Preparation of concentrates of coloring agents

Concentrates of natural coloring agents such as carotene are prepared from organic media, particularly from palm oil, by a process in which the oil, together with a volatile solvent, is subjected to gel permeation chromatography. The concentrated coloring agent may then be used in food products such as margarine and ice cream.

This invention relates to the preparation of concentrates of natural 
colouring agents from naturally derived organic media and in particular 
the separation of carotene from natural oils, oils derived from 
genetically engineered plant material, and from organic media comprising 
fatty acids and fatty acid esters which may have been derived from such 
oils. 
Carotenes are widely used as colouring agents in foods and can be obtained 
by recovery from natural oils or by synthesis. Although they represent a 
high commercial value, the carotenes are destroyed during the oil refining 
process which is necessary to remove components like gums, free fatty 
acids and off-flavours which are undesired in the further oil processing 
and in the food application of natural oils and fats. 
Preparation of concentrates of the colouring agents from the oil without 
degrading the oil has not to date been achieved in a commercially 
successful manner. Attempted methods to recover carotenoids from oil 
depend on either chemical modification of the oil, the use of adsorbent 
materials, progressive fractionation with solvents of the oil, or on 
molecular distillation. 
European patent specification EP 242148A (Lion Corporation) describes a 
method of purifying a carotene-containing concentrate from a source such 
as palm oil by adsorption chromatography in which the palm oil is firstly 
subjected to alcoholysis and is then passed to an adsorption 
chromatography column containing a silica gel or activated alumina filler. 
This process suffers from the disadvantage that the triglycerides in the 
oil are chemically transformed and cannot be used any more in food 
applications like e.g. fat spreads. 
Several methods using adsorbent materials like activated carbon have been 
described (Dutch patent 74349, Ong et al in Palm Oil Prod. Technol. 
Eighties, Rep. Proc. Int. Conf. 1981). 
These methods suffer from the disadvantage that elution of the carotenes 
from the adsorbent can only be achieved effectively with aromatic solvents 
which are not suitable for the production of food ingredients. 
Japanese patent applications J 51144714A and J 51144715A (Riken Vitamin Oil 
KK) describe methods for the purification of tocopherol, another minor 
ingredient of some vegetable oils, by gel permeation chromatography using 
hydrocarbon or alcohol solvents. In the described processes the scum of 
the oil deoderisation process is used as raw material. Unfortunately the 
carotenoids in the oil are destroyed or modified during the deoderisation; 
the described process therefore is not suited for the recovery of 
carotenes. 
Landen & Eitenmiller in J. Assoc. Off. Anal. Chem. (Vol. 62 No. 2, 1979) 
describe the application of high pressure gel permeation chromatography 
for the analyses of the beta-carotene and the retinyl palmitate content in 
oil. The method is based on the simultaneous measurement of the refractive 
index and the absorbance at 340 nm and 436 nm of the effluent of the 
chromatography column. No separation of the oil and the carotenoid 
fraction is achieved however, which makes this process unsuited for the 
recovery of carotenoids from oil. 
It is an object of this invention to prepare concentrates of natural 
colouring agents from naturally derived organic media in such a manner 
that the colouring agent can subsequently be used as a food ingredient and 
in such a manner that the organic medium is not degraded. 
We have now surprisingly found that gel permeation chromatography can be 
used in a commercially successful manner to extract carotene from palm oil 
without first chemically modifying the oil and from other natural oils as 
well as from media comprising fatty acids and/or fatty acid esters derived 
from such oils. 
Thus, according to the invention there is provided a process for the 
preparation of concentrates of natural colouring agents from organic media 
selected from fatty acid glycerides derived from natural sources, 
including genetically engineered plant material, media comprising fatty 
acids and fatty acid esters in which the organic medium, together with a 
volatile solvent, is subjected to gel permeation chromatography. 
To run the process in a commercially successful manner the process is 
preferably carried out at an elevated temperature. We have found that a 
suitable temperature is from 30 to 80.degree. C. such as from 55.degree. 
C. to 65.degree. C., most preferably about 60.degree. C. 
The choice of volatile solvent is linked to the nature of the column 
material. 
For separations with gel permeation the molecular weights of the components 
to be separated should ideally be within the useful molecular weight 
working range of the gel bed. In general a gel bed has to be chosen such 
that the components with the highest molecular weight elute at or near the 
void volume. 
Suitable column materials include cross-linked polystyrene polymers, 
cross-linked dextrans cross-linked agaroses, cross-linked celluloses, 
silica gel and modified silica gels. 
The solvent should be volatile, to enable its removal by distillation 
without degrading the colouring agent being concentrated. Suitable 
solvents include hexane, cyclohexane, butylacetate, iso-propyl alcohol, 
methyl ethyl ketone, acetone, ethanol, water and mixtures thereof. 
Further, the solvent should be such that any traces thereof remaining in 
the colouring agent concentrate are acceptable in food products. 
While the process according to the invention is particularly applicable to 
the extraction of carotene from palm oil, other colouring agents can be 
extracted from other natural oils using the same process. Further 
embodiments of the invention include the extraction of capsanthin and 
capsorubin from paprika oleoresin. 
The process according to the invention is also applicable to the 
concentration of colouring agents from organic media comprising fatty 
acids such as palmitic and stearic acids and mixtures thereof which are 
obtained from a natural oil, such as by conventional fat splitting 
techniques. Organic media comprising esters of such fatty acids, in 
particular methyl and ethyl esters may also be subjected to the process. 
In practice the column material is fully swollen with the same solvent as 
will be used for elution. After swelling the column is packed. The organic 
medium or a mixture thereof with solvent is then added to the top of the 
column. 
We have found that if, when using polystyrene or dextran polymers as column 
material, no solvent is included in the organic liquid at this stage the 
column material tends to shrink, causing unpredictable results. A suitable 
organic medium to solvent ratio is up to 5:1, such as from 2:1 to 1:2, 
preferably about 1:1 by weight. 
As a result of the differential permeation rate of the various molecules in 
the sample, separation occurs in the column upon elution with the solvent 
resulting in a number of fractions. Starting with a natural oil or 
starting with a medium comprising fatty acids and/or fatty acid esters 
containing, for example, up to 1000 ppm colouring agent, a fraction with a 
concentration of at least 0.5% up to 25% by weight can be produced by this 
method. The solvent is removed from this, and other fractions eluted from 
the column, by distillation and may usefully be recycled. 
The invention enables the production of a natural carotene concentrate 
comprising at least 0.5% by weight carotene. 
The concentrated colouring agent may be used as such as a food additive or 
it may be further processed as desired. The concentrate will often be 
enriched in mono-and diglycerides and in tocopherols. These materials may 
give the product added value according to its intended end use. This 
invention also enables the provision of a manufactured food product 
containing the natural carotene concentrate obtained in this way. 
The organic medium from which the colouring agent has been removed may be 
further processed as desired. 
The invention will now be illustrated by the following example.

EXAMPLE 
A gel permeation chromatography column having a diameter of 25 mm. and 
fitted with a heating jacket was used. The column was packed with 75 g 
resin which had been preswollen in ethyl actetate giving a column height 
of 50cm. The resin was a polystyrene polymer with divinylbenzene 
crosslinkages, commercially available as Bio-Beads S-X3 from Bio-Rad 
Laboratories. The column was heated to 60.degree. C. and 12g sample of a 
1:1 mixture of ethyl acetate and crude palm oil was loaded on top of the 
column. The column was eluted with ethyl acetate at a linear flow rate of 
0.65 cm/min. 
The crude palm oil was mainly in the form of tri-, di- and monoglycerides 
but also contains a number of minor ingredients including from 500 to 1000 
ppm carotene. The eluted fraction contained 90% of the total carotenes. 
The ethyl acetate was removed from this fraction by distillation to yield 
a solution of at least 0.5% carotene in an oil base which is suitable for 
use as a food colouring additive for various manufactured food products 
such as margarine and ice cream. 
EXAMPLE 2 
A gel permeation chromatography column, having a diameter of 25 mm and 
fitted with a heating jacket was used. The column was packed with 85 gram 
resin which had been preswollen in methyl ethyl ketone giving a column 
height of 50 cm. The resin was a polystyrene polymer with divinylbenzene 
crosslinkages, commercially available as Bio-Beads S-X4 from Bio-Rad 
Laboratories. 
The column was heated to 60.degree. C. and a 12 gram sample of a 1:1 
mixture of methyl ethyl ketone and crude palm oil was loaded on top of the 
column. The column was eluted with methyl ethyl ketone at a linear flow 
rate of 0.65 cm/min. 
The crude palm oil was mainly in the form of tri-, di- and monoglycerides 
but also contains a number of minor ingredients including from 500 to 1000 
ppm carotene. The eluted fraction contained 90% of the total carotenes. 
The methyl ethyl ketone was removed from this fraction by distillation to 
yield a solution of at least 0.5% carotene in an oil base which is 
suitable for use as a food colouring additive for various food products 
such as margarine and ice cream. 
EXAMPLE 3 
A gel permeation chromatography column, having a diameter of 25 mm and 
fitted with a heating jacket was used. The column was packed with 75 gram 
Bio-Beads SX-3 which had been preswollen in ethyl acetate giving a column 
height of 50 cm. 
The column was heated to 40.degree. C. and a 10 gram sample of a mixture 
comprising ethyl acetate (50%), linoleic acid (12.5%), oleic acid (37.5%) 
and carotene (800 ppm) was loaded on top of the column. The column was 
eluted with ethyl acetate at a linear flow rate of 0.58 cm/min. 
A fraction containing 90% of the total carotenes can be collected. The 
ethyl acetate was removed from this fraction by distillation to yield a 
solution of at least 0.5% carotene which is suitable for use as a food 
colouring additive for various food products such as margarine and ice 
cream.