Materials comprising mainly (mainly meaning more than 40 % preferably
more than 60 %) conjugated isomers of long chain polyunsaturated fatty acids are
known for their health performance, when applied in food products. In general
these products comprise the linoleic acid isomers and from all the different linoleic
acid isomers possible the cis 9 trans 11 and trans 10 cis 12 isomers are most often
the most abundantly present in these materials, in general in a 1:1 weight ratio.
These products with high contents of different conjugated isomers
of the same long chain polyunsaturated fatty acid are useful starting materials
for the preparation of materials with other ratio's of the different conjugated
isomers of the long chain polyunsaturated fatty acids. Such a process could enable
us to prepare products with a limited number of isomers and with very high ratio's
of the different isomers of the conjugated polyunsaturated acids. Therefore such
a process could enable us to take advantage of the different properties of the
different isomers for different purposes.
A process to enrich the mix containing the different conjugated isomers of the
same long chain polyunsaturated fatty acid in one of the isomers is the subject
of our earlier WO patent application WO 97/18320.
The prior art processes for the preparation of above starting materials
rich in conjugated polyunsaturated long chain fatty acids have however a number
of drawbacks.
According to a first prior art method this material can be made by
a process wherein water has to be used as solvent at high pressures and rather
high temperatures, resulting in a product wherein far too many isomers of the
polyunsaturated fatty acid are present.
This means that the product per se, but also the product as a starting material
for the enrichment contains too many components. Therefore the product per se is
less useful as food ingredient, while also the products obtained after the enrichment
process are rather contaminated.
Alternatively the prior art (EP799033) discloses a process, wherein
an organic solvent in this case ethylene glycol has to be used. Ethylene glycol
however has one main drawback, ie it is not foodgrade and it is very difficult
to remove it completely from the reaction product of the isomerisation process.
This means that the product per se, but also later products made from it like the
enrichment products, are not food grade either.
Moreover the yields of desired conjugated polyunsaturated isomers in the reaction
product of the conversion in the presence of base are rather low in that instance.
According to an example 1 of a non-prepublished PCT-application with
an earlier priority date (WO97/46230) conjugated linoleic acids can be obtained
by isomeration of linoleic acid or safflower oil by subjecting the starting material
to base (KOH) in propylene glycol at 180 °C for 20 minutes. When we performed this
process, we found that the reaction product contained relatively large amounts
of other isomers, than the desired conjugated linoleic isomers as well. This probably
is due to the severe reaction, conditions applied.
According to another non-published patent application with an earlier
priority date (EP 839897) conjugated linoleic acids can be obtained by subjecting
fats, containing linoleic acid to base in propylene glycol. However high ratios
of base to solvent (6 mole/l) are applied. Moreover the use of fats as starting
material has the disadvantage over using free fatty acid as staring material, that
a build-up of glycerol in the solvent occurs, when the solvent is recycled in
the reactionsystem.
We found a solution for the above problems that even had another
big unexpected advantage. We found that with our new process not only the yields
were higher at lower temperatures, while the use of a non-foodgrade solvent could
be avoided, but we also found surprisingly that the number of isomers formed was
less and that the isomers formed by a subsequent enzymic enrichment process could
be separated easier than when ethylene glycol was used as a solvent.
Therefore our invention concerns in the first instance a process
for the preparation of materials comprising more than 40 wt % conjugated Linoleic
acid wherein an oil or a free fatty acid composition or an alkyl ester composition
thereof, each, selected from the group consisting of : sunflower oil, rape seed
oil, soybean oil, safflower oil and in particular the free acids derived from these
oils and alkylesters from these free acids. containing at least 25 wt% of at least
one isomer other than the conjugated isomers of Linoleic acid is subjected to a
treatment with a base in a solvent and wherein the solvent is an alcohol with
at least 3 C-atoms and at least two hydroxy groups having:
- a ratio of number of C-atoms: number of OH groups of at least 1.25 but less
than 3.5, preferably from 1.5 to 2.75,
while the reaction is carried out between 100 and 180 °C, more preferably between
120 and 180 °C with the proviso that when the oil is applied as starting material
the base is used in concentrations of 0.25 mole/&litre; to 3.5 mole/&litre; solvent.
This temperature range thus does not include 180 °C per se.
A very suitable solvent is 1.3 dihydroxypropane or 1,2 dihydroxypropane.
These solvents are foodgrade so that traces left in the products are not harmful.
The reaction is preferably performed in the absence of glycerol.
Herefore free fatty acids are preferably used as starting material.
The base could be any base but we found that the best results were
obtained with NaOH or KOH as base. Suitable concentrations for the base are greater
than 0.25 mole/l of solvent, preferably 0.25-3.5 most preferably 1.25-2.75 mole/l.
Using higher amounts of base leads to the formation of products, wherein many isomers
(in particular C18:2
trans/trans-isomers) are present (cf our comparative
example)
The starting materials for our novel process have to contain at least
25 wt% of at least one isomer other than the conjugated isomers of Linoleic acid.
This amount preferably is more than 40 wt %, more preferably even more than 60
wt %. Linoleic acid contains mainly the cis 9 cis 12 diunsaturated carbon chain.
The most preferred products of our novel process are products that
contain the linoleic isomers cis 9 trans 11 and trans 10 cis 12 in about a 1:1
ratio. As disclosed in our earlier WO application 97/18320 these materials can
be converted into materials wherein this ratio cis 9 trans 11: trans 10 cis 12
is changed considerably.
Our products are suitably isolated from the crude reaction mixture by the addition
of diluted acid to the soap formed until an acidic pH is achieved (preferably:
pH 1-3), whereupon the oil is separated from the waterlayer and dried.
According to a last embodiment we can use our oil, or free fatty
acids derived from this oil, or the alkyl esters from these free fatty acids,
comprising mainly conjugated isomers of Linoleic acid for the preparation of a
material comprising mainly conjugated isomers of Linoleic acid in another ratio
by subjecting them to an enzymic enrichment process using an enzyme that has the
ability to discriminate between different isomers of conjugated Linoleic acid.
EXAMPLE I. (=COMPARATIVE)
31 grams of safflower oil were added to a solution of 9.0 grams of
NaOH pellets (dissolved by stirring at 60 oC) in 150 gram of ethylene glycol.
The mixture was heated to 135 oC, while it was stirred in an inert
atmosphere.
Samples of 2 ml were collected after 2,19,25 and 49 hours.
After 49 hours the reaction mix was cooled to 60 oC and the soap
was split with 80 ml of diluted sulphuric acid (diluted 1:10 with distilled water).
The pH of the final mix was 1.5.
The oil was separated from the water phase and dried over Na2SO4.
The oil product was analysed with high resolution FAME GC. All materials
were analysed in the same way.
The intermediate samples removed during the process were worked in
the same way and the oil obtained was also analysed by high resolution FAME GC.
The results are given below.
|
COMPOSITION OF STARTING OIL
component
name
wt %
C18:2
linoleic acid c9,c12
74.8
C18:1
oleic acid
14.1
C18:0
stearic acid
2.7
C16:0
palmitic acid
6.7
others
1.7
|
PRODUCT AFTER 49 HRS
component
wt %
C18:2 c9,t11
28.6
C18:2 t10,c12
28.7
C18:2 others conj
1.6
unidentified
0.3
C18:2 c9,c12
16.4
C18:1
14.2
C18:0
2.7
C16:0
6.9
others
0.6
|
Composition of the samples removed intermediately
time in hrs
c9,t11
t10,c12
C18:2
conversion
2
3.0
2.9
70.4
5.7
19
18.1
18.3
38.4
48.7
25
21.7
22.0
30.9
58.7
49
28.2
28.5
16.3
78.2
Example II
Example I was repeated however 1,2 dihydroxy propane was used as
solvent.
The results are summarized in the tables IV and V
|
PRODUCT AFTER 49 HRS
component
wt %
C18:2 c9,t11
35.6
C18:2 t10,c12
34.9
C18:2 others conj.
2.1
unidentified
0.4
C18:2, c9,c12
2.5
C18:1
14.2
C18:0
2.7
C16:0
6.9
others
0.6
|
composition of the samples removed intermediately.
time in hrs
c9,t11
t10,c12
C18:2
conversion
2
6.5
6.3
63.2
15.5
19
29.8
29.4
15.0
79.9
25
32.8
32.2
8.9
88.1
49
35.3
34.4
2.5
96.7
EXAMPLE III (comparative)
Equipment
60 litre autoclave with electrical heating for 250 deg.C and capable
of pressures more than 50 bar. The autoclave has a gate stirrer. It is made from
316 stainless steel.
Method
30 kgs of a 4 molar ag. solution of sodium hydroxide solution was
made up in the autoclave. The solution was heated to 60 deg.C and then 30 kgs of
Safflower oil were slowly added whilst stirring.
The stirred autoclave was then heated up to 230 deg.C. This took
5 hours and then maintained at 230 deg.C for a further 1.5 hours at which point
the autoclave was cooled in 1 hour to 90 deg.C. The reacted mixture was then run
out of the autoclave into a drum and mixed with an equal quantity of hot water.
To obtain the free fatty acid product, the soap produced in the reactor
was split with acid. With the soap solution at between 90 and 100 deg.C, 1N sulphuric
acid was slowly added and stirred until the pH was less than 3, at which point
the soap reacted to produce free fatty acid which could then allowed to separate
and then decanted off.
Results
The Safflower originally contained 76.6% of linoleic acid (cis-9,cis-12).
Of this more than 90% was conjugated to give the following interpretation on High
Res GLC:
Feed oil
Conjugated
14:0
0.1
0.1
16:0
6.8
6.9
18:0
2.5
2.6
18:1
13.4
13.3
18:2 c9/c12
76.6
4.7
20+
0.6
0.8
CLA c9t11
--
27.9
CLA t10c12
--
20.3
CLA others
--
23.4
Example IV (comparative)
30 grams of safflower oil were added to a solution of 75.1 grams
of KOH pellets (dissolved by stirring at 100 °C) in 150.1 grams of propylene glycol.
(ratio of base: solvent: 9 mole/l).
The mixture was heated to 135 °C, while it was stirred in an inert
atmosphere. After 16.5 hours the reaction mix became very thick and the reaction
was stopped. The sample from the end mixture was taken and the soap was splitted
with diluted sulphuric acid (diluted 1:10 with distilled water) until the pH of
the water phase was 1.5. The oil was separated from the water phase and dried over
Na2SO4. The oil was analyzed by high resolution FAME GC.
|
PRODUCT AFTER 16.5 HRS
component
wt %
C14:0
0.13
C16:0
7.55
C16:1
0.13
C17:0
0.05
C18:0
2.86
C18:1
11.81
C18:2
1.21
C20:0
0.04
C18:3
0.33
C20:1
0.21
C18:2 c9,t11
22.32
C18:2 c11,t13
2.65
C18:2 t10,c12
21.31
C18:2 c,c
4.07
C18:2 t,t
23.48
C18:2 oxid
0.20
C22:0
0.22
others
1.43
EXAMPLE V
30 g of KOH were dissolved in 200 ml of 1,2 dihydroxypropane (=2.7
mole/l). 30 g of free fatty acids from safflower oil were added to this mixture
and were reacted under nitrogen at 135 oC for 47 hrs. The soap formed was worked
up with diluted sulfuric acid (10%). The product obtained was analysed by GLC and
the following product composition was found:
component
wt%
C14:0
0.2
C16:0
4.2
C18:0
1.6
C18:1
22.5
C18:2t
1.6
C18:2c
24.0
C18:2c9t11
20.7
C18:2c11t13
0.6
C18:2t10c12
20.3
C18:2 9,11 cc
0.6
C18:2 10,12 cc
0.6
EXAMPLE VI
210 g of NaOH was dissolved in 2100 ml 1,2 dihydroxypropane. (=2.5
mole/l). 700 g of free fatty acids from sunflower oil were added to this mixture
and were reacted for 47 hrs at 135 oC. The soap formed was worked up by adding
a diluted (10%) sulfiric acid solution until pH=2. The product obtained was analysed
by GLC. The composition of the product was:
component
% in product
in starting FFA
C14:0
0.2
0.2
C16:0
3.8
3.9
C18:0
1.5
1.5
C18:1
22.0
21.9
C18:2 c9c12
7.6
71.5
C18:2 c9t11
30.6
-
C18:2 c11t13
0.5
-
C18:2 t10c12
30.3
-
C18:2 c9c11
0.7
-
C18:2 c10c12
0.7
-
