WO2001040419A2

WO2001040419A2 - Method for commercial preparation of linoleic acid

Info

Publication number: WO2001040419A2
Application number: PCT/CA2000/001308
Authority: WO
Inventors: Martin J. T. Reaney; Ya-Dong Liu; Neil D. Westcott
Original assignee: Her Majesty In Right Of Canada As Represented By The Minister Of Agriculture
Priority date: 1999-12-01
Filing date: 2000-11-08
Publication date: 2001-06-07
Also published as: CA2393403C; CA2393403A1; US6420577B1; WO2001040419A3

Abstract

Methods for simultaneous saponification and quantitative isomerization of glyceride oils containing interrupted double bond systems, with alkali in water to yield soaps with conjugated double bond systems are disclosed. Novel methods of hydrolysis of the soap product with acid to form fatty acid-water emulsions and the breaking of those emulsions are also disclosed. The preferred embodiment uses a vegetable oil rich in linoleic acid such as sunflower or safflower oil, potassium hydroxide, phoshporic acid to neutralize the soaps, and an emulsion breaking compound which can include ethanol or other monohydric alcohol, tannins (either hydrolysable or condensed tannin) or polyethylene glycol. The reaction forms positional and geometric isomers of conjugated linoleic acid and the preferred isomer mixture is controlled by a combination of agitation, precisely controlled heating and rapid initiation and termination of the reaction. The reaction product composition may be enriched by crystallization.

Description

Method for Commercial Preparation of Conjugated Linoleic Acid

Field of Invention

This invention relates to an improved process for preparation of conjugated linoleic acid (CLA) from oils rich in linoleic acid, which overcomes problems with an intractable emulsion that occurs between CLA, and water by treatment with either an alcohol or polyethylene glycol or a tannin The reaction is also unique in that it allows the control of the production of positional isomers in the conjugated linoleic acid The process by-product stream is usable directly as a fertilizer which limits waste disposal costs

Background of the invention

Conjugated linoleic acid is the trivial name given to a series of eighteen carbon diene fatty acids with conjugated double bonds Applications of conjugated linoleic acids vary from treatment of medical conditions such as anorexia (US Patent 5,430,066) and low immunity (US Patent 5,674,901 ) to applications in the field of dietetics where CLA has been reported to reduce body fat (US Patent 5, 554,646) and to inclusion in cosmetic formulae (US Patent 4,393,043)

CLA shows similar activity in veterinary applications In addition, CLA has proven effective in reducing valgus and varus deformity in poultry (US patent 5,760,083), and attenuating allergic responses (US Patent 5,585,400) CLA has also been reported to increase feed conversion efficiency in animals (US Patent 5,428,072) CLA-containing bait can reduce the fertility of scavenger bird species such as crows and magpies (US Patent 5,504, 1 14)

Industrial applications for CLA also exist where it is used as a lubricant constituent (US Patent 4,376,71 1 ) CLA synthesis can be used as a means to chemically modify linoleic acid so that it is readily reactive to Diels-Alder reagents (US Patent 5,053,534) In one method linoleic acid was separated from oleic acid by first conjugation then reaction with maleic anhydride followed by distillation (US Patent 5, 1 94,640)

Conjugated linoleic acid occurs naturally in ruminant depot fats. The predominant form of CLA in ruminant fat is the cιs,trans-9, 1 1 -octadecadιenoιc acid which is synthesized from linoleic acid in the rumen by micro-organisms like Butryvibno fibnsolvens. The level of CLA found in ruminant fat is in part a function of dietary cis, cιs-9, 12-octadecadιenoιc acid and the level of CLA in ruminant milk and depot fat may be increased marginally by feeding linoleic acid (US Patent 5,770,247)

CLA may also be prepared by any of several analytical and preparative methods Paπza and Ha

"pasteurized" a mixture of butter oil and whey protein at 85 C for 5 minutes and noted elevated levels of CLA in the oil (US Patent 5,070,104). CLA produced by this mechanism is predominantly a mixture of cιs,trans-9, 1 1 -octadecadιenoιc acid and trans,cιs-10, 12-octadecadιenoιc acid

CLA has also been produced by the reaction of soaps with strong alkali bases in molten soaps, alcohol, and ethylene glycol monomethyl ether (US Patents 2,389,260, 2,242,230 & 2,343,644)

These reactions are inefficient as they require the multiple steps of formation of the fatty acid followed by production of soap from the fatty acids, and subsequently increasing the temperature to isomerize the linoleic soap The CLA product is generated by acidification with a strong acid (sulfuric or hydrochloric acid) and repeatedly washing the product with brine or CaCI

CLA has been synthesized from fatty acids using SO in the presence of a sub-stoichiometπc amount of soap forming base (US Patent 4,381 ,264). The reaction with this catalyst produced predominantly the all trans configuration of CLA.

Efficient synthesis of cιs,trans-9, 1 1 -octadecadιenoιc from πcinoleic acid has been achieved (Russian Patent 2,021 ,252) This synthesis, although efficient, uses expensive elimination reagents such as 1 ,8-dιazobιcyclo-(5,4,0)-undecene. For most applications the cost of the elimination reagent increases the production cost beyond the level at which commercial production of CLA is economically viable

Of these methods alkali isomeπzation of soaps is the least expensive process for bulk preparation of CLA isomers, however, the use of either monohydπc or polyhydnc alcohols in alkali isomeπzation of CLA can be problematic Lower alcohols are readily removed from the CLA product but they require the production facility be built to support the use of flammable solvents Higher molecular weight alcohols and polyhydnc alcohols are considerably more difficult to remove from the product and residual levels of these alcohols (e.g ethylene glycol) may not be acceptable in the CLA product Water may be used in place of alcohols in the production of CLA by alkali isomeπzation of soaps (US Patents 2,350, 583 and 4, 1 64,505) When water is used for this reaction it is necessary to perform the reaction in a pressure vessel whether in a batch (US Patent 2,350, 583) or continuous mode of operation (US Patent 4, 1 64,505) The process for synthesis of CLA from soaps dissolved in water still requires a complex series of reaction steps Bradley and Richardson {Industrial and Engineering Chemistry February 1942 vol 34 no2 237-242) were able to produce CLA directly from soybean triglyceπdes by mixing sodium hydroxide, water and oil in a pressure vessel Their method eliminated the need to synthesize fatty acids and then form soaps prior to the isomeπzation reaction However, they reported that they were able to produce an oil with up to 40 percent CLA Quantitative conversion of the linoleic acid in soybean oil to CLA would have produced an fatty acid mixture with approximately 54 percent CLA

Commercial conjugated linoleic acid often contains a mixture of positional isomers that may include trans,cιs-8, 10-octadecadιenoιc acid, cιs,trans-9, 1 1 -octadecadιenoιc acid, trans,cιs-10, 12- octadecadienoic acid, and cιs,trans-1 1 , 13-octadecadιenoιc acid (Christie, W W , G Dobson, and F D Gunstone, (1 997) "Isomers in commercial samples of conjugated linoleic acid " J Am Oil Chem Soc 74, 1 1 , 1231 )

Sebedio et al (Inform Volume 10 No 5) prepared highly enriched CLA acids isomers and studied their effects on rat tissue lipids They reported that virtually all of the biological activity of the mixed CLA isomers could be attributed to trans,cιs-1 0, 1 2-octadecadιenoιc acid while very little activity could be ascribed to the cιs,trans-9, 1 1 -octadecadιenoιc acid isomer A commercial reaction to prepare CLA should therefore either produce an enriched fraction of trans, cis 10, 1 2-octadecadιenoιc

The present invention describes a method of production of CLA using water as a reaction medium and a vegetable oil containing more than 60 % linoleic acid The reaction in water produces several isomers but the isomer ratio is controlled by the addition of modifiers and reaction kinetics The reaction products may be specifically enriched in trans,cιs-10, 12-octadecadιenoιc acid the most active CLA isomer in many assays of CLA activity The catalytic reaction in alkali is uniquely useful in producing deuterium and tritium labelled isomers of CLA which is achieved by conducting the reaction in deuterated or tπtiated water

When this product is quantitatively converted to CLA it forms an intractable emulsion that is not readily broken using conventional techniques of resolving emulsions such as freezing, heating and centπfugation Other researchers have used conventional methods to wash and dehydrate the CLA isomers with solutions of CaCI when the CLA content of the fatty acids is below 50 % We have found that these methods are not suitable for breaking emulsions caused by solutions that contain over 60% CLA

Summary of the invention

In the present invention the quantitative production of CLA from linoleic acid rich oils is achieved by hydrolysis and isomeπzation with base in a single step reaction in water, deuterated water or tπtiated water and the physical separation of the fatty acds from the water or isotopically labelled water after isomeπzatioπ A preferred embodiment entails the addition of 45 % aqueous potassium hydroxide as the base/catalyst, thereafter, the reaction mixture is neutralized by a strong acid with 70 % H PO being preferred The selection of H PO as the acid and KOH as the base allow the aqueous salt solution to be disposed of in surface applications as a liquid or solid fertilizer The neutralized reaction mixture forms an emulsion that can be separated only by the addition of demulsifiers The demulsifier used to break the emulsion formed by CLA and water must be inexpensive and suitable for the production of food, cosmetic and medicinal products It has now been found that the emulsions between CLA and water can be broken using polyethylene glycol, ethanol or other monohydnc alcohol or tannin (hydrolysable or condensed) The reaction can also be controlled to minimize the production of undesirable isomers The major element of control is through the vigorous agitation of the CLA reaction mixture during synthesis Refining of CLA with solid phase refining methods is also contemplated The partial enrichment and concentration of specific CLA isomers using crystallization from organic solvent is also contemplated

Thus, by one aspect of the invention there is provided a process for producing a conjugated 'inoleic acid-rich fatty acid mixture comprising reacting a linoleic acid-rich oil with a base in the presence of a catalytic amount of said base in an aqueous medium at a temperature above 170 C, and separating said conjugated lioleic acid-rich fatty acid mixture from said aqueous medium

Brief Description of Drawings

Fig 1 A is the proton spectrum of a standard of cιs,trans-9, 1 1 -octadecadιeπoιc acid, Fig 1 B is the proton spectrum of linoleic acid, Figs 2A-D ar carbon 1 3 NMR traces for selected octadecadienoic acid isomers,

Fig 3 is a trace of carbon NMR of the products of distillation above 226 C,

Fig 4 is a a graph illustrating the visible spectra of bleached and unbleached CLA enriched fatty acids, and

Fig 5 is the proton spectrum of deuterated CLA

Detailed description of the invention

The disclosed process quantitatively converts linoleic acid rich oils to a mixture of fatty acids containing CLA The process involves blending a vegetable oil rich in linoleic acid or a partial glyceπde of such a vegetable oil with 1 -6 moles of base, part of which acts as a reactant and part of which acts as a catalyst, and 10 to 1 00 moles of water per mole of hydrolysable acyl groups The vegetable oil may include cottonseed, cucumber, grape seed, corn, safflower, soybean, sunflower or walnut oil or any other oil, wax or ester that is rich in linoleic acid The reaction will proceed if about 1 mole of a base such as sodium metal, sodium hydroxide, sodium alkoxylate, potassium metal, potassium hydroxide or potassium alkoxylate is used as reactant and up to 5 moles are used as the catalyst The least expensive alkali that does not represent a disposal problem is 45% aqueous potassium hydroxide Furthermore, metallic alkali produces explosive hydrogen gas when added to water and metal alkoxylates are flammable These factors support the use of aqueous potassium hydroxide as the preferred catalyst/reactant The reaction proceeds at temperatures above 1 70 C and the reaction accelerates with increases in temperature Preferred embodiments involve performing the reaction above 1 80 C As the reactor contains water it is necessary to confine the reaction in a sealed pressure vessel

The reaction proceeds very rapidly at temperatures above 200 C and the reaction is sensitive to small changes in temperature The reaction vessel used for the process must have a homogeneous temperature or the reaction will not proceed uniformly Homogeneous temperature is achieved by vigorous stirring or turbulent flow conditions In a preferred embodiment the reaction mixture is prepared with a sub-stoichiometπc level of KOH and heated to the reaction temperature The reactor is then charged with the catalyst under pressure to begin the reaction Using this method the reaction starts in the time required to add the catalyst The reaction is terminated either through the addition of acid or through the rapid cooling of the reaction mixture to prevent the further formation of positional isomers

After the reaction is complete the mixture is cooled to 90-100 C for separation of the reaction by- products Acid is added to the reaction mixture to hydrolyse the soaps in the reactor It is preferred to bring the pH of the contents of the reactor to pH 4 or less through the addition of either a mineral or organic acid Acids that may be used include, but are not limited to, hydrochloric acid, sulfuric acid, phosphoric acid and citric acid It is found that the use of sulfuric and hydrochloric acid is problematic in that these strong acids may react chemically with the CLA during separation The preferred embodiment of this invention involves the use of phosphoric acid or citric acid to hydrolyse the soaps When phosphoric acid is used the waste solution can be neutralized and used as a surface applied fertilizer and there are no disposal costs for discarding this product The hydrolysed soaps form an intractable emulsion in the reactor at 1 00 C The emulsion can readily be broken by the addition of one of several synthetic commercial demulsifiers which, however, are not acceptable for use in food and cosmetic systems This invention includes the use of several natural compounds and one synthetic compound as demulsifiers Ethanol was found to act as a demulsifier when added to the reaction mixture at concentrations between 2 and 1 0 percent Ethanol tended to boil at temperatures required for phase separation and thus ethanol was not a preferred alcohol for phase separation Other higher molecular weight alcohols like n-butanol also effected separation of the fatty acid aqueous emulsion without the problem of the tendency to boil These alcohols tended to partition into the fatty acid phase and it was found necessary to strip these solvents from the fatty acid after processing Polyethylene glycol of widely different molecular weights was effective in separating the emulsion when added at concentrations between 1 and 1 0 percent of the reaction mixture Although polyethylene glycol was effective it accumulated to a concentration of between 1 and 7 percent in the fatty acid phase. This high concentration of PEG could not be effectively removed from the fatty acids by water washing or washing with brine However, as part of the embodiment of this invention we discovered that PEG could be removed from the fatty acid layer by washing the fatty acids with 70 percent aqueous phosphoric acid at between 85 and 1 10 C We found that the emulsion breaking properties and phase partitioning properties of PEG molecules of widely different molecular sizes (PEG 300 and PEG 8,000) to be the same

Surprisingly naturally occurring tannins including tannic acid (0 1 -5%) and quebracho tannin (0 1 - 5 %) were also found to effectively separate the emulsion of fatty acids and water Tannins have previously been shown to be effective at breaking emulsions of crude petroleum and water, for example, US patent 5,693,21 6 reveals methods and compositions for breaking oil and water emulsions in crude oil processing operations using a copolymer of tannin and a cationic monomer, but to our knowledge tannins have never been used to effect the separation of emulsions of fatty acids and aqueous solutions Reaction progress was determined by proton ( H-NMR-400 MHz) nuclear magnetic resonance spectroscopy Figure 1 A is the proton spectrum of a standard of cιs,trans-9, 1 1 -octadecadienoic acid while figure 1 B is the spectrum of linoleic acid As may be concluded from figure 1 the disappearance of interrupted methylene signal at 2 8 PPM is a measure of the disappearance of linoleic acid (Figure 1 ) The concentration of total fatty acid is proportional to the alpha methylene proton peak which is located at 2 2 PPM The initial linoleic acid content was determined from H- NMR spectra by dividing the integrated area of the peak at 2 8 PPM of the vegetable oil by the integrated area of the peak at 2 2 PPM The linoleic acid content of subsequent samples was determined by the same method employed on the reacted fatty acids

Isomer composition of the fatty acids was determined by carbon 13 ( C-NMR 100 MHz) nuclear magnetic resonance spectroscopy Pure standards of cιs,tιans-9, 1 1 -octadecadienoic acid gave four peaks in the C-NMR spectrum that were attributed to the olefinic carbons C-9, C-1 0, C 1 1 and C 1 2 at 130 31 , 1 29 1 0, 1 25 94 and 1 35 22 PPM respectively (Figure 2 A) while mixtures known to contain only cιs,trans-9, 1 1 -octadecadienoic acid and trans,cιs-10, 12-octadecadιenoιc acid gave pairs of peaks in each region (Figure 2 B) The deduced assignment for trans,cιs-10, 12- octadecadienoic acid olefinic carbons C-10, C-1 1 , C-12 and C-13 was 1 34 94, 126 07, 128 99 and

1 3

130 12 PPM respectively C-NMR spectra of samples containing trans, cιs-8, 10 octadecadienoic acid, cιs,trans-9, 1 1 -octadecadienoic acid, trans,cιs-10, 12-octadecadιenoιc acid and cis, trans 1 1 , 1 3- octadecadienoic acid gave quadruple peaks clustered at 1 30, 1 29, 1 25 6 and 1 35 PPM (Figure 2 C) We found that the quadruple peaks located around 1 35 PPM were better suited for analysis of isomer composition than other peaks as they were better separated (Figure 2C)

Examples

Example 1 Simultaneous hydrolysis and isomenzation of sunflower oil to CLA followed by washing, dehydrating and refining the CLA with PEG 300

To commercial sunflower oil (275 mL) and water (121 mL) were added KOH (45% w/w, 204 mL)

The resulting reaction mixture was heated at 21 5 C (reactor pressure 1 75 psi) for 4 hours in a one litre reactor with vigorous agitation After cooling to room temperature, the reaction mixture was transferred to a two litre beaker, acidified with H PO (85%, 204 mL), and the mixture brouqht to

5% by weight PEG 300 The resulting mixture was heated for 0 5 h at 95 C After standing for 0 5 hours at 95°C, the top CLA layer was removed, washed with H PO (85 %, 30 mL) at 95°C for 30 minutes to remove excess PEG and water The dried CLA layer was removed The CLA product contained less than 0 1 % water and less than 0 01 25 % PEG as determined by the method of Muir

1 et al , 1 998 The quantitative conversion of linoleic acid to CLA was confirmed by H-NMR as described above

Example 2-8 Simultaneous hydrolysis and isomeπzation of sunflower oil to CLA followed by washing, dehydrating and refining the CLA with tannic acid or quebracho tannin

To commercial sunflower oil (275 mL) and water ( 121 mL) were added KOH (45% w/w 204 mL)

The resulting reaction mixture was heated at 21 5 C (reactor pressure 175 psi) for 6 hours in a one litre reactor with vigorous agitation After cooling to room temperature, the reaction mixture was transferred to a two litre beaker, acidified with H PO (85%, 204 mL), and tannic acid added accordingly (See Table 1 ) The resulting mixture was heated for 2 5 h at 1 05 1 1 0°C After standing for 0 5 hours 95 C, the upper CLA layer was removed, washed with H PO (85%, 30 mL), and heated at 95 C for 30 minutes The dried CLA layer was separated (See Table 1 )

Taole 1 Separation of CLA emulsions with tannic acid

Ex ample Tannin Tannin CLA Water

(%, w/w oil) (gi ram) (mL) (l mL)

2 8^* 20 240 100

3 0 8* 2 235 0

4 0 1 * 0 25 210 0

5 0 02* 0 025 205 0

6 0 1 * 0.25 1 60 100

7 0 8* ^* 2 240 0 0.8^* * 2 240 100

*Tannιc acid

* *Quebracho tannin

Note

1 Theoretic yield of CLA is ; 250 m L for thi ese reactions

Examples 9-18: Simultaneous hydrolysis and isomerization of sunflower oil to CLA at elevated pressure using lower concentrations of potassium hydroxide.

In a chemical reaction the base performs three discrete functions, hydrolyses the glyceπdes, reacts with water and fatty acids to produce soaps, and catalyses the isomerization reaction During the hydrolysis of ester linkages much of the alkaline base is consumed in the formation of soaps It is only the residual base that remains after the formation of soaps is capable of catalysing the isomerization of linoleic acid to CLA In previous studies of CLA production in water researchers reported using large excesses of alkali to produce CLA (US 2,350,583) Surprisingly, our research shows that the isomerization reaction may be driven by small excess over stoichiometnc addition of KOH Table 2 shows the conversion of CLA based on standard reaction conditions of example 1 with the addition of various concentrations of aqueous KOH at different reaction temperatures In these reactions it was possible to obtain a quantitative conversion of linoleic acid to CLA with only a small excess of KOH if the temperature was raised or reaction time increased

Table 2 Production of CLA with different amounts of added base

Example Sunflower oil 45% KOH Temperature Conversion

(mL) (mL) (°C) (%)

9 275 102 210 38 10 275 102 2116 62

11 275 102 226 86

12 275 122 210 72

13 275 122 227 100

14 275 143 210 85

15 275 143 224 100

16 275 163 213 92

17 275 163 224 100

18 275 204 210 100

Example 19 Refining and enrichment of CLA by fractional distillation

As a counter example, high temperature distillation was used to refine CLA and produce a product free of colour Two hundred grams of fatty acids containing 64% CLA and boiling chips were placed in a 500 mL round bottomed flask with a ground glass fitting and the flask was placed in an electric heating mantle A 20 mm x 470 mm Vigreaux column was inserted in the flask and the column was fitted with a distillation head that incorporated a thermometer for measuring distillate temperature The Vigreaux column was insulated by a 0 25 cm layer of glass wool held in place by aluminum foil The apparatus was completed by the addition of a vacuum adaptor and 250 mL receiving flask also with ground glass fittings Vacuum of approximately 100 mm was applied using a Welch Duoseal Vacuum pump, Model 1400 The temperature of the distillate was monitored throughout the procedure The distillate was removed in three stages determined by boiling temperature The first stage 40 mL was removed between 222 and 226 C and was found to be a colourless liquid that readily crystallized at room temperature The crystalline material was determined to be mostly palmitic acid The second stage of 125 mL was removed between 226 and 232 C consisted of an enriched CLA product The temperature of boiling increased to 245 C and an additional 10 mL of material distilled Thereafter the distillation was terminated

Carbon NMR of the products of distillation at temperatures above 226 C were found to be severely isomeπzed by the distillation process (Figure 3)

Example 20 Refining CLA enriched fatty acids

The fatty acids produced by all of the methods mentioned above have a straw yellow colour and contain some metal ions as determined by inductively coupled plasma spectrometry (Table 3) The yellow colour detracts from marketability and the metal ions may cause the material to be unstable One thousand grams of fatty acid produced as described in example 1 was heated under vacuum in an agitated sealed vessel at 70 C and 10 grams of bleaching clay (Supreme 1 20 FF), was added The mixture was continuously stirred and heated to 105oC, under vacuum, for 30 minutes When the temperature of the mixture had decreased to 60 C, the vacuum was released The mixture was then filtered through a Celite filter bed. A comparison of the visible spectra of the two samples is given in figure 4 to show the removal of colour. Table 3: Metals in reaction mixture and refined oils

Sample* Si Na K Pb Sn Al B Ca Mg Sb

Mixed 1 2 476 1012 12 80 20 52 0 1 33

Bleached 0 2 28 0 0 0 3 1 0 0

Distilled 4 1 4 0 0 0 0 5 0 0

*reactιon mixture contains caustic, sunflower oil and water as described in example 1 Bleached material is treated as described in example 20 while distilled material is distilled as described in example 19

Example 21 : Enrichment of CLA by crystallization in a solvent free system

Five hundred mL of fatty acid produced as described in example 1 was chilled to -20 C and then warmed at 4 C The chilling and thawing treatment resulted in the formation of persistent crystals that were readily removed from the fatty acids by filtration. The fatty acids were filtered over a chilled Buchner funnel at 4 C. One hundred and twenty five grams of fatty acid were retained on the filter while 375 grams passed through. The solid fatty acids retained on the filter contained a substantial amount of the desired CLA product. This product was substantially recovered by washing the solid fatty acids with 50 g of chilled sunflower oil. As the sunflower oil is substantially less valuable than the CLA product its loss in this procedure was acceptable. The final CLA product was enriched by approximately 1 0% in total CLA content.

Example 22: Removal of PEG from CLA by washing with phosphoric acid.

Polyethylene glycol 300 (5g) was dissolved in 100 grams of CLA rich oil produced as described in example 1 and the sample was heated and stirred in 50 mL water at 1 00°C for 1 5 mm The PEG 300 content of upper CLA phase was determined by the method of Muir et al 1 998 It was found that substantial amounts of PEG were detectable in the CLA phase The experiment was repeated in a similar manner except that the water was replaced with 50 mL of phosphoric acid and the mixture was stirred at 1 10 C for 1 5 mm After the latter treatment PEG was not detected in the upper CLA rich phase

Example 23: Production of CLA from an oil that has a high level of trans,cιs-10, 12-octadecadιenoιc acid in a commercial reactor

The reaction that produces CLA can favour the production of specific isomers Sunflower oil, potassium hydroxide and water (386 kg, 466 kg, and 21 0kg) were added to a commercial reactor and the reactor was heated from 30 C to 210 C in 1 36 hours Thereafter the reactor was held at

210 C for 26 minutes The reaction mixture was cooled rapidly to less than 1 50 C by releasing the reactor contents into a non-pressurized stainless steel tank The contents of the holding tank were first acidified with phosphoric acid to pH 4 0 and then 4 kg of quebracho tannin dissolved in 20 L of water was added to the tank The reactor contents were stirred for 30 minutes at 105 C after which the aqueous solution separated from the fatty acid solution The fatty acids were dried under vacuum and bleached to yield a commercial product The carbon-1 3 NMR spectrum revealed that

CLA produced in this manner contained 60% trans, cis 1 0, 1 2 octadecadienoic acid and 40 % cis, trans 9, 1 1 -octadecadienoic acid

Example 24 Synthesis of deuterated CLA

Metallic sodium was added in portions under nitrogen to D20 (80 mL) The resulting NaOD solution was diluted to 1 00 mL with deuterated water to produce a 45% NaOD solution

To sunflower oil ( 140 mL) were added NaOD solution ( 100 mL) and D O (20 mL) The reaction mixture was heated in a teflon lined stirred reactor at 21 0 C for six hours After cooling to room temperature, a portion of the reaction mixture (20 g ) was acidified with H3P04 (cone 85 % 1 0 mL) Quebracho tannin (0 5 g) was added to the acidified mixture and the sample heated at 1 1 5 C for 1 5 hour After cooling to room temperature, the top CLA-d layer was separated and deuterium exchange was determined by 1 H and 13C NMR

Deuterium exchange of the beta carbon protons was detected by the decrease in the proton peak normally observed at 2 3 PPM (Figure 5) Substitution of the protons on the carbon beta to the carbonyl was highly effective as the relative area of the peak at 2 3 PPM should have been approximately the same as the peak observed at 1 7 PPM The area observed was diminished by 86% Protons that were adjacent to the trans bond in the conjugated diene system were also substituted during the reaction

Example 25 Purification and concentration of CLA by crystallization

Conjugated linoleic acid prepared as described in example 1 (22 3g) was dissolved in 25 mL of acetone and the mixture was chilled to -1 8 C where a solid precipitate was observed The precipitate was removed by filtration and washed and weighed and the NMR spectrum recorded The 2 5 g of recovered precipitate was found to contain primarily saturated fatty acids The filtrate was diluted with a further 25 mL of acetone and then chilled to -60 C whereupon a second white precipitate formed The second precipitate was filtered at -60 C and washed twice with acetone chilled to the same temperature Solvent was evaporated from both phases and the weight and NMR spectra recorded The warmed crystals (1 5 1 g) melted to produce a clear solution enriched in conjugated fatty acids while the solution evaporated to yield 4 6 g of a clear but intensely yellow liquid

Example 26 Production of a CLA product enriched in trans,cιs-10,12-octadecadιenoιc acid

To sunflower oil (275 mL) were added KOH (210 mL @45 %w/w), Ca(OH)2 (32 g), water ( 1 10 mL)

The reaction mixture was heated at 210 C for five hours After cooling to room temperature, a portion of the mixture (1 5 g) was acidified with H3P04 (cone 10 mL), water (30 mL) and

Quebracho tannin (0 2 g) added The resulting mixture was heated at 1 1 5 C for two houis, and cooled to room temperature The CLA containing layer was recorded on 1 H and 1 3C The reaction products were found to contain 47 percent trans,cιs-10, 12-octadecadιenoιc acid and 31 percent cis, trans 9 1 1 -octadecadienoic acid This novel isomer ratio has not been previously reported

Example 27 Simultaneous hydrolysis and isomerization of sunflower oil to CLA followed by washing, dehydrating and refining the CLA with humic acid or fulvic acid

The resulting reaction mixture was heated at 21 5 C (reactor pressure 1 75 psi) for 6 hours in a one litre reactor with vigorous agitation After cooling to room temperature, the reaction mixture was transferred to a two litre beaker, acidified with H PO (85 %, 204 mL), and 2 grams of fulvic acid or tannic acid added The resulting mixture was heated for 2 5 h at 1 05 1 10°C After standing for 0 5 hours 95 C, the top CLA layer was removed, washed with H PO (85 %, 30 mL), and heated at

95 C for 30 minutes The dried CLA layer was separated the treatments with either fulvic and humic acid initiated phase separation and 240 mL of CLA rich fatty acids were recovered from the reaction mixture

Claims

What is claimed is

1 A process for producing a conjugated linoleic acid-rich fatty acid mixture comprising reacting a linoleic acid-rich oil with a base, in the presence of a catalytic amount of said base, in an aqueous medium at a temperature above 1 70 C, and separating said conjugated lioleic acid-rich fatty acid mixture from said aqueous medium

2 A process as claimed in claim 1 , wherein said oil is a vegetable oil selected from the group consisting of cottonseed, cucumber, grapeseed, corn, safflower, soybean, sunflower and alnut oil

3 A process as claimed in claim 1 , wherein said base is selected from the group consisting of sodium metal, sodium hydroxide, sodium alkoxylate, potassium metal, potassium hydroxide and potassium alkoxylate

4 A process as claimed in claim 1 including the step of cooling said reaction mixture to a temperature of between about 90 and about 1 00 before said separating step

5 A process as claimed in claim 4 wherein said cooled reaction mixture is treated with an acid so as to hydrolyse soaps therein

6 A process as claimed in claim 5 wherein pH of said cooled reaction mixture is reduced to less than pH 4

7 A process as claimed in claim 6, wherein said acid is selected from the group consisting of hydrochloric, sulfuric, phosphoric and citric acid

8 A process, as claimed in claim 5, wherein the acidified reaction mixture is separated into two phases by the addition of a tannin or condensed tannin

9 A process as claimed in claim 1 wherein said aqueous medium is selected from water, deuterated water, and triturated water

10 A process as claimed in claim 9 wherein said temperature is in the range 170°- 200°C 1 1 A process as claimed in claim 9 wherein said temperature is about 1 80 C

1 2 A process, as claimed in claim 5, wherein the acidified reaction mixture is separated into two phases by the addition of polyethylene glycol with a molecular weight greater than 106 Daltons

1 3 A process, as claimed in claim 5, wherein the acidified reaction mixture is separated into two phases by the addition of a monohydnc alcohol

14 A process as claimed in claim 1 3 wherein said monohydnc alcohol is an aliphatic alcohol containing 1 -5 carbon atoms

1 5 A process, as claimed in claim 1 , wherein phosphoric acid is used to remove water and polyethylene glycol from said fatty acid mixture

16 A process as claimed in claim 1 , wherein the acidified reaction mixture is separated into two phases by addition of an acid selected from the group consisting of fulvic acid and humic acid

1 7 A process as claimed in claim 1 , wherein the fatty acids are separated from said aqueous medium by distillation

1 8 A process as claimed in claim 1 , where the fatty acids are separated from said aqueous medium by solid phase adsorbsion

1 9 A process as claimed in claim 1 , wherein the fatty acids are crystallized to remove saturated fats

20 A process as claimed in claim 1 , wherein at least 85% of said conjugated linoleic acid produced comprises cιs,traπs-9, 1 1 -octadecadienoic acid and trans,cιs-1 0, 1 2-octadecadιenoιc acid

21 A process as claimed in claim 1 , wherein the reaction produces more trans,cιs-10, 12- octadecadienoic acid than cιs,trans-9, 1 1 -octadecadienoic acid

22 A process as claimed in claim 21 wherein an additive selected from the group comprising metal ions and divalent cations is added to said reaction so as to increase production of trans, cιs-10, 12- octadecadienoic acid relative to other isomers.