Title of Invention	METHOD FOR PIGMENT SOLUBILISATION AND A PIGMENT COMPOSITION
Abstract	The present invention relates to an improved method for solubilising an oil-soluble pigment efficiently in oil or fat by extraction of a solid preparation containing the oil-soluble pigment, where the extraction takes place in the presence of water, edible oil or fat and a nonionic surfactant. It also relates to the composition prepared by the extraction, and the use of this composition in animal feed to produce feed pellets. The combination of water and nonionic surfactant increases the amount of pigment that is extracted and solubilised. In detail the process of the invention relates to a method for solubilising an oil- soluble pigment into an oil or fat by extraction of a solid preparation containing the oil-soluble pigment comprising the steps of a) mixing the solid preparation containing the pigment with water, an extraction medium containing an edible oil or fat, and a nonionic surfactant having a hydrocarbyl group, an acyl group or a substituted hydrocarbyl or acyl group containing at least 6 carbon atoms b) optionally centrifugalize the mixture obtained and separate the oil phase. The originally obtained mixture or the separated oil phase may then be added to porous precursor feed pellets to produce feed pellets. These pellets will have a higher amount of oil-soluble pigment available for uptake into the animal.

Title of Invention

METHOD FOR PIGMENT SOLUBILISATION AND A PIGMENT COMPOSITION

Abstract

The present invention relates to an improved method for solubilising an oil-soluble pigment efficiently in oil or fat by extraction of a solid preparation containing the oil-soluble pigment, where the extraction takes place in the presence of water, edible oil or fat and a nonionic surfactant. It also relates to the composition prepared by the extraction, and the use of this composition in animal feed to produce feed pellets. The combination of water and nonionic surfactant increases the amount of pigment that is extracted and solubilised. In detail the process of the invention relates to a method for solubilising an oil- soluble pigment into an oil or fat by extraction of a solid preparation containing the oil-soluble pigment comprising the steps of a) mixing the solid preparation containing the pigment with water, an extraction medium containing an edible oil or fat, and a nonionic surfactant having a hydrocarbyl group, an acyl group or a substituted hydrocarbyl or acyl group containing at least 6 carbon atoms b) optionally centrifugalize the mixture obtained and separate the oil phase. The originally obtained mixture or the separated oil phase may then be added to porous precursor feed pellets to produce feed pellets. These pellets will have a higher amount of oil-soluble pigment available for uptake into the animal.

Full Text	The present invention relates to an improved method for solubilising an oil-soluble pigment efficiently in an oil or fat by extraction of a solid preparation containing the oil-soluble pigment, where the extraction takes place in the presence of water, an edible oil or fat and a nonionic surfactant. It also relates to the composition prepared by the extraction, and the use of this composition in animal feed to produce feed pellets. The combination of water and nonionic surfactant increases the amount of pigment that is extracted and solubilised. In animal feed compositions, pigments are often added to endow an appealing colour to the meat or skin of the animals consuming the feed. The pigments used may be natural, synthesized or fermented, and commonly they are oil-soluble. An example of a class of oil-soluble pigments is the carotenoids, e.g. astaxanthin, canthaxanthin and [beta]-carotene. These are insoluble in water, and have a low solubility in organic solvents, fats and oils. Astaxanthin and canthaxanthin are commonly used in fish feed to improve the flesh colour of salmonids, but also give rise to health benefits. Other uses for pigment in animal feed is for the coloration of the skin of some marine warm-water or tropical freshwater fishes, e.g. of the red seabream, and to endow a pink color to the shell and flesh of prawns. In addition to these aqua-cultural uses, the coloring of egg yolks and of the skin of broilers are other feed application areas. Conventional feed pellets are usually formed from a solid base material, which may be protein-based such as fishmeal or carbohydrate-based such as starch, and where the base material is loaded with a fat or an oil component. The fat or oil, that could be either of animal or vegetable origin, is loaded into pores of the precursor feed pellets formed from the base material. The fat or oil increases the energy content of the feed. Other ingredients of the feed are e.g. vitamins minerals, enzymes and the above-mentioned pigments. These latter ingredients, as well as the fat or oil, are preferably added after the pelletizing step, since the oil component interferes with the pelletizing process and many of the pigments lose their activity when heated. Many of the sensitive pigments are available in coated form, e.g. Carophyll® Pink, which is sold in the form of beadlets. The beadlets of this product consist of a core of astaxanthin emulsified in antioxidants and residing in a matrix of gelatine and carbohydrate, which core is coated by maize starch. The amount of astaxanthin is at least 8% of the beadlet. There is also another astaxanthin product of a similar kind that is called Carophyll® Pink 10% CWS, which differs from the first-mentioned product only in that the matrix is a lignosulfonate. If these beadlets are added as such to the preformed pellets, most of them are deposited only on the pellets surface. Especially for aqua-cultural feed this is a disadvantage, since the beadlets will be washed away when the feed comes into contact with water. Also the bioavailability will be less when the pigments are still contained in the beadlets. In EP 839 004-B1 a method is disclosed for loading bioactive ingredients, such as pigments, into feed pellets. The process includes the steps of removing the gelatin and carbohydrate protective shell around the bioactive ingredient enzymatically or by hydrolysis, mixing the uncoated bioactive ingredient with a fat or an oil and loading porous precursor feed pellets with the resulting mixture to produce the feed pellets. In JP7-16075-A a method is described of making a water-soluble pigment solubilised in fat. Firstly polyols are dissolved in water and mixed with the water- soluble pigment, and then an oil-phase containing an emulsifier is added to obtain an emulsion of the water-soluble pigment in oil. In JP7-23736-A a method is described of making a carotenoid pigment solubilised in water, where pH is made alkaline, cyclodextrins are mixed to the water and the pigment and finally the pH is restored to neutral. EP 682 874-A2 discloses a bioactive feed pellet, where the bioactive ingredient could be for example a pigment. The bioactive ingredient is applied to the pellet in the form of a primary coating dispersion and/or emulsion and/or solution in a fatty component or a mixture of dietary oil, said component or dietary oil comprises a triglyceride and/or fatty acid thereof having a melting point of above 35°C. A second coating layer of an oily product is then applied. In case the bioactive compound to be added is not miscible with the suspending aid, a dispersing or emulsifying agent can be added to improve the mixing properties. Examples of such emulsifiers that are mentioned are distilled monoglycerides, polyunsaturated polyglycerol esters of fatty acids and sorbitan fatty acid esters; saturated monoglycerides are preferred. From the references cited above, it is evident that the extraction of oil-soluble pigments from solid preparations, such as from the above-mentioned beadlets, needs further improvement. Now it has surprisingly been found that when the extraction of a solid preparation, containing an oil-soluble pigment, with a hydrophobic extraction medium, such as an oil or a fat, is performed in the presence of water and a nonionic surfactant, the extraction and solubilisation of the pigment are essentially improved. In detail the process of the invention relates to a method for solubilising an oil- soluble pigment into an oil or fat by extraction of a solid preparation containing the oil-soluble pigment comprising the steps of a) mixing the solid preparation containing the pigment with water, an extraction medium containing an edible oil or fat, and a nonionic surfactant having a hydrocarbyl group, an acyl group or a substituted hydrocarbyl or acyl group containing at least 6 carbon atoms, and b) optionally centrifugalize the mixture obtained and separate the oil phase This method will lead to a more effective extraction of the pigment from the solid preparation and a more effective solubilisation of the pigment into the oil or fat. The originally obtained mixture or the separated oil phase may then be added to porous precursor feed pellets to produce feed pellets. These pellets will have a higher amount of oil-soluble pigment available for uptake into the animal. One embodiment of the process of the invention, where the solid pigment preparation is a coated pigment, such as a beadlet described above, comprises the following steps: a) the coated pigment is agitated in water at a temperature between 4 and 100°C, and the edible oil or fat comprising the nonionic surfactant is added to the mixture obtained at a temperature of from the melting point of the oil or fat to 100°C with agitation, or the coated pigment and the nonionic surfactant are agitated in water at a temperature between 4 and 100°C, and the edible oil or fat is added with agitation to the pigment-surfactant mixture at a temperature of from the melting point of the oil or fat to100°C b) optionally the mixture obtained is centrifugalized and the oil phase is separated c) the mixture obtained by step a) or the separated oil phase obtained by step b) is added to porous precursor feed pellets to produce feed pellets. There are several advantages using the method of the present invention. The method is more effective than the prior art methods in extracting and solubilising the pigments, so that a larger proportion of the pigments will be extracted from the solid preparation and will be present in the oil phase. This is demonstrated in the examples, where the method of EP 839 004-B1 to enzymatically, in the presence of water, break down the gelatin and carbohydrate protective shell around the pigment to set it free from a solid preparation (beadlet) is compared to the method of the present invention where the addition of water and a nonionic surfactant more effectively sets the pigment free. Since the extraction and solubilisation of the pigment are more effective, the method of the present invention will lead to a higher concentration of pigment in the oil. This will in turn lead to a larger proportion of pigment that is kept in the pellets that are loaded with this oil or fat. Further, the oil-phase is not so easily washed away as the untreated beadlets. Also, there will be a better bioavailability when the pigments are solubilised to a larger extent in the oil (see Bjerkeng, B. et al, Bioavailability of all-E-astaxanthin and Z-isomers of astaxanthin in rainbow trout (Oncorhynchus mykiss), Aquaculture 157,63-82). The dispersion or oil phase comprises an edible oil or fat, one or more oil-soluble pigments and one or more nonionic surfactants, and the invention also relates to such a composition suitable for use in loading pellets to be used as animal feed. A suitable composition would be an edible oil or fat comprising 0.25-15%, preferably 2-10% and most preferably 4-10% by weight of one or more nonionic surfactants, where the surfactant is an ester, an alkoxylate of an ester or an alkoxylate of an alcohol, preferably a castor oil ethoxylate with 2-40, preferably 2-25 and most preferably 4-20 moles of ethylene oxide, or a diacetyl tartaric acid ester of mono- and/or diglycerides; 0.0005 to 1% by weight, preferably 0.0005 to 0.3% by weight, more preferably 0.0005 to 0.2% by weight and most preferably 0.0005 to 0.1% by weight, of one or more oil-soluble pigments; and 0-20% by weight of other components including water. The other components could be vitamins, enzymes, anti-oxidants, residues from the beadlets' gelatine and carbohydrates, minerals, prophylactic agents, pharmacologically active compounds, flavouring agents, preservatives and other common feed additives. The water present in the composition is dissolved or emulsified in the oil by the surfactant, and would normally range between 0.1% and 15% by weight. The edible oil or fat may be a fish-oil, such as menhaden oil, herring oil, sardine oil, tobis oil or capelin oil, hydrogenated fish-oil, castor oil, rapeseed oil, hydrogenated rapeseed oil, corn oil, soybean oil, hydrogenated soybean oil, sun flower oil, hydrogenated sun flower oil, olive oil, hydrogenated olive oil, palm oil, hydrogenated palm oil, coconut oil, hydrogenated coconut oil, tallow or lard. Hydrogenated oil is normally needed when the total amount of oil or fat is high, such as for total amounts of oil or fat in fish-feed of 26% (w/w) or higher (counted on the total pellet weight). The amount hydrogenated oil or fat that is present in these pellets is normally between 0.2 to 10% (w/w) of the total amount of oil or fat. The pigment is preferably a carotenoid, which could belong to either of the sub- groups carotenes or xanthophylls. Suitable xanthophylls are lutein, zeaxanthin, canthaxanthin, astaxanthin or p-cryptoxanthin, and suitable carotenes are P- carotene, alfa-carotene and lycopene. Examples of commercial products containing these pigments are Carophyll® Pink (Hoffman LaRoche; min 8% (w/w) astaxanthin), Lucantin® Pink (BASF; min 10% (w/w) astaxanthin), Lucarotin® 10% Feed (BASF; min 10% (w/w) p -carotene), Lucantin® Red (BASF; min 10% canthaxanthin) and Rovimix (Hoffman LaRoche; min 10% (w/w) p-carotene). The surfactant should be a nonionic surfactant, such as an ester, an alkoxylate of an ester or an alkoxylate of an alcohol. Preferred nonionic surfactants are sorbitan esters, ethoxylated sorbitan esters, tartaric acid esters of mono- and diglycerides, alkoxylated fats, oils or other esters, and alkoxylated alcohols. The most preferred nonionic surfactants are castor oil ethoxylates, preferably castor oil ethoxylates with 2-40 moles, more preferably with 2-25 moles, and most preferably with 4-20 moles of ethylene oxide per mol castor oil. Experiments have also been made to use an ionic surfactant, such as a native lecithin, but the results were not as good as when using a nonionic surfactant. Certain nonionics perform better with certain pigments. For example, castor oil ethoxylates are especially suited to be used for the solubilisation of astaxanthin and canthaxanthin, whereas diacetyl tartaric acid esters of mono- and diglycerides are especially suited for the solubilisation of p- carotene. A comparison between some castor oil ethoxylates with different amounts of ethylene oxide, and consequently different HLB-values, and the corresponding mixtures of sorbitan monooleate +20EO and sorbitan monooleate having the same HLB-values show that the castor oil ethoxylates having up to 25 moles of ethylene oxide are much more efficient in solubilising astaxanthin. The porous precursor feed pellets could be manufactured by any known method, e.g. extrusion, and from any commonly used material, such as carbohydrates or protein. When loading the precursor feed pellets, the temperature should be high enough to keep the fat or oil in a liquid state, but not above the decomposition temperature of the pigment. A suitable temperature is between the melting point of the fat or oil and 60°C. The loading of the feed pellets with the pigment-containing oil could be performed by mixing, dipping, spraying, coating or other commonly used means. A suitable pellet composition, obtained by loading precursor feed pellets with the above-mentioned oil or fat composition, has an amount of oil between 1 and 50%, preferably between 3 and 45% and most preferably between 5 and 40%, by weight of the loaded feed pellets. The present invention is further illustrated by the following Examples. Example 1 10% (w/w) of Carophyll® Pink1 was mixed with 90% (w/w) water at a temperature of 60°C with stirring. After cooling to room temperature a formulation was made containing 7.5% (w/w) of the mixture and 92.5% (w/w) of a surfactant/fish-oil mixture (sample type A), the formulation was stirred for 2 minutes at a temperature of ca 45°C and the next day it was centrifugalized at 5000 rpm (G=34000m/s2) for 5 minutes. A sample was taken with a syringe and filtered through a 0.2 urn micropore filter. If the sample was taken after a few days it was not necessary to centrifugalize the formulation, but the sample could be taken directly from the oil- phase and filtered. The sample was then diluted with acetone to a desired concentration, and the absorbance was measured at 474 nm. In Table 1 the absorbance values for different samples are displayed, which is a measure of the abilities of the different surfactants to solubilise the pigment astaxanthin. The formulation could also be made by first mixing the surfactant with the pigment/water mixture, and then mix with the fish-oil (sample type B). Example 2 The pigment used in this example was Carophyll® Pink. The procedure followed was the same as for Example 1. The recovery values are based on the assumption that the product Carophyll® Pink contains 8% astaxanthin, which is the amount astaxanthin that the producer guarantees is present. The amount of surfactant is not very critical within the investigated range. *for an absorbance value of >0.9 there is not a linear relationship between absorbance and concentration Within this range there is a correlation between the amount of surfactant used and the amount of pigment solubilised. Example 3 In this experiment the amount of pigment to be solubilised is varied from ca 25 ppm, counted on the whole mixture, up to 750 ppm, and the amount of surfactant (castor oil +6EO) used is also varied. 5-10% (w/w) of Lucantin® Pink CWD was mixed with 95-90% (w/w) of water at room temperature with stirring. A formulation was made containing ca 0.5-7.5% (w/w) of the mixture and ca 99.5-92.5% (w/w) of the surfactant/fish-oil mixture. The formulation was stirred for 2 minutes at a temperature of 45°C and then centrifugalised at 5000 rpm for 5 minutes. The sample was filtered through a 0.2um micropore filter and 0.400 ml of the filtrate was diluted with acetone to 100 ml. The recovery values are based on the assumption that the product Lucantin® Pink CWD contains 10% astaxanthin, which is the amount astaxanthin that the producer guarantees is present. This experiment shows that for the amounts 23-136 ppm of pigment, the whole amount is easily solubilised even at the low weight ratio of 2/98 of surfactant to oil, whereas for the higher amount of 750 ppm a higher weight ratio is required. However, a weight ratio above 8/92 does not result in any increase of the amount of pigment solubilised. Lucantin® Pink CWD is a product produced by BASF that contains at least 10% (w/w) of the pigment astaxanthin. The recovery values are based on this amount of pigment in the product. Example 5 In this example the castor oil ethoxylates are compared to other kinds of surfactants. The pigment used was Carophyll® Pink. The procedure followed was the same as for Example 1. Example 6 In this example the effect of HLB-values on the solubilising ability is investigated for castor oil ethoxylates and for Tween 808/Span 809 mixtures having the same HLB-values as the castor oil ethoxylates. The pigment used was Lucantin® Pink CWD and the oil was Tobis fish-oil. The procedure was the same as in Example 1 except that the pigment was mixed and stirred with the water at room temperature. All samples were of type A. The formulations contained 2g of the specific surfactant or surfactant mixture, and the weight ratio surfactant:fish-oil was 6:94. The amount of pigment/water mix was 2.57 g. The samples were then further diluted by taking 2.00 ml of the acetone solution and dilute it to 10 ml. For the castor oil ethoxylates there is a marked decline in the solubilising ability around a HLB-value of 11, whereas for the Tween 80/Span 80 mixtures the level is about the same for all mixtures and generally lower than for the castor oil ethoxylates. However, both types of compounds have an effect on the solubilisation of the pigment astaxanthin. Example 11 In this example a comparison is made with the enzymatic method described in the prior art. The procedure for the enzymatic method was the following: 10% (w/w) of Lucantin® Pink was mixed with water, that was buffered to pH 7.5 and that contained 0.5 mg/ml protease (Protease Streptomyces griseus; CAS number 9036-06-0, 5.6 units/mg solid powder), at a temperature of 55°C with stirring. The pigment/water/enzyme mixture was then stirred at 45°C for 90 minutes. After cooling to room temperature a formulation was made containing 7.4%(w/w) of the mixture and 92.6%(w/w) of fish-oil, the formulation was stirred for 2 minutes at a temperature of ca 45°C and the next day it was centrifugalized at 5000 rpm (G=34000m/s2) for 5 minutes. A sample was taken with a syringe and filtered through a 0.2 urn micropore filter. 0.400 ml of the filtrated sample was then diluted with acetone to 100 ml, and the absorbance was measured at 474 nm. The sample according to the invention was treated in the same manner, except that it contained no protease and the formulation was made by mixing with 92.6% (w/w) of castor oil +6EO/fish-oil mixture. In Table below the absorbance values for the different samples are displayed, which is a measure of the abilities of the different methods to solubilise the pigment astaxanthin. The comparison reveals that much more astaxanthin can be solubilised by using the method of the present invention than by using the enzymatic procedure disclosed in the prior art. Example 12 This example is also a comparison with the enzymatic method described in the prior art. The procedure was the same as in Example 11, except that the water was buffered at pH 9.5 and contained 1.0 mg/ml or 0.35 mg/ml of a protease (Protex 6L produced by Genencor International; activity 580000 DU/g) The comparison reveals that also during these conditions with a higher pH, a different protease and a higher concentration of the protease, much more astaxanthin can be solubilised by using the method of the present invention than by using the enzymatic procedure disclosed in the prior art. We Claim: 1. A method for solubilising an oil-soluble pigment into an oil or fat by extraction of a solid preparation containing the oil-soluble pigment, comprising the steps of: a) mixing the solid preparation containing the pigment with water, an extraction medium containing an edible oil or fat, and a nonionic surfactant having a hydrocarbyl group, an acyl group or a substituted hydrocarbyl or acyl group containing at least 6 carbon atoms, and b) optionally, centrifugalize the mixture obtained and separate the oil phase. 2. The method as claimed in claim 1 wherein the solid preparation containing the pigment is a coated pigment; and the coated pigment is agitated in water at a temperature between 4 and 100°C, and the edible oil or fat comprising the nonionic surfactant is added to the mixture obtained at a temperature of from the melting point of the oil or fat to 100°C with agitation, or the coated pigment and the nonionic surfactant are agitated in water at a temperature between 4 and 100°C, and the edible oil or fat is added with agitation to the pigment- surfactant mixture at a temperature of from the melting point of the oil or fat to 100°C. 3. The method as claimed in claim 1 to 2 wherein the nonionic surfactant is an ester, an alkoxylate of an ester or an alkoxylate of an alcohol. 4. The method as claimed in claim 3 wherein the surfactant is a castor oil ethoxylate with 2-25 moles of ethylene oxide or a diacetyl tartaric acid ester of mono- and/or diglycerides. 5. The method as claimed in claim 1-4 wherein the pigment is a carotenoid. 6. The method as claimed in claim 1-5 wherein the pigment is selected from the group astaxanthin, canthaxanthin and β-carotene. 7. An edible oil or fat composition comprising 0.25-15% by weight of one or more nonionic surfactants as claimed in claims 1 to 4, 0.0005 to 1% by weight of one or more oil-soluble pigments, and 0-20% by weight of other components including water. 8. The composition as claimed in claim 7 wherein the pigment is a carotenoid. 9. The composition as claimed in claim 7 to 8 wherein the oil is selected from the group castor oil, a fish-oil, rapeseed oil, corn oil and soybean oil. The present invention relates to an improved method for solubilising an oil-soluble pigment efficiently in oil or fat by extraction of a solid preparation containing the oil-soluble pigment, where the extraction takes place in the presence of water, edible oil or fat and a nonionic surfactant. It also relates to the composition prepared by the extraction, and the use of this composition in animal feed to produce feed pellets. The combination of water and nonionic surfactant increases the amount of pigment that is extracted and solubilised. In detail the process of the invention relates to a method for solubilising an oil- soluble pigment into an oil or fat by extraction of a solid preparation containing the oil-soluble pigment comprising the steps of a) mixing the solid preparation containing the pigment with water, an extraction medium containing an edible oil or fat, and a nonionic surfactant having a hydrocarbyl group, an acyl group or a substituted hydrocarbyl or acyl group containing at least 6 carbon atoms b) optionally centrifugalize the mixture obtained and separate the oil phase. The originally obtained mixture or the separated oil phase may then be added to porous precursor feed pellets to produce feed pellets. These pellets will have a higher amount of oil-soluble pigment available for uptake into the animal.

Full Text

The present invention relates to an improved method for solubilising an oil-soluble
pigment efficiently in an oil or fat by extraction of a solid preparation containing the
oil-soluble pigment, where the extraction takes place in the presence of water, an
edible oil or fat and a nonionic surfactant. It also relates to the composition
prepared by the extraction, and the use of this composition in animal feed to
produce feed pellets. The combination of water and nonionic surfactant increases
the amount of pigment that is extracted and solubilised.
In animal feed compositions, pigments are often added to endow an appealing
colour to the meat or skin of the animals consuming the feed. The pigments used
may be natural, synthesized or fermented, and commonly they are oil-soluble. An
example of a class of oil-soluble pigments is the carotenoids, e.g. astaxanthin,
canthaxanthin and [beta]-carotene. These are insoluble in water, and have a low
solubility in organic solvents, fats and oils. Astaxanthin and canthaxanthin are
commonly used in fish feed to improve the flesh colour of salmonids, but also give
rise to health benefits. Other uses for pigment in animal feed is for the coloration of
the skin of some marine warm-water or tropical freshwater fishes, e.g. of the red
seabream, and to endow a pink color to the shell and flesh of prawns. In addition to
these aqua-cultural uses, the coloring of egg yolks and of the skin of broilers are
other feed application areas.
Conventional feed pellets are usually formed from a solid base material, which may
be protein-based such as fishmeal or carbohydrate-based such as starch, and where
the base material is loaded with a fat or an oil component. The fat or oil, that could
be either of animal or vegetable origin, is loaded into pores of the precursor feed
pellets formed from the base material. The fat or oil increases the energy content of
the feed. Other ingredients of the feed are e.g. vitamins

minerals, enzymes and the above-mentioned pigments. These latter ingredients,
as well as the fat or oil, are preferably added after the pelletizing step, since the oil
component interferes with the pelletizing process and many of the pigments lose
their activity when heated. Many of the sensitive pigments are available in coated
form, e.g. Carophyll® Pink, which is sold in the form of beadlets. The beadlets of
this product consist of a core of astaxanthin emulsified in antioxidants and residing
in a matrix of gelatine and carbohydrate, which core is coated by maize starch.
The amount of astaxanthin is at least 8% of the beadlet. There is also another
astaxanthin product of a similar kind that is called Carophyll® Pink 10% CWS,
which differs from the first-mentioned product only in that the matrix is a
lignosulfonate. If these beadlets are added as such to the preformed pellets, most
of them are deposited only on the pellets surface. Especially for aqua-cultural
feed this is a disadvantage, since the beadlets will be washed away when the feed
comes into contact with water. Also the bioavailability will be less when the
pigments are still contained in the beadlets.
In EP 839 004-B1 a method is disclosed for loading bioactive ingredients, such as
pigments, into feed pellets. The process includes the steps of removing the gelatin
and carbohydrate protective shell around the bioactive ingredient enzymatically or
by hydrolysis, mixing the uncoated bioactive ingredient with a fat or an oil and
loading porous precursor feed pellets with the resulting mixture to produce the
feed pellets.
In JP7-16075-A a method is described of making a water-soluble pigment
solubilised in fat. Firstly polyols are dissolved in water and mixed with the water-
soluble pigment, and then an oil-phase containing an emulsifier is added to obtain
an emulsion of the water-soluble pigment in oil.
In JP7-23736-A a method is described of making a carotenoid pigment solubilised
in water, where pH is made alkaline, cyclodextrins are mixed to the water and the
pigment and finally the pH is restored to neutral.

EP 682 874-A2 discloses a bioactive feed pellet, where the bioactive ingredient
could be for example a pigment. The bioactive ingredient is applied to the pellet in
the form of a primary coating dispersion and/or emulsion and/or solution in a fatty
component or a mixture of dietary oil, said component or dietary oil comprises a
triglyceride and/or fatty acid thereof having a melting point of above 35°C. A
second coating layer of an oily product is then applied. In case the bioactive
compound to be added is not miscible with the suspending aid, a dispersing or
emulsifying agent can be added to improve the mixing properties. Examples of
such emulsifiers that are mentioned are distilled monoglycerides, polyunsaturated
polyglycerol esters of fatty acids and sorbitan fatty acid esters; saturated
monoglycerides are preferred.
From the references cited above, it is evident that the extraction of oil-soluble
pigments from solid preparations, such as from the above-mentioned beadlets,
needs further improvement.
Now it has surprisingly been found that when the extraction of a solid preparation,
containing an oil-soluble pigment, with a hydrophobic extraction medium, such as
an oil or a fat, is performed in the presence of water and a nonionic surfactant, the
extraction and solubilisation of the pigment are essentially improved.
In detail the process of the invention relates to a method for solubilising an oil-
soluble pigment into an oil or fat by extraction of a solid preparation containing the
oil-soluble pigment comprising the steps of
a) mixing the solid preparation containing the pigment with water, an extraction
medium containing an edible oil or fat, and a nonionic surfactant having a
hydrocarbyl group, an acyl group or a substituted hydrocarbyl or acyl group
containing at least 6 carbon atoms, and
b) optionally centrifugalize the mixture obtained and separate the oil phase

This method will lead to a more effective extraction of the pigment from the solid
preparation and a more effective solubilisation of the pigment into the oil or fat.
The originally obtained mixture or the separated oil phase may then be added to
porous precursor feed pellets to produce feed pellets. These pellets will have a
higher amount of oil-soluble pigment available for uptake into the animal.
One embodiment of the process of the invention, where the solid pigment
preparation is a coated pigment, such as a beadlet described above, comprises
the following steps:
a) the coated pigment is agitated in water at a temperature between 4 and 100°C,
and the edible oil or fat comprising the nonionic surfactant is added to the mixture
obtained at a temperature of from the melting point of the oil or fat to 100°C with
agitation, or
the coated pigment and the nonionic surfactant are agitated in water at a
temperature between 4 and 100°C, and the edible oil or fat is added with agitation
to the pigment-surfactant mixture at a temperature of from the melting point of the
oil or fat to100°C
b) optionally the mixture obtained is centrifugalized and the oil phase is separated
c) the mixture obtained by step a) or the separated oil phase obtained by step b) is
added to porous precursor feed pellets to produce feed pellets.
There are several advantages using the method of the present invention. The
method is more effective than the prior art methods in extracting and solubilising
the pigments, so that a larger proportion of the pigments will be extracted from the
solid preparation and will be present in the oil phase. This is demonstrated in the
examples, where the method of EP 839 004-B1 to enzymatically, in the presence
of water, break down the gelatin and carbohydrate protective shell around the
pigment to set it free from a solid preparation (beadlet) is compared to the method
of the present invention where the addition of water and a nonionic surfactant
more effectively sets the pigment free. Since the extraction and solubilisation of
the pigment are more effective, the method of the present invention will lead to a

higher concentration of pigment in the oil. This will in turn lead to a larger
proportion of pigment that is kept in the pellets that are loaded with this oil or fat.
Further, the oil-phase is not so easily washed away as the untreated beadlets.
Also, there will be a better bioavailability when the pigments are solubilised to a
larger extent in the oil (see Bjerkeng, B. et al, Bioavailability of all-E-astaxanthin
and Z-isomers of astaxanthin in rainbow trout (Oncorhynchus mykiss),
Aquaculture 157,63-82).
The dispersion or oil phase comprises an edible oil or fat, one or more oil-soluble
pigments and one or more nonionic surfactants, and the invention also relates to
such a composition suitable for use in loading pellets to be used as animal feed. A
suitable composition would be an edible oil or fat comprising 0.25-15%, preferably
2-10% and most preferably 4-10% by weight of one or more nonionic surfactants,
where the surfactant is an ester, an alkoxylate of an ester or an alkoxylate of an
alcohol, preferably a castor oil ethoxylate with 2-40, preferably 2-25 and most
preferably 4-20 moles of ethylene oxide, or a diacetyl tartaric acid ester of mono-
and/or diglycerides; 0.0005 to 1% by weight, preferably 0.0005 to 0.3% by weight,
more preferably 0.0005 to 0.2% by weight and most preferably 0.0005 to 0.1% by
weight, of one or more oil-soluble pigments; and 0-20% by weight of other
components including water. The other components could be vitamins, enzymes,
anti-oxidants, residues from the beadlets' gelatine and carbohydrates, minerals,
prophylactic agents, pharmacologically active compounds, flavouring agents,
preservatives and other common feed additives. The water present in the
composition is dissolved or emulsified in the oil by the surfactant, and would
normally range between 0.1% and 15% by weight.
The edible oil or fat may be a fish-oil, such as menhaden oil, herring oil, sardine
oil, tobis oil or capelin oil, hydrogenated fish-oil, castor oil, rapeseed oil,
hydrogenated rapeseed oil, corn oil, soybean oil, hydrogenated soybean oil, sun
flower oil, hydrogenated sun flower oil, olive oil, hydrogenated olive oil, palm oil,

hydrogenated palm oil, coconut oil, hydrogenated coconut oil, tallow or lard.
Hydrogenated oil is normally needed when the total amount of oil or fat is high,
such as for total amounts of oil or fat in fish-feed of 26% (w/w) or higher (counted
on the total pellet weight). The amount hydrogenated oil or fat that is present in
these pellets is normally between 0.2 to 10% (w/w) of the total amount of oil or fat.
The pigment is preferably a carotenoid, which could belong to either of the sub-
groups carotenes or xanthophylls. Suitable xanthophylls are lutein, zeaxanthin,
canthaxanthin, astaxanthin or p-cryptoxanthin, and suitable carotenes are P-
carotene, alfa-carotene and lycopene. Examples of commercial products
containing these pigments are Carophyll® Pink (Hoffman LaRoche; min 8% (w/w)
astaxanthin), Lucantin® Pink (BASF; min 10% (w/w) astaxanthin), Lucarotin® 10%
Feed (BASF; min 10% (w/w) p -carotene), Lucantin® Red (BASF; min 10%
canthaxanthin) and Rovimix (Hoffman LaRoche; min 10% (w/w) p-carotene).
The surfactant should be a nonionic surfactant, such as an ester, an alkoxylate of
an ester or an alkoxylate of an alcohol. Preferred nonionic surfactants are sorbitan
esters, ethoxylated sorbitan esters, tartaric acid esters of mono- and diglycerides,
alkoxylated fats, oils or other esters, and alkoxylated alcohols. The most preferred
nonionic surfactants are castor oil ethoxylates, preferably castor oil ethoxylates
with 2-40 moles, more preferably with 2-25 moles, and most preferably with 4-20
moles of ethylene oxide per mol castor oil. Experiments have also been made to
use an ionic surfactant, such as a native lecithin, but the results were not as good
as when using a nonionic surfactant. Certain nonionics perform better with certain
pigments. For example, castor oil ethoxylates are especially suited to be used for
the solubilisation of astaxanthin and canthaxanthin, whereas diacetyl tartaric acid
esters of mono- and diglycerides are especially suited for the solubilisation of p-
carotene. A comparison between some castor oil ethoxylates with different
amounts of ethylene oxide, and consequently different HLB-values, and the
corresponding mixtures of sorbitan monooleate +20EO and sorbitan monooleate

having the same HLB-values show that the castor oil ethoxylates having up to 25
moles of ethylene oxide are much more efficient in solubilising astaxanthin.
The porous precursor feed pellets could be manufactured by any known method,
e.g. extrusion, and from any commonly used material, such as carbohydrates or
protein. When loading the precursor feed pellets, the temperature should be high
enough to keep the fat or oil in a liquid state, but not above the decomposition
temperature of the pigment. A suitable temperature is between the melting point of
the fat or oil and 60°C. The loading of the feed pellets with the pigment-containing
oil could be performed by mixing, dipping, spraying, coating or other commonly
used means.
A suitable pellet composition, obtained by loading precursor feed pellets with the
above-mentioned oil or fat composition, has an amount of oil between 1 and 50%,
preferably between 3 and 45% and most preferably between 5 and 40%, by weight
of the loaded feed pellets.
The present invention is further illustrated by the following Examples.
Example 1
10% (w/w) of Carophyll® Pink1 was mixed with 90% (w/w) water at a temperature
of 60°C with stirring. After cooling to room temperature a formulation was made
containing 7.5% (w/w) of the mixture and 92.5% (w/w) of a surfactant/fish-oil
mixture (sample type A), the formulation was stirred for 2 minutes at a temperature
of ca 45°C and the next day it was centrifugalized at 5000 rpm (G=34000m/s2) for
5 minutes. A sample was taken with a syringe and filtered through a 0.2 urn
micropore filter. If the sample was taken after a few days it was not necessary to
centrifugalize the formulation, but the sample could be taken directly from the oil-
phase and filtered. The sample was then diluted with acetone to a desired
concentration, and the absorbance was measured at 474 nm. In Table 1 the

absorbance values for different samples are displayed, which is a measure of the
abilities of the different surfactants to solubilise the pigment astaxanthin. The
formulation could also be made by first mixing the surfactant with the
pigment/water mixture, and then mix with the fish-oil (sample type B).

Example 2
The pigment used in this example was Carophyll® Pink. The procedure followed
was the same as for Example 1.
The recovery values are based on the assumption that the product Carophyll®
Pink contains 8% astaxanthin, which is the amount astaxanthin that the producer
guarantees is present.

The amount of surfactant is not very critical within the investigated range.

*for an absorbance value of >0.9 there is not a linear relationship between
absorbance and concentration
Within this range there is a correlation between the amount of surfactant used and
the amount of pigment solubilised.
Example 3
In this experiment the amount of pigment to be solubilised is varied from ca 25
ppm, counted on the whole mixture, up to 750 ppm, and the amount of surfactant
(castor oil +6EO) used is also varied.
5-10% (w/w) of Lucantin® Pink CWD was mixed with 95-90% (w/w) of water at
room temperature with stirring. A formulation was made containing ca 0.5-7.5%
(w/w) of the mixture and ca 99.5-92.5% (w/w) of the surfactant/fish-oil mixture. The
formulation was stirred for 2 minutes at a temperature of 45°C and then

centrifugalised at 5000 rpm for 5 minutes. The sample was filtered through a
0.2um micropore filter and 0.400 ml of the filtrate was diluted with acetone to 100
ml.

The recovery values are based on the assumption that the product Lucantin®
Pink CWD contains 10% astaxanthin, which is the amount astaxanthin that the
producer guarantees is present.
This experiment shows that for the amounts 23-136 ppm of pigment, the whole
amount is easily solubilised even at the low weight ratio of 2/98 of surfactant to oil,
whereas for the higher amount of 750 ppm a higher weight ratio is required.
However, a weight ratio above 8/92 does not result in any increase of the amount
of pigment solubilised.

Lucantin® Pink CWD is a product produced by BASF that contains at least 10%
(w/w) of the pigment astaxanthin. The recovery values are based on this amount
of pigment in the product.
Example 5
In this example the castor oil ethoxylates are compared to other kinds of
surfactants. The pigment used was Carophyll® Pink. The procedure followed was
the same as for Example 1.

Example 6
In this example the effect of HLB-values on the solubilising ability is investigated
for castor oil ethoxylates and for Tween 808/Span 809 mixtures having the same
HLB-values as the castor oil ethoxylates. The pigment used was Lucantin® Pink
CWD and the oil was Tobis fish-oil. The procedure was the same as in Example 1
except that the pigment was mixed and stirred with the water at room temperature.
All samples were of type A. The formulations contained 2g of the specific
surfactant or surfactant mixture, and the weight ratio surfactant:fish-oil was 6:94.
The amount of pigment/water mix was 2.57 g.

The samples were then further diluted by taking 2.00 ml of the acetone solution
and dilute it to 10 ml.
For the castor oil ethoxylates there is a marked decline in the solubilising ability
around a HLB-value of 11, whereas for the Tween 80/Span 80 mixtures the level is
about the same for all mixtures and generally lower than for the castor oil
ethoxylates. However, both types of compounds have an effect on the
solubilisation of the pigment astaxanthin.

Example 11
In this example a comparison is made with the enzymatic method described in the
prior art. The procedure for the enzymatic method was the following:
10% (w/w) of Lucantin® Pink was mixed with water, that was buffered to pH 7.5
and that contained 0.5 mg/ml protease (Protease Streptomyces griseus; CAS
number 9036-06-0, 5.6 units/mg solid powder), at a temperature of 55°C with
stirring. The pigment/water/enzyme mixture was then stirred at 45°C for 90
minutes. After cooling to room temperature a formulation was made containing
7.4%(w/w) of the mixture and 92.6%(w/w) of fish-oil, the formulation was stirred for
2 minutes at a temperature of ca 45°C and the next day it was centrifugalized at
5000 rpm (G=34000m/s2) for 5 minutes. A sample was taken with a syringe and
filtered through a 0.2 urn micropore filter. 0.400 ml of the filtrated sample was then
diluted with acetone to 100 ml, and the absorbance was measured at 474 nm. The
sample according to the invention was treated in the same manner, except that it
contained no protease and the formulation was made by mixing with 92.6% (w/w)
of castor oil +6EO/fish-oil mixture. In Table below the absorbance values for the
different samples are displayed, which is a measure of the abilities of the different
methods to solubilise the pigment astaxanthin.

The comparison reveals that much more astaxanthin can be solubilised by using
the method of the present invention than by using the enzymatic procedure
disclosed in the prior art.
Example 12
This example is also a comparison with the enzymatic method described in the
prior art. The procedure was the same as in Example 11, except that the water
was buffered at pH 9.5 and contained 1.0 mg/ml or 0.35 mg/ml of a protease
(Protex 6L produced by Genencor International; activity 580000 DU/g)

The comparison reveals that also during these conditions with a higher pH, a
different protease and a higher concentration of the protease, much more
astaxanthin can be solubilised by using the method of the present invention than
by using the enzymatic procedure disclosed in the prior art.

We Claim:
1. A method for solubilising an oil-soluble pigment into an oil or fat by extraction
of a solid preparation containing the oil-soluble pigment, comprising the steps of:
a) mixing the solid preparation containing the pigment with water, an extraction
medium containing an edible oil or fat, and a nonionic surfactant having a hydrocarbyl
group, an acyl group or a substituted hydrocarbyl or acyl group containing at least 6
carbon atoms, and
b) optionally, centrifugalize the mixture obtained and separate the oil phase.
2. The method as claimed in claim 1 wherein the solid preparation containing the
pigment is a coated pigment; and
the coated pigment is agitated in water at a temperature between 4 and 100°C, and the
edible oil or fat comprising the nonionic surfactant is added to the mixture obtained at a
temperature of from the melting point of the oil or fat to 100°C with agitation,
or the coated pigment and the nonionic surfactant are agitated in water at a temperature
between 4 and 100°C, and the edible oil or fat is added with agitation to the pigment-
surfactant mixture at a temperature of from the melting point of the oil or fat to 100°C.
3. The method as claimed in claim 1 to 2 wherein the nonionic surfactant is an ester,
an alkoxylate of an ester or an alkoxylate of an alcohol.
4. The method as claimed in claim 3 wherein the surfactant is a castor oil ethoxylate
with 2-25 moles of ethylene oxide or a diacetyl tartaric acid ester of mono- and/or
diglycerides.
5. The method as claimed in claim 1-4 wherein the pigment is a carotenoid.
6. The method as claimed in claim 1-5 wherein the pigment is selected from the
group astaxanthin, canthaxanthin and β-carotene.

7. An edible oil or fat composition comprising 0.25-15% by weight of one or more
nonionic surfactants as claimed in claims 1 to 4, 0.0005 to 1% by weight of one or more
oil-soluble pigments, and 0-20% by weight of other components including water.
8. The composition as claimed in claim 7 wherein the pigment is a carotenoid.
9. The composition as claimed in claim 7 to 8 wherein the oil is selected from the
group castor oil, a fish-oil, rapeseed oil, corn oil and soybean oil.

The present invention relates to an improved method for solubilising an oil-soluble
pigment efficiently in oil or fat by extraction of a solid preparation containing the
oil-soluble pigment, where the extraction takes place in the presence of water, edible
oil or fat and a nonionic surfactant. It also relates to the composition prepared by
the extraction, and the use of this composition in animal feed to produce feed pellets.
The combination of water and nonionic surfactant increases the amount of pigment
that is extracted and solubilised. In detail the process of the invention relates to a
method for solubilising an oil- soluble pigment into an oil or fat by extraction of a
solid preparation containing the oil-soluble pigment comprising the steps of a)
mixing the solid preparation containing the pigment with water, an extraction
medium containing an edible oil or fat, and a nonionic surfactant having a
hydrocarbyl group, an acyl group or a substituted hydrocarbyl or acyl group
containing at least 6 carbon atoms b) optionally centrifugalize the mixture obtained
and separate the oil phase. The originally obtained mixture or the separated oil
phase may then be added to porous precursor feed pellets to produce feed pellets.
These pellets will have a higher amount of oil-soluble pigment available for uptake
into the animal.

METHOD FOR PIGMENT SOLUBILISATION AND A PIGMENT COMPOSITION

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