) esterification in the presence of glycerol; or (2) transesterification in the presence of glycerol to produce a composition containing triglycerides of said free fatty acids or the fatty acid moiety of the lower alkyl esters.

6. The formula of claim 5 wherein the lower alkanol is methanol.

7. The formula of claim 5 wherein the lipid mixture is separated into a phase containing phospholipids and sterols and a phase containing the triglycerides and sterols by contacting the lipid mixture with a lower alkanol, separating the phospholipid phase from the triglyceride phase and using the phospholipid phase for the subsequent hydrolysis or transesterification in Step (A) of claim 5.

8. The formula of claim 5 wherein the lipid mixture of Step (A) is a naturally occurring lipid mixture.

9. The formula of claim 5 wherein the lipid mixture is that from the egg of hens.

10. The formula of claim 5 wherein the treatment in Step (A) is alkaline transesterification of the lipids with a lower alkanol to produce lower alkyl fatty acid esters of said lipids.

11. An enteral formula which comprises a nutritionally adequate source or amino nitrogen, carbohydrates, and edible fats, the improvement comprising the inclusion in said formula of a triglyceride composition in an amount sufficient to provide from about 0.1 to 2% of AA and about 0.05% to 0.5%
of DHA, based on the total edible fats in the formula, and wherein the said composition contains less than about 0.1% or phosphorus and less than about 0.5% of cholesterol based on the weight of the said composition wherein said composition is obtained by the process of:
(A) subjecting phospholipids containing AA, DHA and sterols to treatment selected from the group consisting of:
(1) hydrolysis to form a two phase product comprised of a free fatty acid phase containing free fatty acids and sterols and an aqueous phase containing water, glycerol, and phosphorus compounds; and (2) alkaline transesterification with a lower alkanol to produce a two phase product comprised of a lower alkyl fatty acid ester phase containing lower alkyl fatty acid esters and sterols and an aqueous phase containing water, glycerol and phosphorus compounds;
(B) separating the aqueous phase from (1) the fatty acid phase or (2) the lower alkyl fatty acid ester phase formed in Step (A);
(C) reacting the fatty acids with the sterols in the fatty acid phase from Step (B) to form a mixture comprising sterol fatty acid esters and water; and (D) distilling the (1) fatty acids and sterol fatty acid esters from the fatty acid phase of Step (C) or (2) lower alkyl fatty acid esters of Step (3) to separate and recover in the distillate (1) free fatty acids or (2) lower alkyl esters of the fatty acids; and (E) subjecting the: (1) distilled free fatty acids or (2) distilled lower alkyl esters thereof from Step (C) to treatment selected from the group consisting or: (1) reaction of the fatty acids with glycerol to produce a composition containing triglycerides of said free fatty acids and (2) transesterification of the lower alkyl esters of the fatty acids with glycerol to produce a composition containing triglycerides of the fatty acids.

12. An enteral formula which comprises a nutritionally adequate source of amino nitrogen, carbohydrates, and edible fats, the improvement comprising the inclusion in said formula of a triglyceride composition in an amount sufficient to provide from about 0.1 to 2% of AA and about 0.05% to 0.5%
of DHA, based on the total edible fats in the formula, and wherein the said triglyceride composition contains less than about 0.1% of phosphorus and less than about 0.5% of cholesterol based on the weight of the said triglyceride composition wherein said composition is obtained by the process of:
(A) hydrolyzing a lipid mixture containing phospholipids, triglycerides, and sterols to form a two-phase product containing a fatty acid phase comprised of free fatty acids and sterols, and an aqueous phase comprised of water, glycerol, and glycerol phosphoric acid esters;
(B) separating the aqueous phase from the fatty acid phase of the two-phase product formed in Step (A);
(C) reacting the fatty acids with the sterols in the fatty acid phase from Step (B) at a temperature of 150° C to 250° C to form a mixture comprising sterol fatty acid esters and water;

(D) distilling the sterol fatty acid esters formed in Step (C) at a temperature of 130° C to 250° C and a pressure of 1.0 x 10 -3 kPa to 5.3 x 10 -1 kPa, to recover purified fatty acids; and (E) reacting the purified fatty acids prepared in Step (D) with glycerol in a molar ratio of 1:1 to 1:2 moles of fatty acid to each hydroxyl equivalent of the glycerol to produce a triglyceride composition of said fatty acids.

13. The formula of claim 12 wherein the purified fatty acids are essentially free of cholesterol and other sterols, and phosphorus compounds.

14. The formula of claim 12 wherein the hydrolysis is base-catalyzed by an aqueous alkali selected from the group consisting of the sodium, calcium, lithium, and potassium salt of an hydroxide, carbonate, and bicarbonate, to form a metal soap of the fatty acid.

15. The formula of claim 12 wherein the aqueous alkali is added in at least a stoichiometric amount up to two times the stoichiometric amount based on the equivalents of fatty acid groups contained in the lipid mixture.

16. The formula of claim 15 wherein the metal soap of the fatty acid formed in the base-catalyzed hydrolysis of Step (A) is acidified at a pH of less than 4 to a free fatty acid by the addition of a mineral acid.

17. The formula of claim 12 wherein the hydrolysis product of Step (A) additionally contains a lower alkyl alcohol having 1 to 4 carbon atoms in a mass ratio of 0.5:1 to 3:1 alcohol to phospholipids contained in the naturally occurring lipid mixture.

18. The formula of claim 17 wherein the lower alkyl alcohol is present in a mass ratio of 1:1 alcohol to phospholipids contained in the naturally occurring lipid mixture.

19. The formula of claim 18 wherein the lower alkyl alcohol is methanol.

20. The formula of claim 12 wherein a lower alkanol is added to a naturally occurring lipid mixture in a mass ratio of 0.5:1 to 3:1 alcohol to lipid which results in formation of a triglyceride phase and a phospholipid phase.

21. The formula of claim 20 wherein the triglyceride phase is separated from the phospholipid phase by decantation prior to hydrolysis.

22. The formula of claim 12 wherein an esterification catalyst is added in Step (C).

23. An enteral formula which comprises a nutritionally adequate source of amino nitrogen, carbohydrates, and edible fats, the improvement comprising the inclusion in said formula of a triglyceride composition obtained from egg yolk, said triglyceride composition being in an amount sufficient to provide from about 0.1 to 2% of AA and about 0.05% to 0.5%
of DHA, based on the total edible fats in the formula, and wherein the said triglyceride composition contains less than about 0.1% of phosphorus and less than about 0.5% of cholesterol based on the weight of the said triglyceride composition wherein said composition is obtained by the process or (A) adding a lower alkanol to egg yolk solids to form a phospholipid phase containing phospholipids, sterols and alcohol and a triglyceride phase containing triglycerides, at a temperature or 30° C to 65° C, provided that the alcohol is added in a mass ratio of 0.5:1 to 3:1 alcohol to egg yolk solids;
(B) decanting the triglyceride phase;
(C) hydrolyzing the phospholipid phase in the presence of an aqueous alkali to form a soap:
(D) acidifying the soap by the addition of a mineral acid to a pH less than 4 to form a two-phase product containing a fatty acid phase comprised of free fatty acids and sterols, and an aqueous phase comprised of water, glycerol and glycerol phosphoric acid esters;
(E) separating the aqueous phase from the fatty acid phase of the two-phase product;
(F) reacting the fatty acids with the sterols in the fatty acid phase at a temperature of 150° C to 250° C to form a mixture comprising sterol fatty acid esters and water;
(G) distilling the sterol fatty acid esters at a temperature of 130° C to 250° C and a pressure of 1.0 x 10 -3 kPa to 5.3 x 10 -1 kPa, to recover purified fatty acids which are free of cholesterol and other sterols, and phosphorus compounds; and (H) reacting the purified fatty acids prepared in Step (G) with glycerol to produce the triglyceride composition.

24. The formula of claim 23 wherein the purified fatty acids are essentially free of cholesterol and other sterols, and phosphorus compounds and said glycerol is reacted with the purified fatty acids in a molar ratio of 1:1 to 1:2 moles of fatty acid to each hydroxyl equivalent of the glycerol to produce the triglyceride-composition.

25. An enteral formula which comprises a nutritionally adequate source of amino nitrogen, carbohydrates, and edible fats, the improvement comprising the inclusion in said formula of a triglyceride composition obtained from egg yolk, said triglyceride composition being in an amount sufficient to provide from about 0.1 to 2% of AA and about 0.05% to 0.5%
or DHA, based on the weight of total edible rats in the formula, and wherein the said triglyceride composition contains less than about 0.1% of phosphorus and less than about 0.5% of cholesterol based on the weight or the said composition wherein said composition is obtained by the process of:
(A) extracting lipids containing sterols, triglycerides, and phospholipids from egg yolks with methyl alcohol to obtain a solution of lipids in methyl alcohol;
(B) separating insoluble egg yolk components from the lipids in solution;
(C) forming an alkaline reaction medium of the lipids together with a catalytic quantity of an alkaline metal lower alkoxide in the solution to transesterify fatty acid glycerides of the lipids to the methyl esters of said fatty acids to produce a two phase product containing an ester phase comprised of esters and sterols and aqueous phase comprised of water, glycerol and glycerol phosphoric acid esters;
(D) neutralizing the alkaline reaction medium with an acid;
(E) separating the aqueous phase from sterols and methyl esters of said fatty acids;
(F) separating the methyl esters of said fatty acids from sterols by distillation to recover said esters in the distillate;
(G) adding glycerine and an alkaline metal lower alkoxide to the distilled esters and forming an alkaline reaction medium; and (H) subjecting the alkaline reaction medium containing glycerin to transesterification to form a composition containing triglycerides having ester groups of said egg yolk fatty acids including that of AA and DHA.

26 A nutritional supplement comprising a triglyceride composition wherein the said triglyceride composition contains less than 1.0% phosphorus and less than about 5.0%
of cholesterol based on the weight of the said triglyceride composition wherein said composition is obtained by the process of:
(A) subjecting a lipid mixture containing phospholipids, triglycerides and sterols to treatment selected from the group consisting of (1) hydrolysis to form a two phase product containing a fatty acid phase comprised of free fatty acids and sterols and an aqueous phase comprised of water, glycerol and phosphorus compounds, or (2) alkaline transesterification with a lower alkanol to form a two phase product containing a lower alkyl fatty acid ester phase comprised of lower alkyl fatty acid esters and sterols and an aqueous phase comprised of water, glycerol and phosphorus compounds;
(B) separating the aqueous phase from (1) the fatty acid phase containing sterols or (2) the lower alkyl fatty acid ester phase formed in Step (A);
(C) reacting the fatty acids with the sterols in the fatty acid phase from Step (B) to form a mixture comprising sterol fatty acid esters and water; and (D) distilling the (1) fatty acids a d sterol fatty acid esters from the fatty acid phase or (2) lower alkyl fatty acid esters and sterols from the lower alkyl fatty acid ester phase of Step (B) at a temperature of at least about 100° C to separate and recover in the distillate (1) free fatty acids or (2) lower alkyl esters of the fatty acids; and (E) subjecting the said free fatty acids or esters from Step (D) to treatment selected from the group consisting of:
(1) esterification in the presence or glycerol; or (2) transesterification in the presence of glycerol to produce a composition containing triglycerides of said free fatty acids or the fatty acid moiety or the lower alkyl esters.

27. A nutritional supplement according to claim 26 administered to a pregnant and/or lactating female.