Note: Descriptions are shown in the official language in which they were submitted.
NO 5244/00 216 ~ 815
The invention concerns the total enzymatic hydrolysis of
oils rich in polyunsaturated fatty acids.
Fatty acids of the n-6 and n-3 series have nutritional
value, in particular as precursors in the biosynthesis of
prostagl~n~ns. It can be advantageous to have available
fractions enriched in these fatty acids for various
nutritional and cosmetic applications. These fatty acids
are found naturally mainly in the form of triglycerides.
Free fatty acids are obtained industrially from
triglycerides by hydrolysis at high temperatures and
pressures. Free fatty acids may also be obtained chemically
from triglycerides by saponification with a strong base,
followed by liberation of the fatty acids by neutralization
of soaps with the aid of a strong acid and finally by
extraction of the fatty acids from the medium with the aid
of a non polar solvent, for example hexane.
Application of these methods may cause degradation in the
case of polyunsaturated fatty acids.
Enzymatic methods represent an alternative to the preceding
methods since they enable reactions to be carried out under
mild conditions using very little energy and a less
stressed system.
Hydrolysis is usually carried out in a two-phase medium
while maintaining efficient stirring to ensure the best
possible contact between the aqueous phase and the organic
phase.
We have discovered a simple enzymatic method for the total
hydrolysis of the triglycerides of an oil which enables
hydrolysis to be improved in an unexpected manner whilst
obtaining a degree of conversion into fatty acids greater
than the degree obtained by the enzymatic method in a two-
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phase medium, this being under conditions which cause
little degradation and enable an improvement to be obtained
in the quality and stability of polyunsaturated fatty acids
compared with the chemical method.
The invention concerns an enzymatic process for the
preparation of polyunsaturated fatty acids from an oil rich
in these polyunsaturated fatty acids, characterized in that
the oil is hydrolysed by a non stereospecific lipase from
Candida cylindracea in a medium emulsified in the presence
of lecithin as an emulsifying agent.
The process according to the invention is applicable in
particular to the hydrolysis of oils rich in fatty acids of
the n-3 and n-6 series, in particular gammalinolenic acid,
in particular oils from blackcurrant, borage and evening
primrose seeds.
When putting the process into practice, complete enzymatic
hydrolysis is carried out of the triglycerides of oils rich
in polyunsaturated fatty acids in an emulsified medium at a
temperature of 20C to 45C, preferably at room
temperature, at atmospheric pressure and at pH 6 to 8.
These are the control conditions that are advantageous
qualitatively and economically.
The enzymatic reaction takes place at the interface between
the organic phase and the aqueous phase containing the
enzyme and an aqueous buffer solution, at pH 6-8, close to
neutral. The emulsion, of the oil-in-water type, is
produced by vigorous stirring of the oil in the presence of
the aqueous phase and of the emulsifier to produce an
emulsion of oil in the form of fine droplets, for example
with a mean diameter of about 450 nm, dispersed in the
aqueous phase and stabilized by the emulsifier, a lecithin.
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A lecithin that can be used according to the invention may
be, preferably, selected from the following phospholipids:
- A lecithin containing more than 60 ~ by weight of acetone
insoluble matter (hereinafter AIM),
- An oil-free lecithin, containing more than 90 ~ of AIM,
- A lecithin fraction soluble in alcohol, containing more
than 60 ~ by weight of AIM, enriched in phosphatidyl
choline (PC),
- An alcohol soluble, oil-free lecithin, containing more
than 90 ~ by weight of AIM,
- A lecithin fraction containing more than 50 ~ by weight
of PC and
- Lecithins or their preceding fractions modified by
phospholipidase A2 f pancreatic or microbial origin, in
which 10 ~ to 90 ~ by weight of the PC is converted into
lyso-PC (LPC).
The emulsion can be prepared in advance with a view to
subsequent use and for example placed in sterilized
containers.
The composition of the emulsion is determined so as to give
it lasting stability. If the proportion of oil is increased
beyond a certain limit, for example beyond about 20 ~ by
weight, the emulsion is no longer homogeneous and the
degree of hydrolysis obtained is reduced. A lecithin
content of less than about 1 ~ by weight also leads to a
reduction in the degree of hydrolysis. Preferably, the
lecithin content is 0.1 ~ to 5 ~ by weight, advantageously
0.3 ~ to 0.8 ~ by weight and particularly about 0.5 ~ by
weight of the composition. Preferably, the lecithin content
is fixed so as to correspond to 1.5 ~ to 4 ~ by weight, and
particularly about 2.5 ~ by weight of pure PC, based on the
oil content of the emulsion.
The non stereospecific lipase is from Candida cylindracea.
The optimum reaction conditions depend on the nature of the
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substrate to be hydrolysed and the intrinsic properties of
the various lipases.
The reaction takes place between 20C and 45C, preferably
at the lowest temperature, for example about 20C so that
hydrolysis is as complete as possible, which enables in
particular polyunsaturated fatty acids to be handled which
are particularly sensitive to oxidative degradation which
increases with temperature.
The pH does not influence the degree of hydrolysis for
values between 6 and 8. A neutral buffer solution of a salt
is preferably used, such as, for example, a phosphate with
a pH of 6.88, so as to preserve reaction conditions leading
to constant and reproducible results. Under the optimum
conditions described above, complete hydrolysis can be
obtained in 2 to 20 hours.
The degree of hydrolysis depends, under the operating
conditions previously described, on the concentration of
enzyme used, but beyond a certain limit, this parameter no
longer takes part, since the enzymatic reaction takes place
at the interface between the oil and the aqueous medium and
the necessary quantity of enzyme necessary passes through a
maximum depending on the surface area of the available
interface. A concentration of 10 to 100 mg/g of oil is
preferably used.
Once the reaction has finished, the lipase can preferably
be recycled and reused. In order to do this, the emulsion
is centrifuged at a high speed to break it, and the lipid
phase is then recovered by extraction with a solvent,
washing with water, drying over sodium sulphate and
evaporating the solvent. Fatty acids are preferably stored
away from the light, under an inert atmosphere and at a
negative temperature (on the Celsius scale) preferably at
about -25C. The lipase in solution in the aqueous phase
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with glycerol formed during hydrolysis is recovered by
concentrating it, for example by ultrafiltration. The
lipase can then be recycled.
The mixture of fatty acids obtained can be used as a raw
material in a process for the enrichment of specific
polyunsaturated fatty acids or for the preparation of
structured triglycerides in which the fatty acids as well
as their positions in the glycerol molecule are specific.
The products obtained can in their turn be used in
nutritional, cosmetic and pharmaceutical compositions.
The following examples illustrate the invention. In these,
the percentages and parts are by weight, unless otherwise
stated.
Examples 1-7
An oil-in-water emulsion was prepared containing 20~ of
blackcurrant seed oil and 1~2% of soya lecithin (Asol 100
(R), Lucas Meyer) dissolved in 78.8~ of an aqueous solution
of a 0.0 5 M phosphate buffer of pH 6.88 and was passed 5
times through a microfluidizer (110 T, Microfluidics
Corporation, Newton) which led to a mean oil droplet
diameter of about 450 nm.
Lipase was solubilized in the phosphate buffer, and was
then centrifuged at 4000 g for 20 min to remove insoluble
residues. The supernatant was used for experiments. 10 ml
of the preceding emulsion (containing 2g of blackcurrant
seed oil) were placed in a stoppered 25ml Erlenmeyer flask,
in a bath thermostatically maintained at the selected
temperature with magnetic stirring at 250 rpm, and the
enzyme solution was added, corresponding to 0.2g of lipase.
After the reaction, the medium was centrifuged at 4000 g to
break the emulsion and the lipid phase was recovered by
extraction with ether. It was washed with water and dried
215381~
over sodium sulfate and the solvent was then removed by
evaporation. The fatty acids obtained were stored at -25C
away from the light under nitrogen.
The lipase in solution in the aqueous phase as well as the
glycerol formed were recovered by ultrafiltration (YM10
module, cut-off threshold 10,000, Amicon, Denver, USA)
which gave a concentrated solution of lipase which could be
reused.
The degree of hydrolysis, corresponding to the percentage
of free fatty acids liberated during the reaction was
determined by acid-base titration using a Metrohm 692
titroprocessor. The sample analysed, dissolved in 25 ml of
an equivolume mixture of ethanol and ethyl ether was
titrated with an alcoholic solution of KOH at a
concentration of 0.1 N.
The hydrolysis conditions are shown in table I below:
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Table I
Example Candida cylindracea Temperature Duration %
lipase C h hydrolysis
Type B, Biocatalysts Ltd 37 4 99.9
Cardiff, Fngl~n~
2 Type VII, Sigma Chemical 20 20 91.6
Co., St Louis, USA
I0 3 Lot 189, Biogenzia Lemania 37 20 96
SA, T~ nne, Switzerland
4 Type OF, Meito Sangyo Co. 20 8 99.2
Ltd., Tokyo, Japan
Type OF, Meito Sangyo Co. 30 8 96.3
Ltd., Tokyo, Japan
6 Type MY, Meito Sangyo Co. 37 20 91.1
Ltd., Tokyo, Japan
7 Type F5, Enzymatix, 37 20 97.6
Cambridge, Fngl~n~l
As a comparison, hydrolysis in a two-phase system using the
enzymes, temperature conditions and durations of examples 1
to 4, resulted in the following results:
Lipase of example % Hydrolysis in a two-phase
system
1 79
4 81.1
Examples 8-13
By varying the concentration of enzyme, the proportions of
oil and lecithin and the number of successive cycles with
recycled lipase, whilst using the same other conditions as
in example 4, the degrees of hydrolysis shown below in
table II were obtained:
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Table II
Example Composition Concentration Number of % hydrolysis
of emulsion of enzyme 20 h
(æ) mg/g oil hydrolysis
cycles
8 Oil, 30 - - 78
Lecithin, 1.2
Buffer, 68.8
0 9 Oil, 20 - - 89
Lecithin, 1
Buffer, 79
- 20 - 88.6
11 - 50 - 92.7
12 - - 2 96
13 - - 3 94.6
-: Same conditions as in example 4.
Example 14
The quality and stability of the fatty acids obtained by
the enzymatic method were evaluated in comparison with
those obtained by chemical saponification of blackcurrant
seed oil under the following conditions:
100 g of blackcurrant seed oil were mixed with 213 g of an
alkaline solution composed of 47.7~ water, 14.3~ sodium
hydroxide, 37.8~ ethanol and 0.15~ disodium
ethylenediaminetetra-acetate and the mixture was refluxed
at 80-85C for 120 min. After addition of 40 g of water,
and then 80 ml of fuming hydrochloric acid, the mixture was
vigorously stirred for 60 min. The fatty acids formed were
extracted with hexane and, the organic phase was washed
with water and dried over sodium sulphate and the solvent
removed by evaporation.
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Analysis by gas chromatography of the fatty acid of the two
hydrolysates in the form of their methyl esters showed the
perfect agreement of their composition, which enabled it to
be affirmed that the soya lecithin was not hydrolysed.
Analysis by W spectroscopy of the fatty acids in iso-
octane solution showed that the values for the adsorption
maxima of the fatty acids obtained by enzymatic hydrolysis
were lower than those of fatty acids obtained chemically.
Moreover, the values of the molar extinction coefficients,
calculated for several wavelengths and for the two types of
fatty acid, were higher in the case of fatty acids obtained
chemically than in the case of fatty acids obtained
enzymatically.
Finally, a determination of the peroxide index of the fatty
acids enabled the two hydrolysis methods and the quality of
the fatty acids obtained to be compared. The values found
by converting ferric ions into ferric thiocyanate with a
red colour measured at a wavelength of 510 nm on a Hewlett
Packard 845A spectrophotometer, enabled the peroxide index
to be determined expressed in meq of O2/kg. The results
were as follows:
Fatty acids obtained by saponification 7.9
Fatty acids obtained enzymatically 1.4
The preceding analytical results confirmed that the
biocatalysed hydrolysis reaction led to a mixture of fatty
acids having a quality appreciably greater than did
chemical hydrolysis.
