WO2004005353A1 - Complexes of cyclodextrins and carotenoids - Google Patents

Abstract

A complex between a carotenoid, e.g. astaxanthin, and cyclodextrin improves both the pigmentation in tissues of animals (especially fish with colored flesh) and storage ability of the complex in relation to un-complexed carotenoid.

Description

Complexes of cyelo extri-as and oarotenoids

FIELD OF THE INVENTION

The present invention relates to a chemical complex comprising a cyclodextrin and a carotenoid, e.g. astaxanthin. The invention further relates to a composition comprising the complex and methods for producing and using said complex and said composition. The complex of a cyclodextrin and a carotenoid and the composition may be used as a food or feed supplement . The invention further relates to a method of increasing the pigmentation in an animal as well as in tissue or material used as food or feed.

BACKGROUND OF THE INVENTION

Carotenoids belong to the group of highly colored (red, orange, and yellow) lipophilic pigments produced by microorganisms, fungi and plants and some animals (e.g. crustaceans) , which uses the carotenoids as antioxidants and protectants against excessive radiation. Amongst the widely used carotenoids in feed, food, medical preparations or cosmetics are the beta-carotenes: cantaxanthin, astaxanthin, zeaxanthin, lutein and lycopene.

In industrialized food production, the carotenoids may be used as feed additives in the animal production with the aim of achieving natural looking colors of the food derived thereof .

Fish in nature, for example, achieve their pigmentation through their ingestion of carotenoid-producing marine organisms, particularly, salmon and trout species possess the ability of retaining carotenoids in their skin and muscle tissue, thereby rendering their muscle tissue bright red or orange. However, the skin and flesh of farmed fish that do not receive a supplement of carotenoids have a pale red color, which in the opinion of the consumer may appear unattractive. So today it is common to add suitable carotenoids to the feed given to farmed fish in order to achieve red/orange-colored flesh of such fish.

Additionally, the mechanism of uptake for the carotenoids is poorly understood, but it is assumed that it may operate through a passive diffusion, but it may also be facilitated by active transport . In any event it seems clear that the plasma level of carotenoids converges towards a saturation point when astaxanthin in the feed is increased, either as a consequence of an active transport mechanism resulting in a saturation, mechanisms regulating the uptake, or that the transport capacity in the blood is limited. The feed concentration of astaxanthin giving a maximum uptake in the plasma of salmonides seems to lie within the interval 50 to 100 mg/kg.

Furthermore, similar carotenoids may be given to land- dwelling animals to improve the coloration of the food products derived therefrom. For instance the yolk in eggs from hens or other poultry given carotenoids is brighter yellow than normally.

The carotenoids most frequently used as feed additives in the farming of fish are astaxanthin and cantaxanthin.

Astaxanthin may be used in its un-esterified form, i.e. as a diol that is thought to be the form of astaxanthin best absorbed from the intestine [Thorrissen 0.1. et al. Reviews in Aquatic Sciences 1989, vol. 1 pp. 209-225] . However, astaxanthin may also be used in the form of an ester, e.g. astaxanthin dipalmitates, but because of limited capacity of intestinal esterases in the hydrolysis of astaxanthin esters, the un-esterified astaxanthin is more effectively taken up and thus more suitable for use in flesh pigmentation [Foss et al . Aquaculture 1985, 65: 293-305]. However, such carotenoids are unstable, partly because of the presence of free hydroxyl groups that may be prone to oxidation, resulting in a loss of 5-10% w/w or more of the astaxanthin-diol form upon exposure to heat, light and/or air during storage or during fish feeding. Moreover, such carotenoids have a low bioavailability. For example, upon feeding salmon or trout species with commercial available feed formulations comprising astaxanthin, the bioavailability of astaxanthin is in the order of 35% and only about 15% of the amount of astaxanthin entering the gut lumen of the fish is retained in the fish tissue. This is due to limited absorption from the gut and extensive metabolism of astaxanthin [Foss et al Aquaculture 1987, 65: 293-305] .

Accordingly, the overall pigmentation efficacy of such carotenoids may be limited because of this loss of biological activity during storage and metabolism. Thus, animal-feed pigments are very expensive, contributing to up to about 25% of the feed costs in farming marine animals.

Several efforts have been targeted towards improving the efficacy of pigment uptake, thereby reducing the costs of the pigment additive. For example WO 00/62625 relates to providing astaxanthin in the form of a diester of poly- unsaturated fatty acids in order to increase the uptake of astaxanthin in salmon by 41% as compared to commercial pigments ("Carophyll Pink").

Commercial pigment formulations, such as with gelatin and starch, does not allow for a sufficiently high stability, bioavailability and retention of the carotenoids of interest. Thus, the aquaculture industry would benefit economically from pigment formulation that increases the overall efficacy of the pigment.

The present inventors have recognized that one such mean for decreasing the degradation and metabolism of carotenoids of interest, e.g. astaxanthin, is by complexing it with one or more cyclodextrins . The class of compounds generally known as cyclodextrins comprises cyclic oligosaccharides having 6, 7, or 8 glucopyranose units, arranged in a relatively rigid circular structure . The cyclodextrin structure provides a molecule shaped like a segment of a hollow cone with an exterior hydrophilic surface and interior hydrophobic cavity. The hydrophilic surface generates good water solubility for the cyclodextrin and the hydrophobic cavity provides a favorable environment in which 'to fit¹ the drug molecule. This association isolates the drug from the aqueous solvent and may increase the drug's water solubility and stability.

SUMMARY OF THE INVENTION

The present inventor has provided chemical entities comprising a cyclodextrin and a carotenoid that possesses novel properties. Advantageously, complexes of a cyclodextrin and a carotenoid may be more resistant to oxidation, hydrolysis and metabolism than carotenoids in un-complexed form.

Thus, in a first aspect the invention relates to the use of use of a combination of a cyclodextrin and a carotenoid for modifying the pigmentation of an animal tissue and/or an animal body fluid. Advantageously, the combination of a carotenoid and a cyclodextrin is in the form of an inclusion complex.

In a second aspect the invention relates to a complex comprising a cyclodextrin and a carotenoid. Such a complex is preferably an inclusion complex, wherein the astaxanthin or a part of it is enclosed in the cavity of one or more cyclodextri (s) .

In a further aspect, the invention relates to compositions comprising a complex of a cyclodextrin and a carotenoid together with one or more acceptable excipient for preparing a composition, which typically could be food product, a feed product, a food/feed supplement, a pre-mix or a dietary supplement.

DETAILED DESCRIPTION OF THE INVENTION

The present inventor has found that the pigmentation efficacy in fish increases upon administering astaxanthin in a complexed form with certain cyclodextrins .

Thus, in a first aspect the present invention relates to the use of a combination of a cyclodextrin and a carotenoid for modifying the pigmentation of an animal tissue and/or an animal body fluid. In other terms that is to say that the invention relates to a method for modifying the pigmentation of an animal tissue and/or an animal body fluid comprising the steps of complexing a carotenoid with a cyclodextrin and administering the thus formed complex to the animal. According to the invention, the combination of a cyclodextrin and a carotenoid may be in the form of a complex between a cyclodextrin and a carotenoid or a composition comprising said complex together with one or more suitable excipients .

Thus, in efforts to improve the functional utility of astaxanthin, research has led to a new chemical entity, a complex between cyclodextrin and astaxanthin. Accordingly, a further aspect of the invention relates to a complex comprising a cyclodextrin and a carotenoid. Such a complex is preferably an inclusion complex, wherein the astaxanthin or a part of it is enclosed in the cavity of the cyclodextrin.

The term "pigmentation" relates to the coloring of animal tissue of animal body fluids, as determined by spectral reflectance measurement of isolated tissue at wavelengths in the range of 400 nm to 700 nm. Moreover, pigmentation is defined by providing a UN/VIS peak absorption of the supernatant of a tissue sample extracted in heptane in the wavelength range from 360 to 600 nm, preferably in the range of 400 to 550 nm, most preferably in the range of 420 to 500 nm, such as 440 nm to 480 nm.

As used herein, the term "carotenoid" is denoted to mean any one substance or a mixture of substances with a carotenoid backbone structure . A number of carotenoids are of interest according to the invention. Νon-limiting examples of carotenoids are the α-carotenes, β-carotenes, γ-carotenes, δ-carotenes, lutein, lycopene, their isomeric forms, derivatives or salts thereof. In presently preferred embodiments, the carotenoid is a carotenoid with J3- carotenoid structure. Moreover, the term "carotenoid" includes derivatives, such as esters, isomers such as stereoisomers, and salts thereof. Derivatives of carotenoids include any substance derived from a carotenoid, formed by substitution of hydrogen atoms, addition of groups and reduction or oxidation of the carotenoid moiety. Moreover, derivatives of carotenoids include norcarotenoids . Furthermore, the term 'carotenoid' comprises any of various usually yellow to red carotenoids found in nature, for example those produced in plants, animals, crustacean, algae's and which are characterized by having a long aliphatic polyene chain composed of eight isoprene units.

In general, interesting carotenoids of the invention are those exhibiting yellow, orange, red and purple colors when dissolved in methanol . Therefore, interesting carotenoids according to the invention relates to those exhibiting UV/VIS absorption in the range of 460 nm to 700 nm, more specifically in the range of 470 nm to 490 nm, when dissolved in methanol. Furthermore, such carotenoids may be characterized by having a poor solubility in water. Hence suitable carotenoids of the invention has a solubility in water at 25°C of at the most 10 g/1, preferably of at the most 5 g/1, more preferably at the most 1 g/1, even more preferably at the most 0.5 g/1, still more preferably of at the most 0.2, 0.1, 0.05 g/1, preferably of at the most 0.01 g/i.

Typically, the carotenoids according to the invention are chosen among astaxanthin, zeaxanthin, astacin, cantaxanthin, lutein, lycopene, their isomeric forms, enantiomers, derivatives or salts. In one embodiment of the invention, the carotenoid is astaxanthin, its isomers, derivatives or salts.

The term "salts" include base addition salts of for example a hydroxyl group with hydroxides of alkali metals, such as sodium and potassium, alkali earth metals, such as calcium and magnesium, and organic addition salts such as quaternary ammonium cations .

Astaxanthin, its derivatives such as esters and salts thereof may be obtained from natural sources such as plants, algae, crustacean and yeast, by culturing astaxanthin-producing yeast cells, by synthetically means or by gene manipulation techniques. Non-limiting examples of host organisms that may be used directly for producing carotenoids or used with genetic modifications are Phaffia species such as Phaffia rhodozyma or Haematococcus species such as H. pluvialis. Astaxanthin may be extracted from such natural or gene manipulated sources by various conventional extraction methods or just provided in the form of grinded crustacean shells or yeast cells. Thus, the cyclodextrin complexes according to the invention may be prepared with un-purified astaxanthin or other suitable un- purified carotenoids, e.g. in the presence of yeast cell residues and the like. However, in suitable embodiments of the invention, such extracts of astaxanthin or yeast cells comprising astaxanthin may further be subject to purification according to conventional methods, e.g. by preparatory HPLC, super-critical extraction or solvent- /solvent extraction.

The term "complex" is intended to mean a complex wherein at least one moiety of a carotenoid has inserted itself, at least partially, into the cavity of cyclodextrin.

As used herein, the term "cyclodextrin" is denoted to mean any cyclodextrin selected from a α-cyclodextrin, a β- cyclodextrin or a y-cyclodextrin, i.e. the 6-, 7-, or 8- sugar unit macrocycle, or derivatives thereof. The cyclodextrin may be modified such that some or all of the primary or secondary hydroxyls of the macrocycle, or both, may be alkylated or acylated. Methods of modifying these alcohols are well known to the person skilled in the art and many derivatives are commercially available. The cyclodextrin may be modified such that one or more of the primary or secondary hydroxyls of the macrocycle, or both, may be alkylated or acylated. Methods of modifying these alcohols are well known to the person skilled in the art and many are commercially available. Thus, some or all of the hydroxyls of cyclodextrin may have be substituted with an O-R group or an 0-C(0)-R, wherein R is an optionally substituted Cι_₆ alkyl, an optionally substituted C₂-6 alkenyl, an optionally substituted C₂.₆ alkynyl, an optionally substituted aryl or heteroaryl group. R may be methyl, ethyl, propyl, butyl, pentyl, or hexyl group.

Consequently, 0-C(0)-R may be an acetate. Furthermore, R may be such as to derivatize cyclodextrin with the commonly employed 2-hydroxyethyl group, or 2-hydroxypropyl group. Moreover, the cyclodextrin alcohols may be per-benzylated, per-benzoylated, or benzylated or benzoylated on just one face of the macrocycle, or wherein only 1, 2, 3, 4, 5, or 6 hydroxyls are benzylated or benzoylated. The hydroxyl groups of cyclodextrin may be peralkylated or per-acylated such as per-methylated or per-acetylated, or alkylated or acylated, such as methylated or acetylated, on just one face of the macrocycle, or wherein only 1, 2, 3, 4, 5, or 6 hydroxyls are alkylated or acylated, such as methylated or acetylated.

In more specific terms, the cyclodextrin contains at least 5 glucopyranose units, preferably at least 6, more preferably at least 7, most preferably at least 8 glucopyranose units. Furthermore, the term "cyclodextrin" include mixtures of two or several various cyclodextrins .

In a preferred embodiment of the invention, the inclusion complex is between β-cyclodextrin or γ~cyclodextrin and astaxanthin. In a further interesting embodiment, the cyclodextrin is unmodified.

One or more carotenoid molecules of the invention may be included into the cavity of the cyclodextrin molecule. Conversely, one molecule of a carotenoid of the invention may be included into the cavity of one or more cyclodextrin molecules . The inclusion complex may exist in a variety of molar ratios . The molar ratio between a carotenoid and a cyclodextrin is dependent on a variety of physical factors during the formation of the inclusion complex. Furthermore, the molar ratio of the inclusion complex may be transitional and vary during its preparation. Given the inclusion of a carotenoid can result from a variety of interactions with any number of functional groups or moieties of the carotenoid, the depth at which the carotenoid is included within the cavity of a cyclodextrin may vary. Furthermore, the size of the cavity, which depends on the selection of cyclodextrin (α-cyclodextrin, β-cyclodextrin or y-cyclodextrin) and on whether the numerous free hydroxyl groups present on the periphery of the cavity of a cyclodextrin molecule are partially or fully derivatized, will influence the ability for the carotenoid to include itself into the cavity. These factors, amongst others, influence the molar ratio of the inclusion complex. Given the above-stated factors, and that the moiety of the carotenoid molecule which may include itself into the cyclodextrin molecule may vary, the molar ratio between a carotenoid and the cyclodextrin may be in the range of 10:1 to 1:100, preferably in the range selected from the group consisting of 5:1 to 1:80; 5:1 to 1:50; 5:1 to 1:20; 5:1 to 1:10;4:1 to 1:8;2:1 to 8:2; 5:1 to 1:5;2:1 to 1:5;4:1 to 1:4; 2:1 to 1:4,2:1 to 1:3, 2:1 to 1:2 and 1:1. Preferably, a 1:1, 1:2 or 2:1 molar ratio exists between a carotenoid and cyclodextrin.

The term "solubility" in connection with a carotenoid is intended to mean the solubility of the inclusion complex between a carotenoid and cyclodextrin in water. The term "total solubility" relates to the carotenoid concentration in a phase solubility isotherm, namely to the solubility of un-complexed and complexed carotenoid. The "total solubility" is a function of the cyclodextrin concentration.

Given one of the objects of the present invention is to increase the solubility and total solubility of a carotenoid, it is preferred that the solubility of said complex is such that when subjecting the complex to water at 25 °C, the solubility is of the at least 0.5 g/1, preferably of the at least 1 g/1, 2 g/1, 5g/l or lOg/1, more preferably of the at least 15 g/1, 20 g/1 or 25 g/1, even more preferably of the at least 30 g/1, 40 g/1 or 50 g/1, most preferably of the at least 60 g/1, 80g/l or 100 g/i.

Correspondingly, it is preferred that the total solubility of said complexed plus uncomplexed carotenoid is such that when subjecting the complex to water at 25 °C, the solubility is of the at least 0.5 g/1, preferably of the at least 1 g/1, 2 g/1, 5 g/1 or 10 g/1, more preferably of the at least 15 g/1, 20 g/1 or 25 g/1, even more preferably of the at least 30 g/1, 40 g/1 or 50 g/1, most preferably of the at least 60 g/1, 80g/l or 100 g/1.

As stated, one object of the invention is to improve the solubility of a carotenoid in water. According to the invention, said improved solubility corresponds to a relative increase in the solubility, as determined upon dissolving said complexed carotenoid versus dissolving an equivalent carotenoid in un-complexed form in water at 25 °C, by a factor of at the least of 1.1, 1.2 or 1.3, preferably by a factor of at the least of 1.5, 1.8 or 2, more preferably by a factor of at the least of 2.5, 3, 3.5 or 4, even more preferably by a factor of at the least of 5, 7 or 10, still more preferably by a factor of the at least 15, 20, 25, 30, 35 or 40, most preferably by a factor of the at least 45, 50, 60, 70, 80, 90 or 100.

The inclusion complex may exist in the form of a hydrate containing varying amounts of water, such as between about 1% and 25% water. The degree of hydration may vary according to, amongst other reasons, the degree of substitution of the hydroxyls, the method of preparation and the molar ratio of the inclusion complex. The water content of the inclusion complex may depend on the manner in which the inclusion complex is stored, the temperature, pressure and relative humidity. Thus, any discussion on the solid state form of the carotenoid-cyclodextrin inclusion complex comprises the range of hydrates. The hydrate water is part of the crystal lattice and thus modifying the water content may change the crystal lattice and possibly some of the physical properties of the inclusion complex.

As is known to the person skilled in the art, cyclodextrin itself forms an inclusion complex with water. Thus, the cyclodextrin used in the preparation of the carotenoid- cyclodextrin inclusion complex may be in a hydrated form or in an anhydrous form. A further object of the invention is to provide a method for producing an inclusion complex comprising the step of combining cyclodextrin and a carotenoid at a molar ratio of 0.3:1 to 20:1, preferably 1:1, 2:1, 2:3, 3:1, 4:1 or 5:1, 1:2, 2:3, or 3:1, most preferably 2:1 or 3:2.

The term "solution" in connection with cyclodextrin or carotenoids and in connection with the preparation of an inclusion complex is intended to comprise embodiments wherein the solute, namely cyclodextrin or carotenoid, is fully or partially dissolved in the solvent so as to form a homogenous solution, a saturated solution, a supersaturated solution, a slurry or a suspension.

In the preparation of the inclusion complex according to the present invention, the combining of the components may be done using a solution of cyclodextrin, comprising organic solvent or an aqueous solution such as water. In sensible embodiments of the invention, the solvent comprises a mixture of water and an organic solvent . The organic solvent may be selected from any of those commonly used in organic synthesis such as, but not limited to, THF, methylene chloride, diethyl ether, petroleum ether, ethyl acetate, dioxane, DMF, DMSO, acetone, acetonitrile, ethanol, methanol, pyridine, or combinations thereof. Preferably, the organic solvent is miscible with water. Polar solvents are preferred such as water, methanol, ethanol, DMSO, DMF, and pyridine, most preferably water or ethanol, particularly water.

A solution of cyclodextrin, as described supra, in any concentration or degree of homogeneity, may be combined with solid a carotenoid. Alternatively, the cyclodextrin solution may be combined with a solution of a carotenoid. In the embodiment where a solution of cyclodextrin is combined with solid carotenoids, the carotenoids may be in micronized form. In the embodiment where a solution of cyclodextrin is combined with a solution of a carotenoid, the carotenoid may be fully or partly dissolved in an organic solvent or water. Organic solvents may be selected from any of those known to the person skilled in the art such as, but not limited to, THF; methylene chloride, diethyl ether, petroleum ether, ethyl acetate, dioxane, DMF, DMSO, acetone, acetonitrile, ethanol, methanol, pyridine, or combinations thereof.

It follows that a solution of a carotenoid, as described supra, in any degree of homogeneity and in any concentration may be combined with solid cyclodextrin in the preparation of an inclusion complex between cyclodextrin and a carotenoid.

Alternatively, solid carotenoids and solid cyclodextrin may be combined in their solid forms and then combined with water or an organic solvent .

In a preferred embodiment of the invention, a method of producing an inclusion complex comprises the steps of dissolving cyclodextrin in water, optionally with the aid of heating, to form a cyclodextrin solution; dissolving astaxanthin or a source of astaxanthin in a solvent selected from the group comprising of water and ethanol or mixtures thereof, optionally with the aid of heating, to form a astaxanthin solution; combining the cyclodextrin solution and the astaxanthin solution to form a combined solution; stirring the combined solution, preferably while keeping the solution at or below 25°C; filtering the resultant precipitate; washing the precipitate with a solvent selected from the group consisting of water, ethanol, ether and acetone, preferably wherein the solvent is cooled to below 25°C; optionally suspending the resultant solid in a solvent, preferably acetone, and washing the suspended material with a solvent selected from the group consisting of water, ethanol, ether and acetone, preferably wherein the solvent is cooled to below 25°C; removing substantially all of the solvent from the solid material. Preferably, the solvent is removed by spray drying or alternatively by lyophilization.

The method of preparation may further comprise mechanical mixing, agitation or shaking, or heating of the solutions or combined components .

A typical preparation of the carotenoid-cyclodextrin inclusion complex may be as follows : The carotenoid is dissolved in a solvent such as acetone or ethanol . The cyclodextrin is dissolved in water between 20 and 100°C, such as between 30 and 90°C, such as between 40 and 80°C, preferably between 40 and 60°C, such as at or near 40°C, 45°C, 50°C, 55°C or 60°C. The carotenoid solution is added to the cyclodextrin solution and the obtained suspension is stirred at 20-30°C for some hours, such as about 0.5 to 48 hours, then stirred at 2°C for some hours. The crystallized product is isolated and dried. In an alternative process, the carotenoid solution is added to the cyclodextrin solution and the obtained suspension is stirred at temperatures below 25°C.

The crystallised product may be washed with water, acetone and/or any other solvent in order to wash off non-complexed material. The solvent used to wash the crystallised product may be pre-cooled to below 25°C. This crystallised product may be dried over a drying agent such as P₂0₅ or any other known to the person skilled in the art in a vacuum dessicator or cabinet for several hours or days . It may also be cooled in the dessicator during drying, or undergo spray drying or lyophillization.

The cyclodextrins may further be polymerised into aggregates comprising e.g. more than 1 cyclodextrin such as aggregates of 2, 5, 10, 20, 50, 100 or 1000 cyclodextrins. As stated, the present inventors have contributed significantly to the art by improving the functional utility of colored carotenoids such as astaxanthin by complexing it with a cyclodextrin. Such complexes may have numerous applications of which the use as a pigment or a feed supplement for animals may be of particular interest. However, the use of such complexes as a food supplement or a dietary supplement is also anticipated by the present invention. Thus, in various aspects, the invention relates to the use of a complex of the present invention as a pigment, food supplement, feed supplement or a dietary supplement . Such complexes may further comprise one or more component (s) which typically is en excipient, an antioxidant, a pigment, a vitamin, a flavoring agent or a nutrient. Bulk-forming or carrying materials may also be included with the carotenoid/cyclodextrin composition according to the present invention.

Yet another aspect of the present invention relates to a composition comprising i) a complex of a cyclodextrin and a carotenoid, said complex being one of the embodiments mentioned supra; and ii) one or more acceptable excipients for preparing a composition, which typically could be a food product, a feed product, a food/feed supplement, a pre-mix or a dietary supplement .

The composition may further comprise one or more component (s) selected from the group consisting of antioxidants, pigments, vitamins, flavors and nutrients.

The term "food product" is related to products for human intake and consumption.

The term "feed product" is related to products for animal intake and consumption. The term "food supplement" is related to a product for human intake which is not necessarily a food product but is supplementary to the food product by having a supplementary function. The supplementary may be e.g. nutritional (e.g. vitamins, proteins, lipids, minerals, carbohydrates, etc.) or cosmetic such as affecting the pigmentation of the human's skin and/or flesh.

The term "feed supplement" is related to products for animal intake which are not necessarily food but may be supplementary to the food by having a supplementary function. This supplementary function may be e.g. nutritional or cosmetic such as affecting the pigmentation of the animal's skin and/or flesh.

The term "pre-mix" is related to a composition that comprises a complex of the invention and is to be mixed with a food or feed product before use .

The term "dietary supplement" relates to a composition that is a supplement to the normal food diet or feed diet of an animal individual .

The term "excipients" is intended to mean any substance that is required in the formulation of a food/feed product, food/feed supplement, a pre-mix, a cosmetic or a pharmaceutical . Typical excipients are binding agents (e.g. pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen sulphate) ; suspending agents (e.g. sorbitol syrup, cellulose derivatives, dextran or hydrogenated edible fats) ; emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g. fixed oils, sesame oil, ethyl oleate or triglycerides almond oil, oily esters, ethyl alcohol or fractionated vegetable oils) ; and preservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid) , carbohydrates, fats, oils, coating agents (e.g. as described infra) , water, saline, dextrose solution and other aqueous physiologically balanced salt solutions.

The term "antioxidant encompasses some acids, for example ascorbic acid or citric acid, which help diminish oxidative discoloration of fruits and meats, phenolic compounds for example BHA and tocopherols. This category further includes BHA (butylated hydroxyanisole) , BHT (butylated hydroxytoluene) , propyl gallate, TBHQ (tert-butyl hydroquinone) , lecithin, gum and resin gulac, THBP (trihydroxybutyrophenone) , thiodipropionic acid and dilauryl thiodipropionate, glycine; carotenoids as described above (e.g. vitamin A, vitamin E and astaxanthin); selenium and co-enzyme Q10.

The term "vitamin" encompasses vitamin A; vitamin D; vitamin E; vitamin K; vitamin C; thiamin, thiamine, or vitamin Bl; riboflavin or vitamin B2 ; niacin; vitamin B₆; folacin; vitamin B₁₂; pantothenic acid or pantothenate; and biotin. Examples of vitamins which have normal food additive uses include ascorbic acid (vitamin C) , riboflavin and beta carotene (a form of vitamin A) .

The term ' flavoring agent ' comprises additives that give either the complex or the composition a flavor suitable for the given application. This could be feed flavors such as fish feed flavor, cattle feed flavor, pig feed flavor or poultry feed flavor.

The term 'nutrients' encompasses both mineral nutrients and vitamins and molecules that can be metabolized to give energy. Examples are proteins, fats (e.g. polyunsaturates, omega-6 polyunsaturates, linoleic acid, omega-3 polyunsaturates, alpha-linolenic acid, monounsaturates, saturates, trans fatty acids and cholesterol) , carbohydrate (e.g. sugars, sugar alcohols, starch, dietary fiber), vitamin as described above and minerals (e.g. Sodium, Potassium, Calcium, Phosphorus, Magnesium, Iron, Zinc, Iodide, Copper, Chloride, Manganese, Chromium, Selenium and Molybdenum) .

The term "animal" is denoted to mean any kind of an animal including a human. This includes farm animal such as chickens and hens, cattle, pigs, sheep and marine animal as well as animals found in nature. In a particular embodiment of the invention, the animal is a marine animal, such as of a Salmonids species, i.e. salmon and trout.

As stated the complex may be provided in the form of a composition typically including other suitable components. In the composition the complex is typically present in an amount in the range of 0.001% w/w to 99.999% w/w, preferably in the range of 0.001% w/w to 90% w/w, more preferably in the range of 0.001% w/w to 80% w/w, even more preferably in the range of 0.002% to 70% w/w, still more preferably in the range of 0.005% to 60% w/w, most preferably in the range of 0.005% to 50% w/w. Furthermore, the complex is typically in an amount of the at least 0.001% w/w, preferably of the at least 0.005% w/w, more preferably of the at least 0.01%, 0.05% or 0,1% w/w, even more preferably of the at least 0.5%, 1%, 2% or 5%w/w, most preferably of the at least 10%, 20%, 30%, 40% or 50% w/w.

Compositions may be in liquid form, semi-liquid form, solid form or semi-solid form. Given, the use of a complex as a pigment or a feed supplement, the complexes or compositions of the invention is formulated into pellets .

A solid composition may for example be pellets, powders, granules, beads with or without coatings. The coatings may comprise coating agents such as collagen, gelatin, fractionated gelatin, gelatin derivatives, collagen hydrolysates, plant proteins, plant protein hydrolysates, elastin hydrolysates, albumins, agar-agar, gum Arabic, pectins, tragacanth, xanthan, natural and modified starches, dextrans, dextrins, maltodextrin, chitosan, alginates, cellulose derivatives, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid and polymers of methacrylic acid and methacrylic acid esters, lipid and fats (e.g. polyunsaturates, omega-6 polyunsaturates, linoleic acid, omega-3 polyunsaturates, alphalinolenic acid, monounsaturates, saturates, trans fatty acids and cholesterol); and their mixtures.

A composition in a semi-liquid form may comprise a slurry of solid components in a liquid mixture. The liquid part could both be aqueous, organic or a two phase system of an aqueous and an organic phase .

A composition may be all liquid form, and have either an organic solvent or an aqueous solvent or a two phase system consisting of both organic and aqueous solvents or even a mixture of an aqueous solvent and a fully or moderately water miscible organic solvent .

A composition may also be a liquid, semi-liquid or semi- solid material coated with a substantially solid coating, e.g. chosen from list of coating agents above.

In an interesting embodiment the complex or the composition is coated or packaged in such a way that the complex is protected against moisture and does not dissolve when it is contacted with the water but selectively dissolves inside an animal e.g. when reaching the intestines or the gut lumen of e.g. a fish. This may be accomplished by choosing a coating that may be selectively degraded when reaching the target zone of the animal (for example it may be enzymatically degraded by enzymes or dissolve by the acids in the fish gut lumen) .

As stated, the present complexes of the invention possess numerous applications of which some are of particular interest and found advantageous . One such application relates to the modification of pigmentation in an animal in that the presently preferred modification of the pigmentation is to increase the pigmentation. Upon administration of said cyclodextrin complex of a carotenoid, the pigmentation may be increased at least by a factor of 1.2.

An increased pigmentation relatively to the pigmentation following administration of un-complexed carotenoid may be produced, and said pigmentation may be determined by measuring the UV/VIS absorbance at 470 nm of a methanolic extract of 50 g animal tissue in 100 ml methanol. Alternatively the pigmentation may be determined as described in the examples . In further interesting embodiments thereof, the pigmentation is increased by a factor of the at least 1.3, 1.4 or 1.5, preferably of the at least 1.6, 1.7 or 1.8, more preferably of the at least 1.9, 2.0 or 2.5, most preferably of the at least 2.8, 3.0, 3.5 or 4. However, the more applicable aspect of the invention is rather an increased stability of the complex according to the invention (as disclosed infra) . Thus an increased coloration of the flesh and muscle of the fish is a secondary (albeit an advantageous) effect of the complex according to the invention.

The effect of the complex or the composition on the pigmentation may also be measured using a reflectance measurement directly on or in the tissue or body fluid of the animal . The reflectance measurement measures how much light that is absorbed from the surface layers of a materiel . The reflectance measurement should be set up to cover the absorption wavelength range relevant for the given pigment. An increased reflectance means an increased amount of color absorbing pigment. Compared to determination of pigmentation by extraction, the reflectance approach has the advantage that it is better correlated with the color changes in tissue or body fluid as seen by the human eye. A simple alternative for semi-quantification of the pigmentation is use of a fan with strips of increasing color intensity such as the salmofan (TM) from Roche. The strips of the salmofan have an increasingly red intensity. The pigmentation of a salmon fillet is determined by comparing the color of the fillet to the various strips and choosing the strip with the closest match. The pigmentation level is printed on the strip. Preferably the comparison is performed using a standardized light source such as D75 Bulbs (color temperature 7500 K) or using a white, grey or black background.

There exist several issues to be taken into account when considering the uptake of a substance in the tissues of an organism. Some of them are indicated infra. Firstly, the suitability of the complexing agent will have to be proven. Any complexing agent may either form an unsuitable complex (e.g. be harmful or even toxic to the relevant animal) or may render the active compound (carotenoid, e.g. astaxanthin) inoperative. Furthermore, the complexing agent will have to make the complex bioavailable, i.e. the complex will have to be taken up by the gut and must not produce harmful cleavage products. Also, the active agent (carotenoid) will have to be routed to the relevant tissue, and when finally present in this tissue, the agent/complex will have to be active providing its effect at the relevant site in the tissue. All these considerations taken into account, a complex of an active agent may be said to be an improved complex if its effect is not poorer than the previously known, non-complexed one.

Also, environmental factors such as the health, activity, age, species, temperature etc. will have to be taken into account when considering the effect of a substance on a living organism.

Pigmentation of salmonid fishes is affected by factors such as dietary pigment source, the dosage level, duration of feeding, by dietary composition, and the physiological status of the fish. Differences in effects of these factors among salmonid fish species are matters of magnitude rather than matter of principle. Thus, astaxanthin has been regarded as the most effective carotenoid used for pigmentation of salmonid fishes, even though the degree of utilization is different between species. In rainbow trout cantaxanthin results in lower pigmentation-values than astaxanthin at a given concentration. However, recent data indicate that efficacy of cantaxanthin and astaxanthin as muscle pigmentation sources may be different in various species. Thus a negative linear relationship was found between dietary proportions of astaxanthin in a mixture with cantaxanthin and total muscle carotenoid deposition in Atlantic salmon.

In salmonid fishes the dose-response relationship between dietary astaxanthin and muscle pigmentation show a clearly diminishing yield of astaxanthin with increasing dietary dose . Conversely, the retention of astaxanthin and cantaxanthin drops substantially with increasing dose.

Similar dose-response relationships are observed for muscle astaxanthin and cantaxanthin in rainbow trout . As with rainbow trout little extra flesh pigmentation is gained by using doses higher than ca. 50-60 mg/kg in diets for Atlantic salmon. In a 4 weeks pigmentation trial with different carotenoid sources in diets for rainbow trout a good dose-response relationship for muscle carotenoids was found by using two dietary levels of the algae Phaffia rhodozyma (based on an article by Choubert et al . (1995)) .

Thus a result of pigmentation not showing any substantially decreased effect between artificial, natural and complexed astaxanthin would prove the bioavailability and overall utility of the complex between the carotenoid (astaxanthin) and cyclodextrin according to the present invention. A test conducted for investigating the pigmentation of salmon flesh was conducted. When considering the parameters to be investigated, four such parameters are of importance :

1. Pigmentation (Roche scale and video color analysis)

2. Concentration of astaxanthin in muscle

3. Stability of the pigment in storage

4. Retention/bioavailability

1. Pigmentation • Roche scale: It is expected only marginal differences in visual pigmentation/coloration (Roche scale) between the group receiving test feed (CD) and the control group receiving conventional feed with un-complexed astaxanthin after 30 days. The test is conducted during only 2 months and the start and end weights is consequently small compared to ordinary slaughter weight (2-4 kg) .

• In a simulation of pigmentation at 40 or 60 ppm in the feed there is only observed marginal effects on the Roche scale (Fig 1) .

Fig. 1 shows the coloration (Roche scale) at a starting weight of 200 g and 2 months of feeding, 40 or 60 ppm.

• Video color analysis (a*, b*, L*) . The red color intensity (a*) correlates well with the astaxanthin concentration in muscle, but the correlation is curve linear and has partly a large inter-individual variation in Atlantic salmon.

• Preliminary data after 30 days show a tendency of increased red coloration (a* = approx. 48) by the CD-feed as compared to the control feed (a* = approx. 43) . This may indicate a larger retention of astaxanthin in muscle by CD than control feed. 2. Astaxanthin in muscle

• A simulation of uptake of astaxanthin at different dosages according to the setup of the test (Fig. 2) shows that 60 ppm of astaxanthin in the feed gives a 64% higher concentration of astaxanthin in muscle after 12 months in relation to 40 ppm.

• The complexing of astaxanthin with cyclodextrin is expected to give an effect similar to the one being observed after having increased the dosage of astaxanthin in the feed (provided the dosage is in or about the linear dose-response area) . (In comparison the prior art feed gave a 52% increase in the retention of astaxanthin, see item 4. )

Figure 2 shows a simulation of the uptake of astaxanthin in muscle (at a starting weight of 200 g and 2 months feed) at 40, 50 or 60 ppm.

A carotenoid may be more stable, e.g. storage stable at ambient temperatures and stable towards oxidation in air and in water upon providing it as a cyclodextrin complex. Therefore, another aspect of the invention relates to a method for improving the stability of a carotenoid, comprising complexing the carotenoid with cyclodextrin to form a complex of the invention. The invention further comprises such a cyclodextrin-complexed carotenoid per se. In suitable embodiments thereof, the stability of said carotenoid is such that when said cyclodextrin complex of said carotenoid is stored at a temperature in the range of 2-8°C and at a relative humidity of the normal range in the dark for 3 months, the recovery of said carotenoid is at least 99.5, 99.0 or 98.5 % w/w, preferably at least 98, 97 or 95% w/w, more preferably at least 94, 92 or 90% w/w.

Given that the complex of the carotenoid and the cyclodextrin is processed into for example a feed supplement, said complexed carotenoid may be more stable towards degradation during the manufacturing process and the following storage than uncomplexed carotenoids .

The complexed form of a carotenoid may also protect the carotenoid from extensively metabolism upon administering such a complex to an animal such as a human or a fish like a salmon or a trout . In the event where such complexes are administered to a salmon or trout or the like, the bioavailability may be improved, at least in part because of improved stability and/or decreased metabolism of the un-complexed carotenoid. Thus, an interesting aspect of the invention relates to a method for modifying the bioavailability of a carotenoid in an animal comprising the steps of complexing said carotenoid with a cyclodextrin and administering the thus formed complex to the animal .

The term "bioavailability" is intended to mean the molar fraction of a carotenoid that has been absorbed from the gut lumen upon administration of said carotenoid to an animal. As used herein, the active form of a carotenoid relates to an un-metabolized carotenoid or if the metabolized carotenoid has pharmacological or pigmentation properties comparable to that of the un-metabolized carotenoid, the active form may include such a metabolized carotenoid. The bioavailability in humans may typically be determined upon administration of a single dose and measuring the fraction of un-metabolized carotenoid in the urine. In fish such as a salmon or a trout, the bioavailability may be determined as the fraction of the ingested carotenoid that has been recovered in un- metabolized form in the gut-lumen, body-fluids and in the tissues. According to the invention, the said modified bioavailability is such that the relative bioavailability of said carotenoid, as determined upon administration of said complexed carotenoid versus administration of an equivalent carotenoid in uncomplexed form to an animal, is of the at least 1.05, 1.1 or 1.2, preferably of the at least 1.3, 1.4 or 1.5, more preferably of the at least 1.6, 1.7 or 1.8, even more preferably of the at least 1.9, 2.0 or 2.5, still more preferably of the at least 2.8, 3.0, 3.5 or 4, most preferably said relative bioavailability is of the at least 5, 6, 7, 9 or 10.

As follows, upon improving the overall bioavailability of the carotenoids of the invention, the retention of said carotenoids in animal tissues, body fluids and/or egg yolks may also improve upon administering a carotenoid. The term "retention" is intended to mean the molar fraction of a carotenoid in un-metabolized form that is recovered in an animal tissue upon administering a dose of a carotenoid.

Accordingly, a further aspect of the invention relates to improved retention of said carotenoid in an animal tissue. Specifically, said increased retention is such that the relative retention of said carotenoid in an animal tissue, as determined upon administration of said complexed carotenoid versus administration of an equivalent carotenoid in uncomplexed form, is increased a factor of

1.05, 1.1 or 1.2, preferably by a factor of the at least 1.3, 1.4 or 1.5, more preferably by a factor of at least

1.6, 1.7 or 1.8, even more preferably by a factor of the at least 1.9, 2.0 or 2.5, still more preferably by a factor of the at least 2.8, 3.0, 3.5, 4, 4.5. Most preferably said relative retention is increased by a factor of the at least 5, 6, 7, 8, 9 or 10.

As stated the composition according to the invention may be formulated into food product, a feed product, a food/feed supplement, a pre-mix or dietary supplement using one or more acceptable excipient(s) as discussed supra and such compositions may further comprise one or more antioxidants, pigments, vitamins, flavors and/or nutrients. Typically examples of such further components are given above.

However, according to the invention, such complexes may also be applicable for administering to a human. That is to say that complexes or compositions of the invention may be the subject of the preparation of a medicament for treating an animal such as a human against conditions, symptoms and/or diseases related to oxidative stress in an animal, such as eye ailments including age related macular degeneration, cardiovascular diseases including hypercholesterolemia, hypertriglyceridemia, other hyperlipidemias, atherosclerosis, coronary heart disease, angina pectoris, thrombosis, myocardial infarction, hypertension, inflammatory conditions and ageing of skin.

EXAMPLES

Procedure for Muscle Astaxanthin Determination Method:

Concentration range : C > 0,1 mg/kg Sample : Salmon muscle

Equipment :

Analytical balance, Mettler, model AB204 Rotavapor. Heidolph, model LABOROTA 4000 HPLC: Hewlett Packard. Model HP 1100

Spectrophotometer Vis, SPECTRONIC 20 GENESYS M/4001-4 Homogenizador de tejido, DREMEL MULTIPRO Moledora Moulinex Votex TERMOLINE MAXIMIX II

Reagents and solutions

Hexane, grado HPLC Acetone, grado HPLC

Chloroform, grado HPLC Astaxanthin >98%, Sigma Chemical

Procedure Sample preparation:

Mill the samples and be sure of the homogeneity. Weigh 5,0 g of the sample and put it in an adjusted to weight tube of 50 ml, register the weight with four decimals. Add 5 ml of distilled water to the sample. Homogenize the sample, verifying that it is completely disintegrated (the sample must not turn hot) . Weigh 0,5 g of this sample in an adjusted to weight centrifuge tube of 15 ml. Register the weight with 4 decimals. Add 5 ml of chloroform. Shake with Vortex for 30 seconds. Add 3,0 ml of distilled water and shake for 30 seconds in Vortex. Centrifuge for 3 minutes. Verify the phase separation and that the tissues are discolored. Retire the water phase with a pipette and the rest of tissue with a microspatula. Transfer 2 ml of the chloroform phase to a 100 ml round bottom balloon flask and evaporate to dry in a rotavapor at 38°C. Add 4 ml of mobile phase and dissolve again with the Vortex. Filter through membrane of 0,45 μm, then inject to the HPLC.

Standard preparation Put 1 ml of chloroform in a 15 ml tube. Take out with a microspatula the amount of crystallized astaxanthin, approximately the size of an "o" letter. The weight of the crystallized astaxanthin is not necessary, because the standard curve will relate it with the 475 nm solution absorbance. Dissolve the crystalline astaxanthin in 1 ml of chloroform, using Vortex. Use a micropipette to transfer 100 μl, 75 μl, 50 μl and 25 μl of the standard solution to four tubes and dry it under nitrogen (this dry system without heating is recommended, because the heat increases the amount of cis-isomer in the standard) . Add 5 ml of mobile phase (hexane/acetone) to each one of the dried standards. Dissolve using an ultrasonic water bath. Using a 5 ml syringe, filter approximately 2 ml through the 0,2 μm filter and put it in four HPLC vials. With the rest of the solutions, read the absorbance of each one at 475 nm in the spectrophotometer, previously preparing a blank with mobile phase . The four absorbances shall be in the 0,1 and 0,6 range, when the read is made at 475 nm. If this does not happen, adjust the stock solution, and do the describe process again. The standard has to be made quickly, to prevent the formation of excessive amounts of cis-isomer, which will cause a non lineal standard curve.

HPLC Condition

Column: Silica 3 μm, 150 x 4,6 mm

Precolumn: Silica, 3,0 x 4,0 mm Mobile phase: 86:14 hexane/acetone

Flow: 1,0 ml/min.

Temperature : environmental

Injection volume: 20 μl

Detection: 475 nm Retention period: approx. 8-9 min.

Running time: 15 min.

Calibration: Standard external method, peak area.

Calculations

Determine the standard and sample area:

Total Area = trans area + (1.11 x di-cis area) + 1.2x9- cis area) + ( 1.56 x 13 cis area) + ( 1.56 x 15 -cis area)

Standard Concentration del: ppm ( standard) = (absorbance at 457 nm) x 10.000/2100

The results are expressed in mg/Kg.

Preparation Example

A working example of the process for producing an astaxanthin/cyclodextrin complex is given infra.

In the first round of production there was used 150 g "Carophyl Pink" containing 12 g astaxanthin giving 39 g of complex. It was assumed that this starting amount would be sufficient to give the required amount of complex. However, the yield proved to be lower than expected.

In the second round there was used much more "Carophyl Pink" (420 g) for producing as much complex as possible. For the process to be conductible in practice with the available equipment, the volumes were not increased correspondingly so that all concentrations were higher in round 2.

In both the series little color was left after the extraction. This means that there was little loss at the enzyme treatment and the extraction.

After evaporation the amount of dry matter was higher than the theoretical amount of astaxanthin. In the first round the dry weight was 19,7 g in relation to the total amount of astaxanthin being 12 g, and in the second round the amounts were 58 g dry weight and 33,6 g total astaxanthin in the sample, respectively. This means that contaminations were extracted over with the chloroform. It seems that these contaminations do not dissolve in acetone, because when astaxanthin is dissolved in acetone there was still some solids left. In round 1 the residue after the evaporation was dissolved in 2 1 acetone. In this case there were carried some solids into the acetone phase so that this solution had to be filtrated. In round 2 the solid residues were also washed, but in this case the solution was not shaken as intensely so that in this case there was not carried any solids over into the acetone phase. The solids being left was tried to be dissolved in water, and these proved to be partly soluble in water.

The small yield in round 1 was due to loss at the complexing step and wash. At the first centrifuging there was used 2300 g for 5 min., but this proved to be too little, and there was accordingly used 4000 g for 10 min. for all subsequent centrifugings . The supernatant after the first centrifuging was also centrifuged anew at this speed for retaining a little more of the complex being left in this solution.

In round 2 the yield was higher than expected. The cause for this may be that an excess of cyclodextrin was not properly washed out in this case. In both cases there was added a molar excess of cyclodextrin of about 1,7 in relation to astaxanthin. Since there was used the same volume of liquid in the two cases, the concentration may have become too high for obtaining a sufficient wash in the second case .

Chemicals

Round 1.

- 150 g Charophyl Pink

Dissolved in 750 ml water

Added 150 ml enzyme solution Alcalase 2.4 LFG Nitrogen gas was bubbled through the solution for about 5 min. - The solution was treated with ultrasound at 50°C for 30 min. under nitrogen atmosphere The solution was cooled, to ambient temperature Extracted with chloroform - 3 x * 300 ml The chloroform phase was washed with water - 1 x « 1 1

The solvent was evaporated in an evaporator. The evaporation was conducted under a vacuum at 20°C until most of the solvent was removed, and then the temperature was increased to 30°C - Dry weight 19,7 g

Dissolved in 2 1 acetone

Added an aqueous solution of γ-cyclodextrin - 20 g/1

Rest solids (astaxanthin + ?) washed with 1 1 acetone

Filtrated Added 1 1 γ-cyclodextrin solution (20 g/1) The solutions were bubbled with nitrogen for about 5 min.

The containers were stoppered with corks with a slip - The solutions were stored at ambient temperature overnight Centrifuged

Washed twice with water Dry weight : 13 , 8 g

Round 2.

420 g Charophyl Pink

Dissolved in 750 ml water

Added 420 ml enzyme solution Alcalase 2.4 LFG

Nitrogen gas was bubbled through the solution for about 5 min.

The solution was treated with ultrasound at 50°C ' for 30 min. under nitrogen atmosphere

The solution was cooled to ambient temperature

Extracted with chloroform - 3 x « 300 ml - The chloroform phase was washed with water -

1 x « 1 1

The solvent was evaporated in an evaporator.

The evaporation was conducted under a vacuum at 20°C until most of the solvent was removed, and then the temperature was increased to 30°C

Dry weight 58 g

Dissolved in 2 1 acetone

Added an aqueous solution of γ-cyclodextrin - 20 g/1

Rest solids (astaxanthin + ?) washed carefully with 1 1 acetone

Added 1 1 γ-cyclodextrin solution (20 g/1)

The solutions were bubbled with nitrogen for about 5 min.

The containers were stoppered with corks with a slip - The solutions were stored at ambient temperature overnight Centrifuged

Washed twice with water

Dry weight: 139 g

Upscaling

When upscaling it may not be convenient to perform the extractions with a separating funnel. Here two different principles may be used alternatively. The simplest process may be to use a continuous system fist consisting of a mixing vessel, then the phases are moved into a vessel where the phases may be separated and where the chloroform phase may be removed. The further treatment may be conducted in two ways, either be evaporation of the chloroform under vacuum (as presently) wherein the solvent is recirculated, or by a new extraction. By performing a new extraction an energy-demanding evaporation step is avoided. In this extraction it is possible to use an aqueous phase containing cyclodextrin, and then it may be possible to transfer the astaxanthin to the aqueous phase and get the complex formed directly in the same continuous process.

It may also be possible to perform the extraction in a column. In this system the extraction is performed in a closed system, something which may be advantageous when using volatile solvents. However, the process is somewhat more complicated in this instance, and thus harder to optimalize .

With the exception of the enzyme reaction, which is conducted at 50°C, the entire process may be performed at ambient temperature, even when performing an optional evaporation of the solvent which ought to be done at a vacuum and not with an increased temperature. Concerning other factors the oxygen may be removed form the solutions by the aid of nitrogen, and the system may be covered to prevent being affected by light. Example 2 ; Testing¹ of Astaxanthin Content in Salmon from the Complex According to the Invention in Comparison to Commercially Available Astaxanthin.

The test was conducted in the period March 10 to May 22, 2003. The salmon (Salmo salar) being used in the test was hatched in January, and fed initially in March 2002. The fish was kept at a natural day cycle through the summer and autumn, became young fish in the middle of February by the day length initiating the evolvement was speeded up to December 17. Then the day length was increased from 6 hours light: 18 hours darkness to 24 hours light, and the fish received light continuously through the test. The water temperature varied naturally through the summer and autumn, but was adjusted to a constant 12°C prior to the increased length of the light cycle. The fish grew in this period from an average of 41 g to 135 g (December 17, 2002 to March 10, 2003) , which also is the initial weight of the salmon at the beginning of the test . From the starting of the feeding and up to the starting of the test the tested fish received feed without any added pigment with an astaxanthin level in the feed less than 5 mg/kg.

Individually branded fish were divided in 6 circular basins of 500 liters each (80-83 fish per basin) with a water flow adjusted to 20 liters/min. and a current of 19-20 cm/sec. The water flow ensured an oxygen saturation of at least 80% fully saturated water, and has thus not been limiting for the growth. The current speed determines the swimming capacity of the fish. It has been proven that a certain swimming capacity has a positive effect on the growth. This is due to t purely exercising effect (muscle building) , reduced social hierarchies and aggression, and a better distribution of the feed in the basin. If these effects are to exist the swimming speed has to be over 0,5 body lengths per second. In the test the swimming speed was between 0,9 and 0,65 body lengths per second through the growth period of the fish. The water temperature was kept constant at 10°C throughout the test.

The test feed was extruded and has a composition close to the current commercial standard (Table 1) . "Carophyll Pink" (Hoffmann-La Roche) was added prior to the extrusion of the control feed, whereas the cyclodextrin-stabilized pigment according to the present invention was coated on after the extrusion. The added astaxanthin in the feed composition was adjusted for loss over the extruder so that the content thereof was identical in the two types of feed (Table 1) . The fish were fed daily for 6 hours (03.30 - 09.30) . The daily intake of feed was registered at a basin level during the week by feed collectors, as disclosed previously (Bendiksen et al . (2002). The specific growth rate of the fish was calculated based on measurements of the individual weight at the start of the test, at day 28 and at day 65 (test finish) .

Table 1 The composition of the feed types being used in the test described supra. With the exception of the pigment source, the test feeds are practically identical.

When measuring the individual weights during the test (day 28) , half of the fish in each basin were sacrificed for the taking fecal samples, the measuring of organ indexes (liver, gonads and intestines) , as well as the measuring of color in muscle (Roche scale (Salmo-fan) and Minolta Croma Meter CR-200, Minolta Camera Co. Ltd, Osaka, Japan) . The Croma Meter registers the light spectrum being reflected from the surface of the sample by exposure to light with a standard spectral distribution. The color system CIE 1976 L'a*b* space (CIE, 1986), being the one mostly used on fish, was used in the test. In this system the color in a three-dimensional coordinate system wherein the axes represent the brightness (L*) , red/green (a*) and yellow/blue (b*) . The fecal samples were dissected from the rear intestine of all the fish having been sacrificed. Possible changes in the energy metabolism were evaluated by calculating organ indexes for the first 15 individuals, and from 3 of these the content of astaxanthin in their filet was analyzed. Since the amount of astaxanthin may be affected by the size of the fish, the filet samples were collected from fish of different weight to correct the subsequent result for size.

The digestibility of the feeds was evaluated by measuring the remaining amounts of fat (measured by Folch' s method) , protein (Dumas combustion method) , dry matter content by drying to a constant weight, and the content of ashes calculated as a glow rest. The analysis for ash being undissolvable in acid was used to calculate the feed intake being the basis for the digestibility studies.

RESULTS AND DISCUSSION

Fish size and growth All of the fish grew well with little variations throughout the test (Fig. 3) . There were no significant differences in weight between the groups (Fι,ι₂=0, 848, P=0,453) or growth rate (F_1/8=0,041, P=0,844) at any time. In the first part of the test (day 0 to 28) the control group had a daily added growth of 1,96% of the body weight, whereas the group receiving feed according to the invention grew by 2,01%. In period 2 (day 29 to 65) the added growth was somewhat reduced as expected on account of the increased body size, but it was still high (1,41% and 1,45% in the control versus test group, respectively) . The suggestion of a better growth rate in the test group is not significant and is mainly due to a very good growth rate in one replicate. The added growth lies in the upper section of the expected added growth. In studies such as this it is important that the fish takes out its growth potential so that assimilation and metabolizing of the nutrients and other feed ingredients progresses effectively. A stable and high added growth also gives a large weight increase, and fast changes is advantageous when individual treatment effects are to be evaluated.

Figure 3. Weight development in salmon during the pigmentation test . The control group is to the left and the test group is to the right in the figure . Mean values with the variation is shown as the standard deviation.

Feed intake and feed factor

The good added growth reflected a stable and high feed intake in both groups . The control group ate as a mean 1,65% of their body weight per day from the test start to day 28 (19 days of feed assimilation measurements) . The corresponding value for the test group given feed according to the present invention was 1,73% of their body weight (Fig. 4). This gave a factor of 0,84 and 0,86 respectively in the control and the test group. In the second period the results were again consistent with the growth results: The control group ate as a mean value 1,38% of their body weight, whereas the test group again did lie somewhat above this with 1,51% (22 days with feed intake measurements) . This gave a feed factor of 0,99 and 1,04, respectively. The fish has consequently eaten and grown well, and have additionally a relatively good feed factor. There were no significant differences in the feed intake (Fι,₈=0,187, P=0,677) or feed factor (Fι,₈=0,496, P=0,501) between the groups at any time in the test.

Figure 4. Mean feed intake measured at the basin level in salmon provided feed containing two different pigment sources over two periods. Control feed to the left and test feed to the right . The first period is based on 19, whereas the second period is based on 22 measurements of the feed intake . Mean values with variation given as a standard deviation.

The different feed types, which in their composition are very similar, except for the addition of cyclodextrin, gave as expected no significant differences in growth or feed intake throughout the test.

However, given the number of factors influencing the efficacy of fish feed, it may be that given the better consumption of the feed according to the present invention, also a better coloring of the flesh in salmonides may be obtained.

ORGAN INDEXES

Color

Roche scale .

At the test start the fish in both groups had a muscle color lower than the Roche scale for salmon (<20) , and the astaxanthin content was below the detection limit of 0,5 mg/kg. The basis for the test was thus good. After 28 days the control fish had a mean color score on the Roche scale of 21,5, whereas the test group given the feed according to the present invention did lie on 20,7 (Fig. 5) A GLM model revealed statistical differences between the groups

45; P=0,000) wherein the size of the fish contributed significantly, but very poorly to explain the coloring. A corresponding Roche score after 65 days was 26,0 and 23,8 for the control and test group, respectively. Here the difference was significant as well (F_ι#238=144, 53 ; P=0,000), and again the coloring was positively correlated to the fish size, but not in interaction with the treatment. This was neither expected since the size of the fish did not differ between the groups.

Figure 5. Coloration of muscle in salmon fed with two identical diets, but wherein the astaxanthin was bound in different manners. The color measurements have been performed with Roche Salmo-Fan. Control to the left and test group to the right . At day 0 both groups were below the scale (<20) . Asterisks indicate significant differences between the treatments of p< 0, 001 level .

Optical color measurements

Color measurements performed with Roche's Salmo-Fan is very dependent on the light conditions and will additionally be subjective. The results from the Roche scale were consistent all the same with results taken with the optical color-measuring device from Minolta. L* represents how light the sample was with an increasing value when the reflection tends towards white. At time 0 the L* value had a mean value of 47,1. After 28 days this was reduced to a mean of 43,2 and 43,9 in the control and test groups, respectively (Fig. 6) and this difference was reasonably stable throughout the test (41,1 and 41,6 at day 65, respectively) . There were no significant differences between the groups at each point in time (Fι,₈=0,145; P=0,713), but there occurred a general reduction in both groups over time (F₁₍₈=77,56; P=0,000). a* denotes the color on a scale from green towards red, wherein the red color increases with increasing value. At day 0 a* was 0,32. This increased to 4,2 in the control group and 2,9 in the test group during 28 days (Fig. 4) . At the end the red color had increased further, with an a* value of 5,9 and 4,1 in the control and test groups, respectively, b* denotes the color in a scale from blue towards a weakly yellow color with an increasing yellow color when the value rises. At day 0 the samples had a weakly yellow color (5,3), and this increased somewhat in the control group (6,5), while it was relatively stable in the test group (5,6) during the first 28 days of the test (Fig. 4). At the end of the test the component of yellow had increased somewhat in both groups (8,5 and 6,6 in the control and test groups, respectively)

Astaxanthin in muscle

The results for the measured astaxanthin in muscle in the two groups tested (see supra) were the following:

28 days : 1, control mean 1,578 mg/kg, s.d. 0,166 2 , complex mean 0,878 mg/kg s.d. 0,139

65 days :

1, control mean 2,05 mg/kg s.d. 0,725

2 , complex mean 1,478 mg/kg s.d. 0,299

Astaxanthin in faeces

The results of the measured astaxanthin in faeces in the two groups tested (see supra) were the following:

65 days

Control tub 1: 66,0000 mg/kg predrying factor 0,1801 Control tub 3: 60,0000 mg/kg predrying factor 0,1869

Control tub 5: 65,0000 mg/kg predrying factor 0,1821

Mean 63,6667 mg/kg 0,1830 Test tub 2 116,0000 mg/kg predrying factor 0,1879 Test tub 4 101,0000 mg/kg predrying factor 0,1846 Test tub 6 127,0000 mg/kg predrying factor 0,1946

Mean 114,6667 mg/kg 0,1890

STABILITY OF THE ASTAXANTHIN/CYCLODEXTRIN COMPLEX

Complexed astaxanthin and non-complexed astaxanthin was tested for stability in a period of 230 days.

The complex was manufactured as described supra.

The non-complexed astaxanthin was diluted with acetone in a 15 ml test tube with an air tight screw cap.

Complexed astaxanthin was diluted with water in a 15 ml test tube covered with stretchable microfilm on top.

The tubes was stored unprotected in lab on a bench at 20°C, exposed for indoor artificial light and normal natural daylight exposed through window.

Measurements of stability were carried out on a Shimadzu UV spectrophotometer. Samples were diluted to a final content with 1 part acetone and 1 part distilled water before measuring. As blank the same dilution with 1:1 of acetone and distilled water was used. Diluted samples were measured in 1 cm measuring cell and scanned at each evaluation. Maximum absorption in the region 450 - 500 nm was the value used for stability. Calculations of absorption were done according to original concentration.

The stability of complexed astaxanthin was superb when compared to non-complexed. The results were obvious as the characteristic red distinct colour made it possible to follow the stability test by visual inspection as well . The non-complexed astaxanthin was completely opaque transparent with out any signs to reddish or other colours at all when inspected visually, while the complexed astaxanthin was still after 230 days emitting a distinct reddish colour. The results were convincing and complexed astaxanthin showed a remarkable stability compared to non-complexed astaxanthin under the given conditions .

Example 3

The stability of astaxanthin complexed with cyclodextrin as produced according to Example 2 , was compared with commercially formulated feed ( "Carophyll Pink" , Hoffmann - La Roche)

During storage of feed for 105 days at 10°C, in the dark in plastic foiled paper bags, there was surprisingly no loss of astaxanthin in the feed containing the complexed astaxanthin compared with feed containing the commercial pigment. In the latter the loss after 105 days was 10% and hence significant .

The results from this example is shown in Fig. 7.

Claims

1. A complex of a cyclodextrin and a carotenoid.

2. The complex according to claim 1, wherein the carotenoid is selected from the group consisting of - carotenes, β-carotenes, γ-carotenes, δ-carotenes, lutein, lycopene and their isomeric forms and derivatives.

3. The complex according to any one of claims 1 or 2 , wherein the β-carotene is selected from the group consisting of R-carotene, astaxanthin, zeaxanthin, astacin, cantaxanthin and their isomeric forms and derivatives.

4. The complex according to claim 1, wherein the carotenoid is selected from the group consisting of astaxanthin, zeaxanthin, astacin, cantaxanthin, lutein, lycopene and their isomeric forms and derivatives .

5. The complex according to claim 1, wherein the carotenoid is selected from the group consisting of astaxanthin, zeaxanthin, astacin, cantaxanthin and their isomeric forms and derivatives.

6. The complex according to any one of the preceding claims, wherein the carotenoid has a solubility in water at 25°C of at the most 10 g/1, preferably of at the most 5 g/1, more preferably at the most 1 g/1, even more preferably at the most 0.5 g/1, still more preferably of at the most 0.2, 0.1, 0.05 g/1, preferably of at the most 0.01 g/1.

7. The complex according to any one of the preceding claims, wherein the carotenoid is astaxanthin, an isomer or a derivative thereof .

8. The complex according to any one of the preceding claims, wherein the carotenoid is a mixture of carotenoids.

9. The complex according any one of the preceding claims, wherein the cyclodextrin is selected from the group consisting of α-cyclodextrins, β-cyclodextrins, y- cyclodextrins and derivatives thereof .

10. The complex according to any one of the preceding claims, wherein the complex is an inclusion complex.

11. The complex according to any one of the preceding claims, wherein the carotenoid and the cyclodextrin is in a molar ratio in the range of 10:1 to 1:100, preferably in the range selected from the group consisting of 5:1 to

1:80; 5:1 to 1:50; 5:1 to 1:20; 5:1 to 1:10; 4:1 to 1:8; 2:1 to 8:2; 5:1 to 1:5; 2:1 to 1:5; 4:1 to 1:4; 2:1 to 1:4, 2:1 to 1:3, 2:1 to 1:2 and 1:1.

12. The complex according to any one of the preceding claims, wherein the complex has a solubility at 25°C in water of at the least 0.5 g/1, preferably of at the least 1, 2, 5 or 10 g/1, more preferably of at the least 15, 20 or 25 g/1, even more preferably of the at the least 30, 40 or 50 g/1, most preferably of at the least 60, 80 or 100 g/1.

13. The complex according to any one of the preceding claims, further comprising one or more component (s) selected from the group consisting of excipients, antioxidants, pigments, vitamins, flavors, nutrients and therapeutically active agents.

14. A composition comprising

i) a complex of a cyclodextrin and a carotenoid; and

ii) one or more acceptable excipient for preparing a composition selected from the group consisting of a food product, a feed product, a food/feed supplement, a pre-mix and a dietary supplement .

15. The composition according to claim 14, wherein the complex is as defined by any one of claims 1 to 13.

16. The composition according to any one of claims 14 or 15, further comprising one or more component (s) selected from the group consisting of antioxidants, pigments, vitamins, flavors and nutrients.

17. The composition according to any one of the claims 14 to 16, wherein the complex is in an amount in the range of 0.001% w/w to 99.999% w/w, preferably in the range of 0.001% w/w to 90% w/w, more preferably in the range of

0.001% w/w to 80% w/w, even more preferably in the range of 0.002% to 70% w/w, still more preferably in the range of 0.005% to 60% w/w, most preferably in the range of 0.005% to 50% w/w.

18. The composition according to any one of the claims 14 to 17, wherein said formulation is in liquid form, semi- liquid form, solid form or semi-solid form.

19. Use of a combination of a cyclodextrin and a carotenoid for modifying the pigmentation of an animal tissue and/or an animal body fluid.

20. The use according to claim 19, wherein the combination of a cyclodextrin and a carotenoid is a cyclodextrin complex of a carotenoid as defined in any one of claims 1 to 13.

21. The use according to any one of claims 19 to 20, wherein said pigmentation is increased such that upon administration of said cyclodextrin complex of a carotenoid, the pigmentation is increased at least by a factor of 1.2 relatively to the pigmentation following administration of un-complexed carotenoid, said pigmentation is determined by measuring the UN/VIS absorbance at 470 nm of a methanolic extract of 50 g animal tissue in 100 ml methanol.

22. The use according to claim 21, wherein said increase of the pigmentation is by a factor of the at least 1.3, 1.4 or 1.5, preferably of the at least 1.6, 1.7 or 1.8, more preferably of the at least 1.9, 2.0 or 2.5, most preferably of the at least 2.8, 3.0, 3.5 or 4.

23. The use according to any one of claims 19 to 22, wherein said animal tissue and/or an animal body fluid is from a marine animal .

24. The use according to claim 23, wherein the marine animal is of a Salmonids species .

25. Use of the complex as defined in any one of claims 1 to 13 as a pigment.

26. Use of the complex as defined in any one of claims 1 to 13 as a food/feed supplement.

27. Use of the complex as defined in any one of claims 1 to 13 as a dietary supplement.