i

New process for the manufacture of feed additives of carotenoids

Summary of the invention

The present invention is directed to a process for the manufacture of a feed additive comprising the following ingredients a) to e), a) at least a carotenoid; b) at least a lignosulfonate; c) at least a compound selected from hexose-dimers, modified hexose-dimers, hexose-oligomers, modified hexose-oligomers, hexose-polymers, modified hexose-polymers, and any mixture thereof, whereby further optionally at least one hexose may be present; d) at least an antioxidant, preferably a fat-soluble antioxidant; e) at least an absorbent; wherein the amount of ethoxyquin (= 6-ethoxy-2,2,4-trimethyl-1,2- dihydroquinoline) in the feed additive is ≤ 0.5 weight-%; wherein the amount of butylated hydroxytoluene in the feed additive is ≤ 0.5 weight-%; whereby both amounts are based on the total weight of the feed additive; whereby said process comprises the following steps: i) Providing a matrix by dissolving the lignosulfonate(s), the compound(s) c) and optionally a water-soluble antioxidant in water; ii) Suspending the carotenoid(s) into the matrix obtained in step i) to obtain a dispersion, preferably a suspension; iii) Milling the carotenoid in the dispersion (preferably the suspension) obtained in step ii); iv) optionally emulsifying a fat-soluble antioxidant(s) into the dispersion (preferably the suspension) obtained in step iii); v) Drying the dispersion (preferably the suspension) obtained in step iii) or iv) in presence of an absorbent to obtain the feed additive. The present invention is also directed to a feed additive obtainable by such a process. Further objects of the present invention are feed comprising such feed additive, as well as methods for pigmentation and corresponding uses of such feed additives and feed.

When such feed additive according to the present invention or a feed comprising such feed additive is administered to an animal, the administration thereof results in a desired level of muscle retention in said animal. The desired level of the carotenoid retained in the muscle leads to a pleasant, consumer-appealing flesh color similar to wild counterparts.

Background of the invention

Carotenoids are organic pigments ranging in color from yellow to red that are naturally produced by certain bioorganisms, including photosynthetic organisms (e.g., plants, algae, bacteria such as cyanobacteria), and some fungi. Carotenoids are responsible for the orange color of carrots, as well as the pink color in flamingos and salmon, and the red color in lobsters and shrimp. Animals, however, cannot produce carotenoids and must receive them through their diet. Carotenoid pigments (e.g. β-carotene and astaxanthin) are used industrially as ingredients for food and feed stocks, both serving a nutritional function and enhancing consumer acceptability. For example, astaxanthin is widely used in salmon aquaculture to provide the pink/ red pigmentation characteristic of their wild counterparts. Some carotenoids provide potential health benefits, for example as vitamin A precursors or antioxidants. Some carotenoids such as β- carotene, lycopene, astaxanthin, zeaxanthin and lutein are currently sold as nutritional supplements.

Astaxanthin is a red to reddish-orange pigment and is produced naturally in the freshwater microalgae Haematococcus pluvialis and the yeast fungus

Xanthophyllomyces dendrorhous (also known as Phaffia). When the algae is stressed by lack of nutrients, increased salinity, or excessive sunshine, it produces astaxanthin. Animals who feed on the algae such as red sea bream, flamingos and crustaceans (ie. shrimp, krill, crab, lobster and crayfish) and carnivorous fish consuming small crustaceans, subsequently reflect the red to reddish-orange astaxanthin pigmentation to various degrees. Objects of the invention

There is a need to provide a sustainable process for the manufacture of a feed additive, whereby no organic solvent is used. By avoiding the use of a solvent the energy of removing it later in the process again is saved rendering said process environmentally friendly and economic.

Furthermore, there is a need to provide a feed additive comprising at least a carotenoid or a feed comprising such feed additive which results in a desired level of muscle retention in the animal it is administered to. There is especially a need to provide a feed additive comprising astaxanthin or any derivative thereof which results in a muscle retention of at least 7% of astaxanthin in an aquatic animal as defined below; i.e. 7% of the astaxanthin amount that has been ingested by the aquatic animal is retained in the muscle. Furthermore, there is a need to provide a stable feed additive comprising at least one carotenoid which can be used for the pigmentation of animals, especially aquatic animals.

Aquatic animals in the context of the present invention encompass crustaceae and fish, preferably farmed Crustacea such as shrimp and carnivorous species of farmed fish such as salmons, rainbow trout, brown trout (Salmo trutta) and gilthead seabream.

There is a further need to provide a feed additive comprising astaxanthin or any derivative thereof which results in a muscle retention of at least 7% of astaxanthin in salmon; i.e. 7% of the astaxanthin amount that has been ingested by the salmon is retained in the muscle. Moreover, there is a need to provide a feed additive comprising astaxanthin or any derivative thereof which results in the desired astaxanthin level of 7 mg/kg in salmon, to which feed is administered which comprises the feed additive of the present invention.

There is also a need to provide a feed additive comprising astaxanthin or any derivative thereof which results in a muscle retention of at least 13%, preferably of at least 14%, of astaxanthin in rainbow trout; i.e. at least 13%, preferably at least 14%, of the astaxanthin amount that has been ingested by the rainbow trout is retained in the muscle.

Besides, there is a need to provide a feed additive comprising astaxanthin or any derivative thereof which results in the desired astaxanthin level in shrimp, to which feed is administered which comprises the feed additive of the present invention.

In addition, there is a need to provide a feed additive which is “animal-free" meaning that it does not comprise any ingredient from animal origin. Detailed description

This need is fulfilled by the present invention, which is directed to a process for the manufacture of a feed additive comprising the following ingredients a) to e), a) at least a carotenoid; b) at least a lignosulfonate; c) at least a compound selected from hexose-dimers, modified hexose-dimers, hexose-oligomers, modified hexose-oligomers, hexose-polymers, modified hexose-polymers, and any mixture thereof, whereby further optionally at least one hexose may be present; d) at least an antioxidant; e) at least an absorbent; wherein the amount of ethoxyquin (= 6-ethoxy-2,2,4-trimethyl-1,2- dihydroquinoline) in the feed additive is ≤ 0.5 weight-%; wherein the amount of butylated hydroxytoluene in the feed additive is ≤ 0.5 weight-%; whereby both amounts are based on the total weight of the feed additive; whereby said process comprises the following steps: i) Providing a matrix by dissolving the lignosulfonate(s), the compound(s) c) and optionally a water-soluble antioxidant in water; ii) Suspending the carotenoid(s) into the matrix obtained in step i) to obtain a dispersion; iii) Milling the carotenoid in the dispersion obtained in step ii); iv) Optionally emulsifying a fat-soluble antioxidant(s) into the dispersion obtained in step iii); v) Drying the dispersion obtained in step iii) or step iv) in presence of an absorbent to obtain the feed additive.

Since no organic solvent is used in this process of the present invention, the process is sustainable, economic and environmental-friendly. Furthermore, the feed additive obtainable by such a process fulfills the needs in the market. The feed additive of the present invention is especially resulting in the desired muscle retention level in the animal to which it is administered in form of a feed comprising such feed additive according to the present invention.

The feed additive of the present invention is also resulting in the desired astaxanthin level of 7 mg/ kg in rainbow trout, to which feed is administered which comprises the feed additive of the present invention.

Furthermore, the feed additive of the present invention shows the necessary stability.

Besides, the feed additive of the present invention shows a low filtration residue, preferably a filtration residue ≤ 5%, more preferably ≤ 3%, most preferably ≤ 1% (see Table 3). The filtration residue is used to assess the quality of the feed additive by re-dispersing ca. 1 g of feed additive in water, filtering it through a filter paper (pore size 4-12 μm) and a filter aid, and washing the filter with water. The fraction remaining in the filter is recovered and determined by spectrophotometry (K. Schiedt and S. Liaaen-Jensen, Isolation and Analysis. In: G. Britton, S. Liaaen- Jensen, H. Pfander (Eds.). Carotenoids, Volume 1A: Isolation and Analysis;1995 Birkhauser Verlag Basel, Switzerland).

Further objects of the present invention are feed comprising such feed additive, as well as methods for pigmentation and corresponding uses of such feed additives and feed.

Advantageously, the feed additives of the present invention do not comprise any ingredient from an animal source such as e.g. beeswax which is in discussion because of increasing levels of pesticide residues. Thus, the feed additive of the present invention is animal-free.

Preferably the lignosulfonate(s) and the compound c), preferably being (modified) starch hydro lysate(s), form a dense and glassy matrix as mixture of compounds of varying molecular size and featuring different functional groups.

The single compounds of the feed additive according to the present invention and their amounts may be determined as follows:

Compound a): Carotenoid(s) The carotenoid can e.g. be extracted and analyzed by HPLC-DAD (High Performance Liquid Chromatography Diode Array Detection) or HPLC-FL (High Performance Liquid Chromatography-Fluorescence Detection) according to the following published method:

W. Schüp, J. Schierle, Carotenoids, Volume 1A: Isolation and Analysis; Editors: G. Britton, S. Liaaen-Jensen, H. Pfander; Birkhauser Verlag Basel (CH), 1995.

Compound b): Lignosulfonate(s) The lignosulfonate(s) can be spectrophotometrically determined in formulations e.g. according to a procedure disclosed by G. Jayne and E. Pohl in Das Papier, 1967, 21, Vol. 10A, pages 645-653 (“Nachweis der Ligninsulfonsaure in grosser Verdünnung (Abwasser von Sulfitzellstoff-Fabriken)").

Compound c): Starch hvdrolvsate(s)

Starch hydrolysates can be analyzed by size-exclusion chromatography; see e.g. White DR Jr, Hudson P, Adamson JT in Journal of Chromatography A 2003, 997(1-2), pages 79-85 (“Dextrin characterization by high-performance anion-exchange chromatography— pulsed amperometric detection and size-exclusion chromatography— multi-angle light scattering-refractive index detection.").

Compound d): Antioxidant(s)

Antioxidants can be analyzed by HPLC-DAD/FL (High Performance Liquid Chromatography- Diode Array Detection/ Fluorescence Detection) as e.g. disclosed by Paula Becker Pertuzatti, Marla Sganzerla, Andressa Carolina Jacques, Milene Teixeira Barcia, Rui Carlos Zambiazi, in LWT - Food Science and Technology 2015, Volume 64, Issue 1, pages 259-263 (“Carotenoids, tocopherols and ascorbic acid content in yellow passion fruit (Passiflora edulis) grown under different cultivation systems").

Compound e): Absorbent(s)

The coating with the absorbent can be qualitatively characterized using microscopic techniques coupled with spectroscopic techniques such as FTIR (Fourier Transformation Infrared) for identification of starches and X-ray fluorescence for silicium dioxide; see e.g. P.V. Kowsik, N. Mazumder, Microsc. Res. Tech. 2018, 81, pages 1533-1540 (“Structural and chemical characterization of rice and potato starch granules using microscopy and spectroscopy.") and M. Mutsuga, K. Sato, Y. Hirahara, Y. Kawamura, Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 2011, 28(4), pages 423-427 (“Analytical methods for SiO and other inorganic oxides in titanium dioxide or certain silicates for food additive specifications"). For the determination of the other absorbents corresponding analytical methods are known to the person skilled in the art. Details of the single compounds and their amounts in the feed additive according to the present invention are given below.

Compound a): carotenoids

In the present invention, examples of the carotenoid include naturally-occurring carotenoids obtained by extraction from natural sources such as plant materials as well as synthetic carotenoids obtained by conventional methods such as chemical synthesis or fermentation. Examples of carotenoids include hydrocarbons (carotenes) and oxidized alcohol derivatives thereof (xanthophylls).

Examples of carotenoids include actinioerythrol, astaxanthin, bixin, canthaxanthin, capsanthin, capsorbin, β-8'-apo-carotenal (apocarotenal), β-12'-apo-carotenal, β- 4'-apo-carotenal, C _1-5 alkyl-8'-apo-β-caroten-8'-oate such as preferably ethyl-8'-apo-β-caroten-8'-oate, α-carotene, β-carotene, γ-carotene, α-cryptoxanthin, β- cryptoxanthin, lutein, lycopene, violerythrin, zeaxanthin, citranaxanthin, phytoene, phytofluene, crocin, crocetin, rubixanthin, violaxanthin, rhodoxanthin, and any mixture thereof, as well as derivatives such as esters (e.g. fatty acid esters) of hydroxyl- or carboxyl-containing compounds selected from the above.

“ C _1-5 alkyl" in the context of the present invention encompasses straight C _1-5 alkyl as well as branched C _3-5 alkyl and cyclopentyl.

Preferably, all carotenoids already being used in feed may be used in the feed additive according to the present invention. These carotenoids may be used for pigmentation, especially for coloring the skin and fat of poultry, for egg yolk pigmentation or for the pigmentation of aquatic animals such as e.g. fish and crustaceae. Preferred examples of such carotenoids are: astaxanthin and its derivatives such as fatty acid esters thereof, canthaxanthin, ethyl-8'-apo-β- caroten-8'-oate (“apo-ester"), β-carotene, lutein and its derivatives such as fatty acid esters thereof, as well as any mixture thereof. Thus, preferably the following carotenoids are used in the feed additive of the present invention: astaxanthin, canthaxanthin, ethyl-8'-apo-β-caroten-8'-oate (= ethyl-2,6,11,15-tetramethyl-17-(2,6,6-trimethyl-1-cyclohexen -1-yl)-2,4,6,8,10,12,14,16- heptadecaoctaenoate), β-carotene, lutein, as well as astaxanthin derivatives, lutein derivatives and any mixture thereof. More preferably single carotenoids are used, whereby astaxanthin, astaxanthin derivatives, canthaxanthin and ethyl-8'-apo-β- caroten-8'-oate are especially preferred. Most preferred are astaxanthin and its derivatives. “Derivatives" are structural analogs of a compound that are derived from a similar compound by a chemical reaction. The term “derivatives" especially encompasses esters, preferably fatty acid esters. The fatty acids in these esters are preferably linear or branched, saturated or unsaturated fatty acids having 8 to 22 carbon atoms.

Astaxanthin and its derivatives

Carotenoids particularly preferably used in the present invention include the free form of astaxanthin and/or its derivatives such as esters of astaxanthin (hereinafter, these are generically referred to as "astaxanthins").

Astaxanthin diesters as disclosed in WO 2003/066583 could also be used in the feed additives of the present invention, i.e. compounds of the following formula (l) wherein R and R* are independently from each other -NH-CH(R ¹ )-COOR ² or OR ³ or -(Y)n-Z, whereby R ¹ signifies hydrogen or the residue of a protein-forming amino acid, R ² signifies C _1-6 -alkyl or C _3-8 -cycloalkyl, R ³ signifies C _1-12 -alkyl or C _3-8 -cycloalkyl,

Y signifies C _1-7 -alkylene or C _2-7 -alkenylene, n signifies 0 or 1, and Z, when n=0, signifies — C _3-8 -cycloalkyl, — CH(C ₆ H ₅ )OR ⁴ with R ⁴ being H or acetyl, —COR ⁵ with R ⁵ being hydrogen or C _1-6 -alkyl, or — CH ₂ N ⁺ (CH ₃ ) ₃ X- with X- being a halogen ion, or Z, when n=1, signifies amino, — O(CO)R ⁶ with R ⁶ being C _1-6 -alkyl, aryl or heteroaryl, — OR ⁷ with R ⁷ being hydrogen, C _1-6 -alkyl or acetyl or— SR ⁸ with R ⁸ being C _1-6 -alkyl; or Z, regardless of whether n is 0 or 1, signifies alternatively aryl, heteroaryl, — COOR ⁵ with R ⁵ being hydrogen or C _1-6 -alkyl or a group — CH(CH ₃ )OR ⁴ with R ⁴ being H or acetyl. Preferably R and R* are the same group.

In the above definition of the astaxanthin derivatives of the formula (l) any alkyl or alkenyl group containing three or more carbon atoms can be straight chain or branched. This also applies to the C _1-7 -alkylene or C _2-7 -alkenylene (divalent) group signified by Y; thus the alkylene group can be for example methylene or di-, tri-, tetra-, penta-, hexa- or heptamethylene, or, respectively, ethylidene, propylidene (ethylmethylene), 1- or 2-methyl substituted ethylene and further mono- or multi- branched alkylene groups containing altogether up to seven carbon atoms. In addition, for the straight chain or branched C _2-7 -alkenylene group, this is understood to encompass alkenylene groups with one or (from C ₄ ) more double bonds; examples of such alkenylene groups are those of the formulae — CH=CH— , ~CH=CH-CH ₂ -, -CH=CH-(CH ₂ ) ₃ - and — (CH=CH) ₂ — .

Any aryl group (a significance of Z or of R ⁶ in the group — O— COR ⁶ signified by Z when n is 1) can be unsubstituted phenyl, naphthyl or a further multiring aromatic hydrocarbon group, or such a group featuring one or more substituents, particularly those substituents selected from C _1-4 -alkyl, C _1-4 -alkoxy, halogen and benzyloxy. Halogen indicates fluorine, chlorine, bromine or iodide. Examples of substituted phenyl groups are p-tolyl, 3-methoxyphenyl, 4-methoxyphenyl, 2,5- dimethoxyphenyl, 3,4-dimethoxyphenyl and 4-benzyloxyphenyl.

The expression "heteroaryl", also a significance of Z or of R ⁶ in the group — 0- (CO)R ⁶ , means a heterocyclic group of aromatic character featuring as ring member(s) one or more heteroatoms selected from oxygen, sulphur and nitrogen. Examples of such heteroaryl groups are 2- or 3-furyl, 2- or 3-thienyl and 4-pyridyl. As in the case of the aryl groups, the heteroaryl groups can be unsubstituted or substituted by one or more substituents as indicated hereinabove for the substituted aryl groups.

As regards the expression "residue of a protein-forming amino acid" (the significance of R ¹ when not signifying hydrogen), this means that the group -NH- CH(R ¹ )-COOR ² in which R1 has this significance is derived from any amino acid H ₂ N- CH(R ¹ )-COOH, R ¹ signifying the variable part of the amino acid molecule. Where the amino acid is e.g. glycine, the group signifies -NH-CH ₂ -COOR ² , R ¹ being hydrogen and R ² being any C _1-6 -alkyl or C _3-8 -cycloalkyl group. In the case of phenylalanine and methionine, the group signifies -NH-CH(C ₆ H ₅ )-COOR ² and phenyl (C ₆ H ₅ ), and -NH- CH(CH ₂ CH ₂ SCH ₃ )-COOR ² and 2-methylthioethyl (CH ₂ CH ₂ SCH ₃ ), respectively.

Finally, the halogen ion X- can be a fluoride, chloride, bromide or iodide ion, preferably a chloride ion, Cl-.

The astaxanthin derivatives of formula (l) can be in any possible isomeric form or in the form of mixtures of isomers, e.g. racemate mixtures. Examples of specific astaxanthin derivatives of the formula (l) (with the appropriate significance of R) are: astaxanthin-diethyldicarbonate (R is ethoxy), astaxa nth in-diethyldioxa late (R is ethoxycarbonyl), astaxanthin-di(N-acetylglycinate) (R is acetylaminomethyl), astaxanthin-dimaleinate (R is -CH=CH-COOH), astaxanthin-disuccinate (R is -CH ₂ - CH ₂ -COOH), astaxanthin-dimethyldisuccinate (R is -CH ₂ -CH ₂ -COOCH ₃ ), astaxanthin- diethyldisuccinate (R is -CH ₂ -CH ₂ -COOC ₂ H ₅ ), astaxanthin-diethyldiglycine- dicarbamate (R is -NH-CH ₂ -COOC ₂ H ₅ ), astaxanthin-dinicotinate (R is 3-pyridyl), astaxanthin-dimethioninedicarbamate (R is -NHCH(CH ₂ CH ₂ SCH ₃ )COOC ₂ H ₅ ), astaxanthin-diacetyldiglycolate (R is acetyloxymethyl), astaxanthin-diphenyl- alaninedicarbamate (R is -NHCH(CH ₂ C ₆ H ₅ )COOC ₂ H ₅ ), astaxanthin-diethyldifumarate (R is -CH=CH-COOC ₂ H ₅ ), astaxanthin-di(2-furoate) (R is 2-furyl), astaxanthin- dimethyldimalonate (R is -CH ₂ -COOCH ₃ ), astaxanthin-di(3-methylthiopropionate) (R is 3-methylthioethyl), astaxanthin-dimethoxyacetate (R is methoxymethyl), astaxanthin-di-[(2-thienyl)acetate] [R is (2-thienyl)methyl], astaxanthin-dilactate (R is 1-hydroxyethyl), astaxanthin-di(acetylmandelate) (R is a-acetyloxybenzyl) and astaxanthin dibetainate [R is -CH ₂ N ⁺ (CH ₃ ) ₃ Cl-]. Each of the above-named astaxanthin derivatives is preferably in the (all-E)-3,3'-rac isomeric form. The six astaxanthin derivatives astaxanthin-diethyldicarbonate, -dimethyldi-succinate, - diethyldisuccinate, -dinicotinate, -dimethoxyacetate and -di-[(2-thienyl)-acetate] are especially preferred ones.

Further astaxanthin diesters that could be used in the feed additives of the present invention are disclosed in WO 2010/100229. These are astaxanthin esters of the formula (l) as given above, whereby R and R* are independently from each other -A-(CO)OR ^x with A being -CH ₂ -CH ₂ - or -CH=CH- and R ^x being C _1-4 -alkyl, whereby R and R* are preferably the same group.

Astaxanthin monoesters with the groups as given above are also encompassed by the expression “astaxanthin derivatives".

The chemical name of the free form of astaxanthin is 3,3'-dihydroxy-3,3-carotene- 4,4'-dione. Astaxanthin has three isomers: 3S,3S'-form, 3S,3R'-form (meso form), and 3R,3R'-form depending on the steric configuration of the hydroxyl group at the 3(3') -position of the ring structures present at both ends of the molecule. Astaxanthin also has cis and trans geometrical isomers with respect to the conjugated double bond system of the polyene chain at the center of the molecule. Examples include the 9-cis isomer, the 13-cis isomer, 15-cis isomer and the all-E isomer. This also applies for the astaxanthin derivatives. The hydroxyl group at the 3(3')-position can form an ester with a fatty acid. For example, astaxanthins obtained from krill contain a relatively large amount of a diester having two fatty acids bounded thereto. Astaxanthin obtained from Haematococcus pluvialis, in which astaxanthin is in the 3S,3S'-form, contain a relatively large amount of a monoester having one fatty acid bonded thereto.

Astaxanthin obtained from Phaffia Rhodozymo is the 3R,3R'-form which has a structure reverse to the 3S,3S'-form generally found in nature. This is also present in the non-ester form without forming any ester with a fatty acid, in other words, in the free form.

The feed additive of the present invention may contain an astaxanthin-containing oil, which is separated or extracted from astaxanthin-containing natural products. Examples of such an astaxanthin-containing oil include extracts obtained from cultures of a red yeast, Phaffia, a green alga Haematococcus, marine bacteria, or other organisms; and extracts from antarctic krill or the like. The astaxanthin that can be used in the present invention may be the extracts mentioned above, products obtained by appropriate purification of the extracts as needed, or chemically synthesized products. Chemically synthesized astaxanthin as commercially available from DS Nutritional Products AG (CH) is especially preferred. As regards the amount of astaxanthin in the feed additive of the present invention, the amount of the free form of astaxanthin is calculated directly, but the amount of a fatty acid ester of astaxanthin is calculated in terms of the free form of astaxanthin. The amount of the carotenoid is chosen in such a way so that its final amount in the feed additive is preferably ranging from 0.5 to 25 weight-%, more preferably its final amount is ranging from 2.0 to 20 weight-%, even more preferably its final amount is ranging from 5.0 to 20 weight-%, most preferably its final amount is ranging from 8 to 16 weight-%, based on the total weight of the dry matter of the feed additive. These preferences also apply to the preferred carotenoids as given above.

Compound b): Lignosulfonate(s)

The lignosulfonate(s) present in the feed additives according to the present invention are especially industrially produced products which contain lignosulfonates having the widest variety of cations. Sodium, calcium, magnesium and ammonium lignosulfonate are especially preferred. A feed additive according to the present invention can contain a single lignosulfonate or a mixture of several lignosulfonates as ingredient b). Furthermore, the lignosulfonate(s) present in the feed additives according to the present invention can be part of an industrially produced product which contains further components in addition to the lignosulfonate(s).

As is known, the biopolymer lignin occurs together with cellulose in plants, especially in wood. Wood, depending on the type, contains about 16 to 37 weight-% of lignin. Considered chemically, lignin consists of irregular polymers of methoxylated phenylpropane monomers (p-coumaryl alcohol, coniferyl alcohol, sinapyl alcohol etc.) having a molecular weight estimated to be at least 20 kD. In a first step in the production of cellulose the wood is decomposed, which is achieved in most cases by treatment with sulfite lyes at 125°-180°C. Thereby, the cellulose is liberated and the lignin is converted into a water-soluble derivative, lignosulfonate (also known as “sulfite lignin"). On a smaller scale, the decomposition of wood is also achieved by treating the wood with sodium hydroxide and disodium tetrasulfide (the “Kraft process"). The lignin obtained in this process is referred to as “Kraft lignin" or “sulfate lignin" and is not water-soluble at neutral pH. More recent processes for the production of cellulose use organic solvents e.g. alcohol, also mixed with water, for the decomposition of wood, and the thus-produced lignin is referred to as “organosolv lignin". This form of lignin is likewise not water-soluble. At present, primarily lignosulfonates and Kraft lignins are commercially available.

Frequently, after the decomposition of the wood, the cellulose is separated and the resulting lignosulfonate-containing solution is concentrated to about 50% solid content and sold in this form. Most producers also offer pulverous products which have been obtained by spray-drying the solutions, and these solid forms also contain various saccharides in considerable amounts in addition to lignin. Some producers manufacture lignosulfonates having a relatively high content of lignosulfonate(s) from the primary (crude) lignosulfonates by enzymatic removal of the saccharides and, if necessary, by purification, for example by ultracentrifugation. The Kraft lignins, which are also offered, can be sulfonated in order to achieve water- solubility and the sulfonation products are suitable as lignosulfonates for use in the preparations in accordance with the invention. Commercial lignosulfonate products typically consist of about 40-90% lignosulfonate and smaller amounts of various saccharides, ash, carbohydrates, acetates, formates, resins etc., with the composition depending very much on the quality of the wood which is used. Such water-soluble lignosulfonate products are also suitable for use in the feed additives in accordance with the invention. In general, not only the crude products having a relatively high content of saccharides and additional byproducts but also the aforementioned purified lignosulfonate(s) can be used in the feed additives in accordance with the invention, provided that such lignosulfonate(s) are water- soluble or at least water-dispersible.

Preferred examples of well-suited lignosulfonate(s) are: sodium lignosulfonate, ammonium lignosulfonate, magnesium and calcium lignosulfonate. Sodium lignosulfonate and calcium lignosulfonate are especially preferred. Most preferred is calcium lignosulfonate.

Suppliers of lignosulfonate(s) are: Borregaard Industries Limited, Norway; Burgo Group, Rayonier Advanced Materials, Wuhan Xinyingda Chemicals, Shenyang Xingzhenghe Chemical, Abelin Polymers, GREENAGROCHEM, Harbin Fecino Chemical, Karjala Pulp, Nippon Paper Industries, Pacific Dust Control, Sappi, The Dallas Group of America, Venki Chem and Xinyi Feihuang Chemical.

Especially suitable de-sugared calcium lignosulfonate is available from Borregaard Industries Limited, Norway under the tradenames Borrebright CY22P, Borresperse Na220 and Borrement CA120, whereby Borrebright CY22P is especially preferred.

This is manufactured by cutting spruce timer into chips and feeding it into a digester together with cooking calcium bisulfite solution. During the cooking at high temperature (130-140°C) the lignin in the wood is depolymerized and sulfonated, which makes water-soluble lignosulfonates. At the end of the cooking the sulfite liquor contains calcium lignosulfonate and sugars. The sulfite liquor (calcium lignosulfonate and sugars) is separated from the cellulose pulp by filtration. The sulfite lye is concentrated to about 53% in an evaporation plant. The concentrated liquor is fed into a spray dryer to produce lignosulfonate powder (inlet temperature ranging from 200 to 250° C).

The amount of the lignosulfonate(s) is chosen in such a way so that its final amount in the feed additive is preferably ranging from 35 to 70 weight-%, more preferably its final amount is ranging from 35 to 60 weight-% and especially from 37 to 57 weight-%, even more preferably its final amount is ranging from 40 to 55 weight-%, most preferably its final amount is ranging from 45 to 53 weight-%, based on the total weight of the dry matter of the feed additive.

In a preferred embodiment of the present invention the weight ratio of the lignosulfonate(s) b) to the carotenoid(s) a) is ranging from 1:1 to 15:1, preferably ranging from 1:1 to 10:1, more preferably ranging from 2:1 to 7:1.

In further preferred embodiments of the present invention the weight ratio of the compound(s) c) to the lignosulfonate(s) b) is ranging from 2:1 to 1:12, preferably ranging from 1:1 to 1:15, more preferably ranging from 1:1 to 1:10, most preferably ranging from 1:2 to 1:8.

Compound c)

The compound c) is selected from hexose-dimers, modified hexose-dimers, hexose oligomers, modified hexose oligomers, hexose-polymers, modified hexose- polymers and any mixture thereof. A hexose may also be present. That means that mixtures of hexoses and hexose-dimers are also encompassed. An example of a mixture of hexose and hexose-dimers is invert sugar (glucose + fructose + sucrose).

A hexose is a monosaccharide with six carbon atoms. Hexoses are classified by functional group, with aldohexoses having an aldehyde at position 1, and ketohexoses having a ketone at position 2.

Preferably the compound c) is selected from aldohexose-oligomers, such as starch hydrolysates as defined below, preferably dextrins, glucose syrups and/or dried glucose syrups, or modified aldohexose polymers, such as preferably OSA starch, or any mixture thereof.

The hexose in the hexose-dimers may be one single hexose or two hexoses being distinct from each other. Examples of hexose-dimers are sucrose (glucose-fructose- dimer), lactose (glucose-galactose-dimer), maltose (glucose-dimer with an α-(1-4)- linkage), isomaltose (glucose-dimer with an α-(1-6)-linkage), trehalose (glucose- dimer with an α-(1-1)-linkage) and nigerose (glucose-dimer with an α-(1-3)-linkage), as well as any mixture thereof. One preferred example of a hexose-dimer where the two hexoses are distinct from each other is sucrose, a glucose-fructose-dimer.

The hexose in the hexose-oligomers may be one single hexose or several hexoses being distinct from each other. Preferably the hexose is glucose. More preferred examples of hexose-oligomers are hydrolysed starch products such as glucose syrups, dried glucose syrups or dextrins. Such glucose syrups, dried glucose syrups and dextrins are classified according to their “dextrose equivalents" and may further contain hexoses, hexose-dimers and hexose polymers.

“Dextrose" is a synonym for “glucose". The term “dextrose equivalent" (DE) denotes the degree of hydrolysis and is a measure of the amount of reducing sugar calculated as D-glucose based on dry weight; the scale is based on native starch having a DE close to 0 and glucose having a DE of 100.

Maltodextrin is a dextrin with a DE in the range of from 3 to 20; hydrolysed starch products with a DE > 20 are called “glucose syrups" or “dried glucose syrups" - depending on their water content. “Glucose syrups" or “dried glucose syrups" may be used in form of powders, micro-granulates or granulates. Glucose syrups consist in general of a mixture of glucose, maltose and oligo- and polysaccharides with varying amounts of these ingredients.

Commercially available hexose-oligomers that also contain hexoses and hexose- dimers are e.g. commercially available under the tradenames Glucidex 21 (from Roquette), Glucidex IT 47 (from Roquette), Dextrose Monohydrate ST (from Roquette), Sirodex 331 (from Tate & Lyle), Glucamyl F 452 (from Tate & Lyle) and Raftisweet I 50/75/35 (from Lebbe Sugar Specialties), whereby Glucidex 21 and Glucidex 47 are especially preferred.

The hexose in the hexose-polymers and modified hexose-polymers may be one single hexose or a mixture of many hexoses. Preferably one hexose or two hexoses being distinct from each other are present in the hexose-polymers or modified hexose-polymers of the present invention. More preferably the modified hexose- polymers are modified food starches such as starches modified with octenyl succinic acid (so-called “OSA-starches"), which are in fact a mixture of glucose, glucose- dimers, glucose-oligomers, glucose-polymers (= starch) and OSA-modified glucose- dimers, glucose-oligomers and glucose-polymers (= OSA-modified starch).

Further suitable modified hexose-polymers are the OSA-starches modified according to the processes as disclosed in WO 2020/093962, WO 2020/093919, WO 2020/093960 and CN-A 109517080.

The most preferred compounds c) are starch hydrolysates, such as e.g. dried glucose syrups and dextrins, with a DE ranging from 10 to 50, more preferably with a DE ranging from 15 to 40, even more preferably with a DE ranging from 15 to 30, most preferably with a DE ranging from 15 to 25.

Commercially available examples of such starch hydrolysates are dried glucose syrups as e.g. Glucidex 21 and Glucidex IT47, and dextrins such as Yellow dextrin. Most preferred examples are also their mixtures with modified food starches.

Glucidex 21 is a dried glucose syrup with a DE ranging from 20 to 23 in the form of a fine powder with at least 50% of the particles being greater than 40 μm and at most 10% of the particles being greater than 250 μm. Glucidex 21 contains 3% glucose, 7% maltose and 90% oligo- and polysaccharides.

Glucidex IT 47 is a dried glucose syrup with a DE ranging from 43 to 47 in the form of micro-granulates with at least 95% of the particles being greater than 40 μm and at most 5% of the particles being greater than 500 μm . Glucidex IT 47 contains 5% glucose, 50% maltose and 45% oligo- and polysaccharides.

Further preferred compounds c) are starch hydrolysates that have a maximum amount of 10 weight% of reducing sugars, based on the total weight of the starch hydrolysate, as for example specific dextrins.

Dextrins

Dextrins are a group of low-molecular-weight carbohydrates produced by the hydrolysis of starch or glycogen. Dextrins are mixtures of polymers of D-glucose units linked by α-(1→4) or α-(1→6) glycosidic bonds.

Dextrins can e.g. be produced from starch using enzymes like amylases or by applying dry heat under acidic conditions (“pyrolysis» or “roasting»). Dextrins produced by heat are also known as «pyrodextrins». The starch hydrolyses during roasting under acidic conditions, and short-chained starch parts partially re-branch with α-(1,6) bonds to the degraded starch molecule. They have a low viscosity.

Preferably commercially available Yellow Dextrin from Roquette is used in the feed additives of the present invention.

The amount of the compound c) is chosen in such a way so that its final amount in the feed additive is at least 5 weight-%, preferably its final amount is ranging from 5 to 30 weight-%, more preferably its final amount is ranging from 5 to 25 weight- %, even more preferably its final amount is ranging from 8 to 20 weight-%, most preferably its final amount is ranging from 8 to 15 weight-%, based on the total weight of the dry matter of the feed additive.

Compound d): Antioxidants

The regulatory approval to use ethoxyquin in feed is suspended in the European Union. Therefore, it is advantageous that the feed additive of the present invention is essentially free of ethoxyquin. “essentially free of" in the context of the present invention means that the amount of ethoxyquin is ≤ 0.5 weight-%, preferably ≤ 0.2 weight-%, more preferably ≤ 0.1 weight-%, based on the total weight of the dry matter of the feed additive. Most preferably no ethoxyquin is added during the manufacture of the feed additive of the present invention. Thus, most preferably no ethoxyquin is present in the feed additive of the present invention.

Advantageously the feed additive of the present invention is also essentially free of butylated hydroxytoluene such as 2,6-di-tert-butyl-p-cresol (lUPAC name = 2,6-di- tert-butyl-4-methylphenol).

“essentially free of" in the context of the present invention means that the amount of butylated hydroxytoluene is ≤ 0.5 weight-%, preferably ≤ 0.2 weight-%, more preferably ≤ 0.1 weight-%, based on the total weight of the dry matter of the feed additive. Most preferably no butylated hydroxytoluene is added during the manufacture of the feed additive of the present invention. Thus, most preferably no butylated hydroxytoluene is present in the feed additive of the present invention.

In a most preferred embodiment of the present invention neither ethoxyquin nor butylated hydroxytoluene are present in the feed additive of the present invention.

The feed additive may comprise an antioxidant or a mixture of antioxidants. Preferably a mixture of a fat-soluble antioxidant and a water-soluble antioxidant is used.

If an antioxidant or a mixture of antioxidant is present, its/their total amount is chosen in such a way so that its/their final amount in the feed additive is preferably ranging from 1 to 20 weight-%, more preferably its final amount is ranging from 3 to 15 weight-%, most preferably its final amount is ranging from 5 to 12 weight-%, based on the total weight of the dry matter of the feed additive.

Fat-soluble antioxidants Examples of suitable fat-soluble antioxidants are tocopherols and analogues thereof such as e.g. compounds of formula (ll) wherein R ^1a and R ^2a are independently from each other H or C _1-11 -alkyl or (CH ₂ ) _n — OH with n being an integer from 1 to 4, or R ^1a and R ^2a represent together a keto group, A is CHR ^3a or C(=O), and wherein R ^3a , R ^4a and R ^6a are independently from each other H or C _1-4 -alkyl, and wherein R ^5a is H or OH or C _1-4 -alkyl or C _1-4 -alkoxy, as disclosed in WO 2019/185894.

Further suitable fat-soluble antioxidants are compounds of formula (ll), wherein one of the two substituents R ^1a and R ^2a is C _12-2 ralkyl and the other of the two substituents R ^1a and R ^2a is either hydrogen or C _1-5 -alkyl or (CH ₂ ) _n -OH with n being an integer from 1 to 5, and wherein A is CH(R ^3a ), and wherein R ^3a , R ^4a and R ^6a are independently from each other H or C _1-4 -alkyl, and wherein R ^5a is H or OH or C _1-4 - alkyl or C _1-4 -alkoxy, as disclosed in WO 2019/185938.

Compounds of formula (ll), wherein A is CH ₂ , R ^1a is C _1-5 -alkyl, R ^2a is either H or C _1-2 - alkyl, R ^5a is either H or C _1-4 -alkoxy or C _1-4 -alkyl, and R ^4a and R ^6a are independently from each other either H or C _1-4 -alkyl, with the preferences as disclosed in WO 2019/185900 are also suitable antioxidants in the feed additives of the present invention.

Preferred examples of the antioxidants of formula (l l) as disclosed in WO 2019/185894 are the following compounds of formula (1)-(11) with “Me" being methyl:

Further examples of suitable antioxidants that can be used in the feed additives of the present invention are compounds of formula (ill) and (IV), wherein R ^1b and R ^2b are independently from each other H or C _1-11 -alkyl or (CH ₂ ) _n — OH with n being an integer from 1 to 6 or R ^1b and R ^2b together represent a keto group, and wherein R ^3b , R ^4b , R ^5b , and R ^6b are independently from each other H or C _1-6 -alkyl or C _1-6 -alkoxy, and R ^7b is H or C _1-6 -alkyl, as disclosed in WO 2019/185898.

“alkyl" and “alkoxy" hereby encompass linear alkyl and branched alkyl, and linear alkoxy and branched alkoxy, respectively. Preferred examples of compounds of formula (III) and (IV) are the following compounds (12)-(19):

Further suitable antioxidants are compounds of formula (V), whereby R ¹ , R ² and R ³ are independently from each other H or linear C _1-6 -alkyl or branched Cs s-alkyl, whereby preferably R ¹ is H or methyl or ethyl or n-propyl or iso-propyl or tert-butyl and R ² and R ³ are independently from each other H or methyl or ethyl, with the further preferences as disclosed in WO 2019/185940.

Also, the compounds of formula (VI) with n being 1 or 2, R ^1b and R ^3b being independently from each other H or C _1-5 -alkyl, and R ^2b being either H or C _1-5 -alkyl or C _1-5 -alkyloxy, preferably with the proviso at least one of R ^1b , R ^2b and R ^3b being H, as disclosed in WO 2019/185904 can be used as antioxidants in the feed additives of the present invention.

Hereby the following compounds of formulae (VI-1) and (VI-2) are especially preferred:

The asterisks * mark each a chiral/stereogenic center, i.e. all possible isomers having any configuration at said centers are encompassed by the term “compound of formula (VI-1)" and “compound of formula (VI-2)", respectively.

Further suitable antioxidants are gallic acid derivatives such as the ones disclosed in WO 2008/080152, hydroxycinnamic acids such as e.g. ferulic acid (= 3-(4-hydroxy- 3-methoxyphenol)prop-2-enoic acid), hydroxycoumarines, hydroxybenzoic acids such as e.g. gallic acid (= 3,4,5-trihydroxybenzoic acid) and syringic acid (= 4-hydroxy- 3,5-dimethoxy-benzoic acid), propyl gallate, rosmarinic acid and carnosic acid.

Also suitable fat-soluble antioxidants are compounds of the following formulae (VIl) and (VIll) with R ^1c , R ^2c and R ^3c being independently from each other H or C _1-4 -alkyl as published in WO 2019/185942 and WO 2019/185888, respectively. Preferred examples thereof are tocotrienols and tocopherols of the formulae (20) to (27) as shown below.

The asterisks * mark each a chiral/stereogenic center. The term “compound of formula (VIl)/(VIll)" encompasses all possible isomers having any configuration at said centers.

Especially preferred examples of the compound of formula (VIl) are the following compounds of formulae (20) (= alpha-tocotrienol), (21) (= beta-tocotrienol), (22) (= gamma-tocotrienol) and (23) (= delta-tocotrienol), whereby all possible diastereomers and enantiomers are included.

Especially preferred examples of the compound of formula (VIll) are the following compounds of formulae (20) (= alpha-tocopherol), (21) (= beta-tocopherol), (22) (= gamma-tocopherol) and (23) (= delta-tocopherol), whereby all possible diastereomers and enantiomers are included.

The asterisks * mark each a chiral/stereogenic center. The term “compound of formula (20)/(2l)/(22)/(23)/(2 )/(25)/(26)/(27)" encompasses all possible isomers having any configuration at said centers.

The most preferred fat-soluble antioxidant is a-tocopherol, especially DL- a- tocopherol.

Water-soluble antioxidants In general any water-soluble antioxidant being allowed in feed and known to the person skilled in the art may be used.

Preferred examples of water-soluble antioxidants are ascorbic acid and salts thereof, such as alkali and earth alkali salts of ascorbic acid and ascorbyl-2- phosphate salts as disclosed in EP-A 972777.

Especially preferred are alkali and earth alkali salts of ascorbic acid. The most preferred water-soluble antioxidant is sodium ascorbate. Most preferred antioxidants in the feed additive according to the present invention Most preferred is a mixture of alpha-tocopherol and sodium ascorbate, whereby a weight ratio of alpha-tocopherol to sodium ascorbate ranging from 5:1 to 1:5 is especially preferred, a weight ratio of alpha-tocopherolto sodium ascorbate ranging from 3:1 to 1:3 is more preferred, a weight ratio of alpha-tocopherol to sodium ascorbate ranging from 3:1 to 1:1 is even more preferred, a weight ratio of alpha- tocopherolto sodium ascorbate ranging from 2.5:1 to 1:1 is especially more preferred, and a weight ratio of alpha-tocopherolto sodium ascorbate of 2:1 is most preferred.

Feed additives according to the present invention

The composition of the feed additives according to the present invention is shown in the following Table 1, where the ingredients and their amounts are given. The amounts are given in weight-% and are based on the total weight of the feed ingredient comprising an absorbent. The amounts of all ingredients sum up to a total weight of 100%.

It is understood that each single preferred amount of one ingredient may be combined with each preferred single amount of any other ingredient.

In further preferred embodiments of the present invention the weight ratio of the compound(s) c) to the lignosulfonate(s) b) is ranging from 2:1 to 1:20, preferably ranging from 1:1 to 1:15, more preferably ranging from 1:1 to 1:10, most preferably ranging from 1:2 to 1:8.

A preferred feed additive is one, wherein the total amount of the compounds a) to d) is at least 90 weight-%, preferably at least 95 weight-%, preferably at least 97 weight-%, based on the total weight of the dry matter of the feed additive without the weight of the absorbent.

Table 1: Composition of a solid feed additive according to the present invention, whereby the feed additive comprises an absorbent. All amounts are based on the total weight of said feed additive.

Processes of the present invention The present invention is also directed to a process for the manufacture of a feed additive with all the preferences as cited above comprising the following steps: i) Providing a matrix by dissolving the lignosulfonate(s), the compound(s) c) and optionally a water-soluble antioxidant in water; ii) Suspending the carotenoid(s) into the matrix obtained in step i) to obtain a dispersion; iii) Milling the carotenoid in the dispersion obtained in step ii); iv) Optionally emulsifying a fat-soluble antioxidant(s) into the dispersion obtained in step iii); v) Drying the dispersion obtained in step iii) or in step iv) in presence of an absorbent to obtain the feed additive.

The single steps of the process of the manufacture of the feed additive are disclosed in more detail below.

Step i)

The amounts of the lignosulfonate(s) b), the compound c) and the water-soluble antioxidant d), if present, are chosen so that the final amounts of these compounds in the resulting feed additive after having performed steps i) to v), respectively, is as described above.

Preferably this step is performed at a temperature ranging from 25 to 70° C, more preferably at a temperature ranging from 30°C to 65° C, even more preferably at a temperature ranging from 0°C to 62°C, most preferably at a temperature ranging from 50°C to 60°C.

Step ii)

The amount of the carotenoid(s) a) is chosen so that the final amount in the resulting feed additive after having performed steps i) to v) is as described above.

Step iii) Preferably this step is performed at a temperature ranging from 15 to 70° C, more preferably at a temperature ranging from 20 to 68° C, even more preferably at a temperature ranging from 30° C to 65° C, most preferably at a temperature ranging from 35 to 60°C to obtain a dispersion. Preferably the dispersion is a suspension.

The water content of the dispersion is hereby ranging from 30 to 70 weight-%, preferably ranging from 35 to 65 weight-%, more preferably ranging from 40 to 60 weight-%, based on the total weight of the dispersion.

Advantageously, the water amount is reduced after the milling by using a rotary evaporator or a thin film evaporator cascade. Other methods known to the person skilled in the art are also applicable. Alternatively, this step may be performed after step iv).

Step iv)

Preferably a fat-soluble antioxidant is present and this step is carried out.

The emulsification of the fat-soluble antioxidant can be achieved by using a rotor- stator device or a high-pressure homogenizer or both. Other devices known to the person skilled in the art may also be used.

If rotor-stator device and/or a high-pressure homogenizer is used, a pressure drop ranging from 70 to 1000 bar, more preferably ranging from 100 to 300 bar, is preferably applied.

Step v)

The drying can be carried out after step iii) or step iv), preferably it is carried out after step iv). The drying can preferably be achieved by any method known to the person skilled in the art where an absorbent is used, preferably by a powder-catch technique, whereby the sprayed dispersion droplets are caught by an absorbent (so-called “catch media") such as starch and dried. Suitable absorbents include corn starch, as well as starches from other botanical sources, silica, modified silica, tricalcium phosphate, calcium carbonate, sodium carbonate, potassium carbonate, magnesium carbonate, calcium oxide, magnesium oxide, dicalcium diphosphate, calcium silicate, magnesium silicate, magnesium trisilicate, sodium aluminum silicate, talc, kaolin, calcium stearate, magnesium stearate, cellulose or mixtures thereof. Especially preferred are starch (i.e. corn starch as well as starches from other botanical sources), silica, tricalcium phosphate and hydrophobically modified silica, whereby corn starch or starches from other botanical sources such as waxy corn, wheat, tapioca, pea and potato are especially preferred.

In another embodiment of the present invention, the conversion to the solid form can be achieved by any method known to the person skilled in the art where no absorbent is used, e.g. by spray-drying, spray-drying in combination with fluidized bed granulation. The resulting feed additive does not comprise an absorbent; the amounts of the ingredients and their ranges are the same as given above, but based on the total weight of the feed additive without the weight of the absorbent. Characteristics of the feed additives of the present invention

Preferably the inner phase of the feed additive according to the present invention, i.e. the inner phase of the liquid feed additive after step iv) or the inner phase of the solid feed additive after step v), when re-dispersed in deionized water, has a average particle size ranging from 50 to 600 nm, preferably ranging from 70 to 500 nm, more preferably ranging from 90 to 450 nm, even more preferably ranging from 100 to 400 nm, most preferably ranging from 110 to 360 nm, measured via dynamic light scattering using especially the particle analyzer Delsa™ Nano S (Beckmann Coulter, New Castle, DE, USA); i.e. the particles have an average diameter according to normalized intensity distribution within the ranges as given above.

In a preferred embodiment the particle size distribution of the feed additive of the present invention is as follows, i.e. the inner phase of the solid feed additive after step v), when re-dispersed in deionized water, has a D (10%) ranging from 40 to 200 nm, preferably ranging from 50 to 180 nm, more preferably ranging from 60 to 160 nm, even more preferably ranging from 80 to 140 nm, most preferably ranging from 80 to 110 nm, and/or a D (50%) ranging from 60 to 340 nm, preferably ranging from 80 to 320 nm, more preferably ranging from 100 to 300 nm, even more preferably ranging from 120 to 290 nm, most preferably ranging from 120 to 230 nm, and/or a D (90%) ranging from 100 to 1300 nm, preferably ranging from 150 to 1100 nm, more preferably ranging from 200 to 900 nm, more preferably ranging from 250 to 700 nm, most preferably ranging from 260 to 500 nm, measured via dynamic light scattering according to normalized intensity distribution using especially the particle analyzer Delsa™ Nano S (Beckmann Coulter, New Castle, DE, USA).

In a more preferred embodiment of the feed additive of the present invention the D (10%) and the D (50%) and the D (90%) are as given above, whereby all possible combinations of the values and ranges for D (10%), D (50%) and D (90%) are encompassed.

An especially preferred feed additive e.g. has a D _V (10) ranging from 40 to 200 nm and a D _v (50) ranging from 60 to 340 nm and a D _v (90) ranging from 100 to 1300 nm. The most preferred feed additive has a D _V (10) ranging from 80 to 110 nm and a D _v (50) ranging from 120 to 230 nm and a D _v (90) ranging from 260 to 500 nm.

Feed according to the present invention

The present invention is also directed to feed comprising the feed additive according to the present invention with the preferences as given above. Feed (or ‘feedingstuff' ) means any substance or product, including additives, whether processed, partially processed or unprocessed, intended to be used for oral feeding to animals.

Feed in the context of the present invention is especially feed for aquatic animals in case of the carotenoid being astaxanthin or a derivative thereof. Aquatic animals in the context of the present invention encompass crustaceae and fish, preferably farmed Crustacea such as shrimp and carnivorous species of farmed fish such as salmons, rainbow trout, brown trout (Salmo trutta) and gilthead seabream. Thus, the feed is preferably for crustaceae and farmed fish, more preferably for salmonids, most preferably for salmons.

A typical composition for fish feed is e.g. shown in Table 4.

The feed additive may be added to the feed according to processes known to the person skilled in the art. The feed additive according to the present invention may e.g. be added to the feed pre-extrusion with the other micro ingredients, preferably in an amount ranging from 0.01 to 0.1 weight-%, especially in an amount so that the amount of the carotenoid in the feed as given below is reached.

The feed additive according to the present invention may also be added to the feed post-extrusion. In this case the feed additive is added to the oil that is coated onto the feed pellets after they are extruded.

For pigmenting the aquatic animal, the feed comprises the supplemented carotenoid, especially astaxanthin or a derivative thereof, preferably in an amount ranging from 5 to 250 mg, more preferably in an amount ranging from 20 to 200 mg, based on 1 kg of feed.

If the aquatic animal is a salmonid such as salmon or rainbow trout or salmon trout, the feed comprises the supplemented carotenoid, especially astaxanthin or a derivative thereof, preferably in an amount ranging from 5 to 150 mg, more preferably in an amount ranging from 20 to 100 mg, even more preferably in an amount ranging from 30 to 80 mg, most preferably in an amount ranging from 40 to 60 mg, based on 1 kg of feed.

If the aquatic animal is shrimp, the feed comprises the supplemented carotenoid, especially astaxanthin or a derivative thereof, preferably in an amount ranging from 50 to 250 mg, more preferably in an amount ranging from 100 to 230 mg, even more preferably in an amount ranging from 130 to 220 mg, most preferably in an amount ranging from 150 to 200 mg, based on 1 kg of feed, whereby the amount of the astaxanthin derivative is calculated in terms of astaxanthin.

When such feed additive according to the present invention or a feed comprising such feed additive is administered to an animal, the administration thereof results in a desired level of muscle retention in said animal. The desired level of the carotenoid retained in the muscle leads to a pleasant, consumer-appealing flesh color similar to wild counterparts of said animal. The feed additive according to the present invention comprising astaxanthin or any derivative thereof results especially in a muscle retention of at least 7% of astaxanthin in Atlantic salmon or at least 13% of astaxanthin in rainbow trout; i.e. 7% of the astaxanthin amount that has been ingested by Atlantic salmon and 13% of the astaxanthin amount that has been ingested by the rainbow trout, respectively, is retained in the muscle.

Furthermore, the feed additive according to the present invention comprising at least one carotenoid which can be used for the pigmentation of animals, especially aquatic animals, is stable per se and in feed.

Usually fish feed is stored for a maximum of 4-6 weeks. As shown in Table 6 the maximum loss of the carotenoid is below 10% of the initial concentration after 6 weeks which fulfills the requirements of the market.

Use according to the present invention

The present invention is further directed to the use of the feed additive or the feed according to the present invention for pigmentation of an animal excluding humans. Especially in case of the carotenoid being astaxanthin or a derivative thereof, the animal to be pigmented is an aquatic animal.

Aquatic animals in the context of the present invention encompass crustaceae and fish, preferably farmed Crustacea such as shrimp and carnivorous species of farmed fish such as Atlantic and Pacific salmon (particularly Salmo salar and Oncorhynchus kisutch), rainbow trout ( Oncorhynchus mykiss), brown trout ( Salmo trutta) and gilthead seabream (Sparus aurata ). Thus, the feed additive or the feed according to the present invention is preferably used for pigmentation of crustaceae and farmed fish, more preferably for pigmentation of salmonids, most preferably for pigmentation of Atlantic salmon and rainbow trout.

To achieve the desired level of pigmentation the feed additive has to be eaten by the animal for a time period known to the person skilled in the art. In case an aquatic animal is to be pigmented, the aquatic animal needs to consume the feed additive for at least 3 months before slaughtering.

In case the aquatic animal is salmon or salmon trout or rainbow trout, the aquatic animal needs to consume the feed additive for at least 2.5 months, preferably of at least 3 months, before slaughtering.

Method for pigmentation

Another embodiment of the present invention is a method of pigmenting an animal excluding humans by administering a feed additive or a feed according to the present invention to said animal. Especially in case of the carotenoid being astaxanthin or a derivative thereof, said animal is an aquatic animal.

Aquatic animals in the context of the present invention encompass crustaceae and fish, preferably farmed Crustacea such as shrimp and carnivorous species of farmed fish such as Atlantic and Pacific salmon (particularly Salmo salar and Oncorhynchus kisutch), rainbow trout ( Oncorhynchus mykiss), brown trout ( Salmo trutta) and gilthead seabream (Sparus aurata). Thus, the present invention is preferably directed to a method of pigmenting crustaceae or farmed fish, more preferably to a method of pigmenting salmonids, most preferably to a method of pigmenting Atlantic and Pacific salmons. Hereby the astaxanthin or the astaxanthin derivative is preferably used in an amount ranging from 5 to 150 mg, more preferably in an amount ranging from 20 to 100 mg, even more preferably in an amount ranging from 30 to 80 mg, most preferably in an amount ranging from 40 to 60 mg, based on 1 kg of feed, whereby the amount of the astaxanthin derivative is calculated in terms of astaxanthin.

If the aquatic animal is shrimp, the supplemented astaxanthin or astaxanthin derivative is preferably used in an amount ranging from 50 to 250 mg, more preferably in an amount ranging from 100 to 230 mg, even more preferably in an amount ranging from 130 to 220 mg, most preferably in an amount ranging from 150 to 200 mg, based on 1 kg of feed, whereby the amount of the astaxanthin derivative is calculated in terms of astaxanthin.

The invention is now further illustrated in the following non-limiting examples.

Examples

Examples 1-3: Preparation of the feed additive according to the present invention

The used ingredients and their amounts are given in Table 2.

Table 2: Ingredients and their amounts of the feed additives according to example

1.2 and 3

The lignosulfonate, Dextrin Yellow and sodium ascorbate are dissolved in deionized water to obtain the so-called “matrix" including an adjustment to pH 7.0 using aqueous sodium hydroxide (32% weight/weight).

Subsequently, astaxanthin is suspended into the aforementioned matrix with a rotor-stator device to achieve deagglomeration. 1.25 kg of the resulting suspension is wet-milled using an agitated ball mill of type LabStar, milling chamber LS1 (Netzsch, Selb, Germany; milling beads ≤ 0.5 mm) for 0.5 hours (example 1), 2 hours (example 2) and 6.5 hours (example 3), respectively with a pin rotor speed ranging from 8-15 m/sec.

After wet-milling, the astaxanthin suspensions are concentrated by reducing the water content of 50-55% to ca. 45% with a thin film evaporator (“concentration"). Subsequently, D/L-α-tocopherol is emulsified into the suspension using a rotor- stator device (“emulsification"). Alternatively, the emulsification may be carried out first and subsequently the concentration.

Then the emulsion is sprayed into fluidized corn starch. The obtained beadlets remained in the corn starch for 45 to 60 minutes. The beadlets are sieved and further dried by an air stream in a glass sinter strainer. The dried beadlets are sieved (160- 630 μm) and stored at 8°C under inert gas (Argon) until further analyses and application in feed via pre-extrusion for the pigmentation trial.

The following Table 3 shows the relevant parameters of the obtained beadlets.

Table 3

Examples 4-6: Fish trials using a feed additive according to examples 1. 2 and 3. respectively a) Production of the feed

All diets are formulated to contain 55 mg/kg of astaxanthin. The level of astaxanthin inclusion is as typically used in the salmonid industry (40-60 mg/kg). Astaxanthin is added to the mash pre-extrusion. The ingredients and the basal composition of the diets are presented in Table 4.

Table 4

Dry ingredients (all except oils) are mixed into a mash and extruded to produce pellets (4 mm in diameter) using a Bühler twin-screw extruder. After extrusion, pellets are vacuum-coated together with the fish and rapeseed oil mixture heated to 45° C using a Forberg vacuum coater. Experimental diets are stored at 4°C for the duration of the feeding trial. b) Fish trial Rainbow trout is used as fish. Per each treatment 75 fish are randomly distributed into 3 tanks with 25 fish per tank. The average body weight at the start of the trial is 121.0 ± 0.1 g. Treatments are randomly assigned to each tank and fish are fed the experimental diets for 86 days. At the end of the trial fish are anesthetized, individually weighed, and both fillets removed for the determination of fillet pigmentation.

Fillets are taken from the 10 fish closest to the average weight of each replicate tank. Fillet color is analyzed in 3 replicate locations within 1-2 hours of euthanasia using a Minolta chroma meter. Color readings are taken of the Norwegian Quality Cut (NQC) region (Fig. 1) approximately identified by each number in Fig. 2. Readings are taken for mean redness, yellowness, lightness, hue, and chroma (see Table 5).

For analysis of muscle astaxanthin, circa 50 g NQC samples are taken from the 10 selected fillets. The NQC includes a complete dorsal to ventral section, as shown in

Fig. 2. c) Results

Pigmentation efficacy is determined by 1) assessment of fillet color space measured by a chroma meter and, 2) measurement of muscle astaxanthin and astaxanthin retention.

In all treatments, astaxanthin sufficiently accumulates in the fillet to exceed the industry target minimum of 7 mg/kg fillet astaxanthin.

Table 5: Mean ± standard deviation (“SD") of fillet color characteristics, fillet astaxanthin and astaxanthin retention offish from day 0 to day 86.

Thus, it is shown that rainbow trout fed with a feed comprising the feed additive according to the present invention shows the desired muscle retention.

Furthermore, the feed additive also has the necessary stability in feed as shown in Table 6 since the loss of astaxanthin is less than 10% of the initial concentration after 6 weeks of storage at 4°C. Table 6