Fatty acid fractionation for polyunsaturated fatty acid fraction enrichment

A mixture of fatty acids rich in polyunsaturated fatty acids is esterified by enzymatic catalysis and then the reaction mixture is saponified, which provides an organic phase containing esters and an aqueous phase which contains fatty acids, the phases are separated and the fatty acids of the aqueous phase are extracted by a non-polar solvent to obtain the fatty acids in the solvent, after which the solvent may be removed from the fatty acids.

BACKGROUND OF THE INVENTION 
The present invention relates to fatty acid fractionation for 
polyunsaturated fatty acid fraction enrichment, and the present invention 
also relates to employing enzymatic hydrolysis and enzymatic 
esterification in the course of obtaining the fractions. 
Fatty acids of the n-6 and n-3 series have a nutritional value, in 
particular as precursors in the biosynthesis of prostaglandins. It may be 
advantageous to have available fractions enriched in these fatty acids for 
various nutritional and cosmetic applications. These fatty acids are found 
in nature principally in the form of triglycerides. Free fatty acids are 
obtained industrially from triglycerides by hydrolysis at a high 
temperature and under a high pressure. In order then to fractionate these 
fatty acids, several methods have been developed, for example 
crystallization, distillation, the formation of inclusion complexes or 
chromatographic techniques. Application of these methods may bring about 
degradation in the case of polyunsaturated fatty acids or prove to be too 
costly to be applied industrially. 
Enzymatic methods represent an alternative to the preceding methods since 
they enable reactions to be carried out under mild conditions using little 
energy and equipment which is less stressed. 
Enzymatic processes are known, for example from Matthew J. Hills et al in 
JAOCS, Vol.67, no.9, p.561-563, for fractionating fish oil and evening 
primrose oil fatty acids based on the fact that the kinetics of the 
esterification by butanol catalysed by rape lipases and Mucor miehei would 
be a function of the degree of saturation of the acid to be esterified. 
After esterification of fatty acids with a higher degree of saturation, 
the esters are separated from the fatty acids which have not reacted, by 
thin layer chromatography. 
SUMMARY OF THE INVENTION 
The object of the invention is to provide a process for the fractionation 
of fatty acids from oils rich in polyunsaturated fatty acids by enzymatic 
esterification which is applicable industrially, avoiding chromatographic 
separation. 
The invention thus concerns an essentially enzymatic process for the 
fractionation of polyunsaturated fatty acids, in which esterification of a 
mixture of fatty acids is carried out by enzymatic catalysis, the esters 
formed are then separated from the fatty acids which have not reacted and 
a fraction is obtained enriched in the desired polyunsaturated fatty 
acids, characterized in that the esters of the fatty acids are separated 
by controlled saponification of the fatty acids, extraction of the soaps 
formed with water, acidification of the aqueous phase and extraction of 
the acids formed with a non-polar solvent.

DETAILED DESCRIPTION OF THE INVENTION 
In order to put the process according to the invention into practice, the 
mixture of fatty acids used is derived, preferably, from the total 
enzymatic hydrolysis of the triglycerides of an oil rich in 
polyunsaturated fatty acids. This hydrolysis preferably takes place in a 
medium emulsified by a non regio-specific lipase of Candida cylindracea, 
preferably at room temperature, at neutral pH or close to neutrality and 
at atmospheric pressure. 
As the starting oil, any natural oil may be cited of animal or vegetable or 
synthetic origin containing polyunsaturated fatty acids, in particular 
with a degree of unsaturation of three and more, including fatty acids of 
the n-3 and n-6 series, for example blackcurrant seed oil, borage oil, 
evening primrose oil, rich in gamma-linolenic acid (GLA), or "TGA" oil 
rich in arachidonic acid. 
Esterification takes place in the presence of methanol and a solvent, for 
example hexane or a small quantity of water. The reaction lasts 5 to 70 h 
and preferably 10 to 40 h, preferably at room temperature. The enzyme used 
may be immobilized or not. It is preferably immobilized so that it can be 
reused. It may be regio-specific or not. 
After reaction, a mixture is obtained of free fatty acids and the methyl 
esters of fatty acids. The esterified fraction is impoverished in fatty 
acids with a high degree of unsaturation, for example in the case of 
blackcurrant seed oil particularly in GLA and in stearidonic acid (SA), 
which indicates that these acids are hardly esterified at all and 
consequently enriched in the free fatty acids fraction. 
According to the invention, separation of free fatty acids from the esters 
takes place by saponification of fatty acids under mild conditions. These 
conditions are characterized by a reaction with a weak base in an aqueous 
medium, for example carbonates, phosphates, citrates of sodium, potassium 
or ammonium or their mixtures, preferably sodium carbonate, at a 
temperature starting from room temperature, for example 20.degree. C. up 
to about 80.degree. C., with stirring and preferably increasing stirring 
and progressively the temperature from 45.degree. C. up to about 
75.degree. C. Separation is then observed between an aqueous phase and an 
organic phase and this separation can be accentuated, for example by 
addition of a saturated solution of sodium chloride. 
It is possible by this process to react only the fatty acids which, in the 
form of soaps, become water soluble. The aqueous phase obtained can be 
then separated easily from the organic phase containing the liposoluble 
esters after decantation, for example by centrifuging. The free fatty 
acids can be recovered by acidification of the aqueous phase by an acid, 
for example concentrated hydrochloric acid, extracted by a solvent, for 
example hexane, and the solvent can then be removed, for example by 
evaporation. 
EXAMPLES 
The following examples illustrate the invention. In these, the percentages 
and parts are by weight, unless indicated to the contrary. As regards the 
quantitative analysis of free fatty acids, this was carried out, following 
methylation by acetyl chloride, by gaseous phase chromatography (GPC), and 
thus on the basis of the methyl esters. 
Example 1 
1.1. Preparation of the starting mixture of fatty acids. 
A mixture of fatty acids was used as starting material derived from the 
enzymatic hydrolysis of a blackcurrant seed oil by means of a nonspecific 
lipase of Candida cylindracea. 
To obtain the mixture of fatty acids, and oil-in-water emulsion was 
prepared containing 20% of blackcurrant seed oil and 1.2% of soya lecithin 
(ASOL 100 Lucas Meyer) dissolved in 78.8% of an aqueous solution 20 of 
0.025M phosphate buffer of pH 6.88 and carrying out 5 passes through a 
microfluidizer (110T, Microfluidics Corporation, Newton), which led to a 
mean diameter of the oil droplets of about 450 nm. 
Lipase of Candida cylindracea type B, Biocatalysts Ltd., Cardiff, England, 
was solubilized in the phosphate buffer, and then centrifuged at 4000 g 
for 20 min. to remove the insoluble residues. The supernatant was used for 
the experiments. 10 ml of the preceding emulsion (containing 2 g of 
blackcurrant seed oil) were placed in a 25 ml stoppered Erlenmeyer flask, 
in a bath thermostatically controlled to a temperature of 37.degree. C. 
with magnetic stirring at 250 rpm to which the enzyme solution was added 
corresponding to 0.2 g of lipase. 
After reacting for 4 h, the medium was centrifuged at 4000 g to break the 
emulsion and the lipid phase was recovered by extraction with ether. The 
extract was washed with water and dried over sodium sulphate, and the 
solvent then was eliminated by evaporation. The fatty acids obtained were 
stored at -25.degree. C. protected from the light and under nitrogen. 
The lipase in solution in the aqueous phase was recovered as well as the 
glycerol formed by ultrafiltration (module YM 10, cut off threshold 
10,000, Amicon, Denver, U.S.A.), which gave a concentrated solution of 
lipase which could be reused. 
The degree of hydrolysis, corresponding to the percentage of fatty acids 
liberated during the reaction corresponded to 99.9%, determined by 
acid-base titration with the aid of a Metrohm titroprocessor 692. The 
sample to be analysed, dissolved in 25 ml of an equivolume mixture of 
ethanol and ethyl ether, was titrated with an alcoholic solution of KOH 
with a concentration of 0.1N. 
The mixture of fatty acids had the following composition determined by GPC 
in the form of methyl esters 
______________________________________ 
Fatty Acids 
% 
______________________________________ 
C 16:0 7.1 
C 18:0 1.7 
C 18:1 13.3 
C 18:2 45.6 
C 18:3 gamma 
15.5 
C 18:3 alpha 
12.2 
C 18:4 2.9 
C 20:1 0.8 
C 20:2 0.2 
Others 0.8 
______________________________________ 
1.2 Esterification 
The fatty acids were esterified by methanol using 900 mg of the mixture of 
fatty acids in a mixture of 11 ml of hexane and 1 ml of methanol and 1200 
mg of immobilised enzyme, Lipozyme TM 20 from Mucor miehei, Novo Nordisk, 
A/S Denmark. This enzyme has an increased specificity for positions 1 and 
3 of the glycerol skeleton of the triglycerides compared with position 2. 
The reaction was carried out at room temperature in a glass flask provided 
with a magnetic bar and placed on a mechanical stirrer for 20 h. After 
reaction, the enzyme was filtered off which, after rinsing and rehydration 
with 10% water by volume, could be reused. A mixture of free fatty acids 
and methyl esters of fatty acids was obtained. 
1.3. Separation of fatty acids and methyl esters. 
20 g of the preceding mixture of fatty acids and fatty acid methyl esters 
were heated at 40.degree. C., and then 1.1 g of sodium carbonate dissolved 
in water was added with stirring. The rate of stirring was then increased 
and the temperature raised to 75.degree. C. When this latter temperature 
was reached, heating was discontinued and 20 ml of saturated salt solution 
was added. The formation of an organic phase and an aqueous phase was then 
noted. Everything was then centrifuged for 10 min at 3000 rpm, two phases 
were separated and the aqueous phase which contained soaps was then 
acidified with a solution of hydrochloric acid. The fatty acids formed 
were then extracted with hexane and the hexane was then evaporated off. 
1.5 g of fatty acids were thus recovered enriched in GLA and in SA having 
the following composition (GPC of methyl esters): 
______________________________________ 
Fatty Acids 
% 
______________________________________ 
C 16:0 1.3 
C 18:1 2 
C 18:2 6.5 
C 18:3 gamma 
73.4 
C 18:3 alpha 
2.4 
C 18:4 13.6 
Others 0.8 
______________________________________ 
Example 2 
The procedure was as in example 1, except that in the esterification stage 
(corresponding to 1.2 of example 1), a mixture of solvents was used 
consisting of 9 ml of methanol and 1 ml of water. 
64.6% of GLA and 11.8% of SA were thus obtained in the enriched phase 
(consisting of the non-esterified part obtained from the soaps). 
Examples 3-7 
The procedure was as in example 1, except for the fact that, in the 
esterification stage (corresponding to 1.2 in example 1), the enzyme 
reaction time was varied as indicated, and with the results of the % GLA 
and SA in the enriched phase indicated below (determined by GPC of the 
methyl esters): 
______________________________________ 
Example 3 4 5 6 7 
______________________________________ 
Reaction 5 10 30 40 70 
time, h 
% GLA 70 73.5 77.6 75.6 74.8 
% SA 12 13 12.6 13.3 12.8 
______________________________________ 
Examples 8-15 
The esterification of a mixture of blackcurrant seed oil fatty acids was 
carried out as in example 1 (corresponding to 1.2 of example 1) with 
enzymes of various origins and regio-specificities by reaction at room 
temperature for 20 h. The results obtained were evaluated by determining 
the composition in fatty acids of the esterified fraction (by GPC in the 
form of methyl esters): 
__________________________________________________________________________ 
Example 
8 9 10 11 12 13 14 15 
Enzyme of 
Rhizopus Penicill- 
Penicill- 
Geotri- 
Lipo- 
Candida 
Rhizopus 
Asper- 
javanicus 
ium ium chum 
mod cylin- 
javanicus, 
gilus niger 
Bio- cyclop- 
roque- 
candi- 
mixture 
dracea 
Type type 
catalyst ium forti 
dum of lipases 
TYPE OF 
F-AP 15 
AP 6 
__________________________________________________________________________ 
C 13:0 
-- -- -- 0.5 -- -- -- -- 
C 16:0 
7.6 5.8 8.8 11.2 
8.4 7.9 8.1 7.6 
C 16:1 
-- 0.1 -- -- -- -- -- -- 
C 18:0 
1.2 1.1 1.8 1.8 1.5 1.4 1.3 1.4 
C 18:1 
15.8 15.7 16.3 12.8 
16.9 15.9 15.4 17.4 
C 18:2 
57 59 54.9 48.7 
56.8 56.2 56.1 57.7 
C 18:3 
2.1 1.6 2.2 1.2 1.8 2 5.1 2 
gamma 
C 18:3 
14.5 15.3 13.5 7.2 12.3 14.4 12.9 13 
alpha 
C 18:4 
-- -- -- 1.8 1 0.3 -- -- 
C 20:1 
0.6 0.6 0.8 0.6 -- 0.8 0.6 0.8 
C 20:2 
-- 0.2 -- -- -- 0.2 -- -- 
Others 
1.3 0.6 1.8 14.2 
1.4 0.8 0.5 0.1 
__________________________________________________________________________ 
--: not quantifiable 
As may be seen from the Table for Examples 8-15, and as may be seen from 
the preceding descriptions of Examples 1-7, a mixture of unsaturated fatty 
acids, which comprise mono-unsaturated, di-unsaturated and polyunsaturated 
fatty acids, is esterified in the presence of methanol with a lipase which 
is suitable for esterifying mono- and di-unsaturated fatty acids so that a 
reaction product of mono- and di-unsaturated fatty acid methyl esters and 
of free polyunsaturated fatty acids is obtained, and after addition of a 
weak base to the reaction product, a second reaction product is obtained 
which comprises an aqueous fraction phase and an ester fraction phase. The 
tabular results of Examples 8-15 show that in all cases the esterified 
fraction was impoverished in GLA and in SA, which indicates that these two 
acids were hardly esterified at all and hence that the free fatty acids 
fraction was enriched in these acids. 
Example 16 
As a raw material, use was made of a total hydrolysate of a synthetic TGA 
oil from Suntory Ltd. Tokyo, Japan, extracted from Mortierella fungus, 
very rich in arachidonic acid. After hydrolysis of the triglycerides, the 
mixture of free fatty acids was esterified as in example 1 (corresponding 
to 1.2 of Example 1) with LIPOZYME 20 for 20 h at room temperature. The 
methyl esters of the free fatty acids were then separated by thin layer 
chromatography, and the composition of the mixture of free fatty acids was 
then analysed. The composition of the starting mixture of fatty acids and 
that of the mixture of free fatty acids obtained after esterification 
(determined by GPC of the methyl esters) are indicated below: 
______________________________________ 
Composition of fatty Of the esterified 
acids % Of the TGA oil 
fraction 
______________________________________ 
C 14:0 0.7 -- 
C 16:0 17.2 5.6 
C 16:1 0.2 -- 
C 17:0 0.3 0.2 
C 18:0 9.2 5.5 
C 18:1 22.3 7.7 
C 18:2 8.3 2.4 
C 18:3 gamma 1.7 6.5 
C 18:3 alpha 1 -- 
C 20:0 0.9 0.9 
C 20:1 1 0.7 
C 20:2 0.7 0.5 
C 20:3 3.9 13.1 
C 20:4 20.2 40.2 
C 22:0 3.5 4.2 
C 22:6 8.2 11.2 
Others 0.7 1.5 
______________________________________ 
--: not quantifiable 
The preceding results show that, as for blackcurrant seed oil, it was 
especially the fatty acids that were tri-unsaturated which were more 
enriched in the free fatty acid fraction. It will also be noted that, in 
spite of its low percentage of TGA oil, GLA was selectively enriched 
relative to alpha linolenic acid (ALA).