AU2020365042A1

AU2020365042A1 - New feed additives of carotenoids

Info

Publication number: AU2020365042A1
Application number: AU2020365042A
Authority: AU
Inventors: Sebastien RIDER; Christian Schaefer; Roland SCHEX; Viviane VERLHAC; Thomas Zwick
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2019-10-11
Filing date: 2020-10-12
Publication date: 2022-04-07
Also published as: CL2022000886A1; EP4040980A1; CN114502009A; CN114513963A; AU2020364020A1; TW202128022A; WO2021069733A1; CL2022000885A1; EP4040981A1; WO2021069753A1

Abstract

The present invention is directed to a feed additive comprising at least a carotenoid and a process for its manufacture. 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.

Description

New feed additives of carotenoids

Summary of the invention

The present invention is directed to a feed additive comprising a) at least a carotenoid in an amount ranging from 0.5 to 25 weight-%; b) at least a lignosulfonate in an amount ranging from 35 to 60 weight-%; and 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 in an amount ranging from 5 to 25 weight-%, whereby further optionally at least one hexose may be present; d) at least an antioxidant in an amount ranging from 0 to 20 weight-%; e) at least an absorbent in an amount ranging from 1 to 20 weight-%; and residual moisture in an amount ranging from 0 to 10 weight-%; wherein the lignosulfonate b) and the compound c) form a matrix in which the carotenoid is encapsulated; 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 all amounts sum up to 100 weight-% and are based on the total weight of the feed additive.

The present invention is also directed to a process for the manufacture of such feed additive. 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 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 feed additive comprising a) at least a carotenoid in an amount ranging from 0.5 to 25 weight-%; b) at least a lignosulfonate in an amount ranging from 35 to 60 weight-%; and 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 in an amount ranging from 5 to 25 weight-%, whereby further optionally at least one hexose may be present; d) at least an antioxidant in an amount ranging from 0 to 20 weight-%; e) at least an absorbent in an amount ranging from 1 to 20 weight-%; and residual moisture in an amount ranging from 0 to 10 weight-%; wherein the lignosulfonate b) and the compound c) form a matrix in which the carotenoid is encapsulated; wherein the amount of ethoxyquin (= 6-ethoxy-2,2,4-trimethyl-1,2- dihydroquinoline) in the feed additive is < 0.5 weight-%; and wherein the amount of butylated hydroxytoluene in the feed additive is ≤ 0.5 weight-%; whereby all amounts sum up to 100 weight-% and are based on the total weight of the feed additive.

The feed additive of the present invention is 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 especially resulting 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. Furthermore, the feed additive of the present invention shows the necessary stability which is proven by the stress tests and per-se stability tests performed (results shown in Tables 7, 8 and 10-12).That means that the feed additives according to the present invention, especially those containing astaxanthin or a derivative thereof, are stable for at least 1 month, preferably for at least 2 months, more preferably for at least 3 months, most preferably for at least 6 months at 40° C and 75% relative humidity, "stable” means that the loss of the carotenoid due to degradation etc. is less than 7 weight-% on the basis of the initial concentration, preferably less than 5 weight-%.

Besides, the feed additive of the present invention shows a low filtration residue (see Tables 6 and 13). 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).

Advantageously, the feed additives of the present invention do not comprise beeswax which is in discussion because of increasing levels of pesticide residues.

Preferably the lignosulfonate(s) and the compound c) form a dense and glassy matrix as mixture of compounds of varying molecular size and featuring different functional groups.

"encapsulated” means that the carotenoid is embedded in the matrix of lignosulfonate(s) b) and the compound c) and thereby protected against oxidation and degradation. The precursor of the feed additive, i.e. the dispersion obtained after having performed step iv) of the process for the manufacture of the feed additive according to the present invention, forms an oil-in-water type dispersion whereby the carotenoid is the oil being located in the internal phase and the lignosulfonate acts as emulsifier. The compound c) is the filler and additional emulsifier in the matrix contributing to its stability. After “powder catching" of the dispersion, the matrix comprising the carotenoid is coated by the absorbent. "Coated” in the context of the present invention means that the absorbent surrounds the matrix.

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. 10 A, pages 645-653 ("Nachweis der Ligninsulfonsäure in grosser Verdünnung (Abwässer von Sulfitzellstoff-Fabriken)”).

Compound c)

Modified hexose-polymers such as e.g. OSA starches can be qualitatively identified using wet chemistry methods such as e.g. described in the monograph "Modified Starches” (FAO JECFA Monograph 16). The quantification of the OSA-starches can be performed by polarimetry (see e.g. Gorden A. Mitchel, Methods of Starch Analysis, 1990, 42, pages 131-134). Mixtures of hexoses, hexose-dimers, hexose-oligomers and/or hexose-polymers; i.e. e.g. 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 chromatogrchromatography--pulsed amperometric detection and size-exclusion chromatography--multi-angle light scattering— refractive index detection.”).

Hexoses and hexose-dimers; i.e. e.g. sugars such as sucrose, can be analyzed by HPLC (High Performance Liquid Chromatography) with Rl (Refractive Index)- detection or pulsed amperometric detection; see e.g. Diana Duarte-Delgado, Carlos-Eduardo Narvaez-Cuenca, Luz-Patricia Restrepo-Sanchez, Ajjamada Kushalappa, Teresa Mosquera-Vasquez, Journal of Chromatography B 2015, Volume 975, pages 18-23 ("Development and validation of a liquid chromatographic method to quantify sucrose, glucose, and fructose in tubers of Solanum tuberosum Group Phureja”).

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 SiO₂ 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, b-s'-apo-carotenal (apocarotenal), p-12'-apo-carotenal, b- 4’-apo-carotenal, 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.

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 and canthaxanthin 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 (I) wherein R and R* are independently from each other -NH-CH(R¹)-COOR² or OR³ or -(Y)_n-Z, whereby R¹ signifies hydrogen or the residue of a protein-forming amino acid, R² signifies C_1-6-alkyl or C_3-8-cycloalkyl, R³ 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(C₆H₅)OR⁴ with R⁴ being H or acetyl, —COR⁵ with R⁵ being hydrogen or C_1-6-alkyl, or — CH₂N'(CH₃)₃ X- with X- being a halogen ion, or Z, when n=1, signifies amino, — O(CO)R⁶ with R⁶ being C_1-6-alkyl, aryl or heteroaryl, — OR⁷ with R⁷ being hydrogen, C_1-6-alkyl or acetyl or— SR⁸ with R⁸ being C_1-6-alkyl; or Z, regardless of whether n is 0 or 1, signifies alternatively aryl, heteroaryl, — COOR⁵ with R⁵ being hydrogen or C_1-6-alkyl or a group — CH(CH₃)OR⁴ with R⁴ being H or acetyl. Preferably R and R* are the same group.

In the above definition of the astaxanthin derivatives of the formula (I) 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 C₄) more double bonds; examples of such alkenylene groups are those of the formulae — CH=CH— , ~CH=CH-CH₂-, -CH=CH-(CH₂)₃- and -(CH=CH)₂- .

Any aryl group (a significance of Z or of R⁶ in the group — 0— COR⁶ 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 R⁶ in the group — O- (CO)R⁶, 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 R¹ when not signifying hydrogen), this means that the group -NH- CH(R¹)-COOR² in which R1 has this significance is derived from any amino acid H₂N- CH(R¹)-COOH, R¹ signifying the variable part of the amino acid molecule. Where the amino acid is e.g. glycine, the group signifies -NH-CH₂-COOR², R¹ being hydrogen and R² being any C_1-6-alkyl or C_3-8-cycloalkyl group. In the case of phenylalanine and methionine, the group signifies -NH-CH(C₆H₅)-COOR² and phenyl (C₆H₅), and -NH- CH(CH₂CH₂SCH₃)-COOR² and 2-methylthioethyl (CH₂CH₂SCH₃), 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 (I) 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 (I) (with the appropriate significance of R) are: astaxanthin-diethyldicarbonate (R is ethoxy), astaxanthin-diethyldioxalate (R is ethoxycarbonyl), astaxanthin-di(N-acetylglycinate) (R is acetylaminomethyl), astaxanthin-dimaleinate (R is -CH=CH-COOH), astaxanthin-disuccinate (R is -CH₂- CH₂-COOH), astaxanthin-dimethyldisuccinate (R is -CH₂-CH₂-COOCH₃), astaxanthin- diethyldisuccinate (R is -CH₂-CH₂-COOC₂H₅), astaxanthin-diethyldiglycine- dicarbamate (R is -NH-CH₂-COOC₂H₅), astaxanthin-dinicotinate (R is 3-pyridyl), astaxanthin-dimethioninedicarbamate (R is -NHCH(CH₂CH₂SCH₃)COOC₂H₅), astaxanthin-diacetyldiglycolate (R is acetyloxymethyl), astaxanthin-diphenyl- alaninedicarbamate (R is -NHCH(CH₂C₆H₅)COOC₂H₅), astaxanthin-diethyldifumarate (R is -CH=CH-COOC₂H₅), astaxanthin-di(2-furoate) (R is 2-furyl), astaxanthin- dimethyldimalonate (R is -CH₂-COOCH₃), 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 -CH₂N⁺(CH₃)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 (I) as given above, whereby R and R* are independently from each other -A-(CO)OR^x with A being -CH₂-CH₂- 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-β,β-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 DSM 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 treatingthe 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 60 weight-%, more preferably its final amount is ranging 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 40 to 54 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 , even more preferably ranging from 3:1 to 6:1, most preferably ranging from 3.5:1 to 5.2:1.

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 1:10, preferably ranging from 1:1 to 1:7, more preferably ranging from 1:1.5 to 1:6, most preferably ranging from 1:2 to 1:5.5.

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-ketohexose-dimers, aldohexose-oligomers or modified aldohexose polymers 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 a-(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).

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, whereby a weight ratio of dried glucose syrup or dextrin to modified food starch ranging from 1:3 to 3:1, preferably from 1:2 to 2:1, most preferably of 1:1 is especially preferred. More details are given below.

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 pm. 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 pm. 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.

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 25 weight-%, most preferably its final amount is ranging from 10 to 22 weight-%, based on the total weight of the dry matter of the feed additive.

In a preferred embodiment of the present invention, the compound c) is a mixture of at least 5 weight-% of a hexose oligomer such as e.g. dextrins and at least 5 weight-% of a modified hexose polymer such as e.g. modified food starch, both amounts based on the total weight of the feed additive.

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. “Modified food starch"

A modified food starch is a food starch that has been chemically modified by known methods to have a chemical structure which provides it with a hydrophilic and a lipophilic portion. Preferably the modified food starch has a long hydrocarbon chain as part of its structure (preferably C5-C18).

At least one modified food starch is preferably used to make a feed additive of this invention, but it is possible to use a mixture of two or more different modified food starches in one feed additive.

Starches are hydrophilic and therefore do not have emulsifying capacities. However, modified food starches are made from starches substituted by known chemical methods with hydrophobic moieties. For example, starch may be treated with cyclic dicarboxylic acid anhydrides such as succinic anhydrides, substituted with a hydrocarbon chain. A particularly preferred modified food starch has the following formula (I) wherein St is a starch, R is an alkylene radical and R^' is a hydrophobic group. Preferably R is a lower alkylene radical such as dimethylene or trimethylene. R^' may be an alkyl or alkenyl group, preferably having 5 to 18 carbon atoms. A preferred compound of formula (I) is an "OSA-starch” (starch sodium octenyl succinate). The degree of substitution, i.e. the number of esterified hydroxyl groups to the number of free non-esterified hydroxyl groups usually varies in a range of from 0.1% to 10%, preferably in a range of from 0.5% to 4%, more preferably in a range of from 2% to 3%. The term “OSA-starch" denotes any starch from any natural source that was treated with octenyl succinic anhydride (OSA). The degree of substitution, i.e. the number of hydroxyl groups esterified with OSA to the number of free non- esterified hydroxyl groups usually varies in a range of from 0.1% to 10%, preferably in a range of from 0.5% to 4%, more preferably in a range of from 2% to 3%. "Modified food starch" is a synonym often used for OSA-starches.

The natural source of the starch may be corn, waxy corn, wheat, tapioca, pea and potato or synthesized.

The term “OSA-starches” encompasses also such starches that are commercially available e.g. from Ingredion under the tradenames HiCap 100, Capsul (octenylbutanedioate amylodextrin), Capsul HS, Purity Gum 2000, Cleargum COA1, Cleargum CO03, UNI-PURE, HYLON VII; from Ingredion and Roquette, respectively; from Cargill under the tradename C*EmCap or from Tate & Lyle.

In a preferred embodiment of the present invention a commercially available modified food starch such as Capsul and Capsul HS from Ingredion or Cleargum COA1 from Roquette, respectively, is used.

The terms "modified starches" and "OSA-starches” encompass further also modified starches/OSA-starches that were partly hydrolysed enzymatically, e.g. by glycosylases (EC 3.2; see http://www.chem.qmul.ac.Uk/iubmb/enzyme/EC3.2/), as well as to modified starches/OSA-starches that were partly hydrolysed chemically by known methods (so-called acid degradation).

The enzymatic hydrolysis is conventionally carried out at a temperature of from about 5°C to about < 100°C, preferably at a temperature of from about 5°C to about 70°C, more preferably at a temperature of from about 20°C to about 55°C.

The glycosylases can be from fruit, animal origin, bacteria or fungi. The glycolase may have endo-activity and/or exo-activity. Therefore, enzyme preparations of endo- and exo-glycosylases or any of their mixtures may be used. Preferably the glycosylases have pectolytic and/or hemicelluloytic activity. Usually the glycosylases show also unknown side activities, but which are not critical for the manufacture of the desired product.

Examples of glycosylases are the commercially available enzyme preparations from the suppliers Novozymes, Genencor, AB-Enzymes, DSM Food Specialities, Amano, etc.

The glycosylase is added to provide a concentration of from about 0.01 to about 10 weight-%, preferably of from about 0.1 to about 1 weight-%, based on the dry weight of the modified starch/OSA-starch. In a preferred embodiment of the process of the invention, the enzyme is added at once. The enzymatic hydrolysis may also be carried out stepwise. For instance, the glycosylase or a mixture of glycosylases is added to the incubation batch in an amount of e.g. 1 % whereupon, e.g. after 5 to 10 minutes (at a temperature of 35 °C) further glycosylase or a mixture of glycosylases which may by the same or different from the first added glycosylase or mixture of glycosylases is added, e.g. in an amount of 2 % whereupon the incubation batch is hydrolysed at 35°C for 10 minutes. Using this procedure, starting modified starches/OSA-starches having a degree of hydrolysis of approximately zero can be used.

The duration of hydrolysis may vary between about a few seconds and about 300 minutes. The exact duration of the enzymatic treatment may be determined in an empirical way with respect to the desired properties of the modified starch/OSA- starch, such as emulsifying stability, emulsifying capacity, droplet size of the emulsion, depending strongly on parameters like enzyme activities, or composition of the substrate. Alternatively, it may be determined by measuring the osmolality (W. Dzwokak and S. Ziajka, Journal of food science, 1999, 64 (3) 393-395).

The inactivation of the glycosylase is suitably achieved by heat denaturation, e.g. by heating of the incubation batch to about 80 to 85°C for 5 to 30 minutes, especially for 5 to 10 minutes. 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 2 to 15 weight-% and especially from 2 to 12 weight-%, even more preferably its final amount is ranging from 3 to 10 weight-%, most preferably its final amount is ranging from 3 to 8 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 (II) wherein R^1a and R^2a are independently from each other H or C_1-11-alkyl or (CH₂)_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(=0), 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 (II), wherein one of the two substituents R^1a and R^2a is C_12-21-alkyl and the other of the two substituents R^1a and R^2a is either hydrogen or C_1-5-alkyl or (CH₂)_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 (II), wherein A is CH₂, 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 (II) 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 (III) and (IV), wherein R^1b and R^2b are independently from each other H or Ci-n-alkyl or (CH₂)_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 R¹, R² and R³ are independently from each other H or linear C_1-6-alkyl or branched C_3-8-alkyl, whereby preferably R¹ is H or methyl or ethyl or n-propyl or iso-propyl or tert-butyl and R² and R³ 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 (VII) and (VIII) 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 (VII)/(VllI)” encompasses all possible isomers having any configuration at said centers.

Especially preferred examples of the compound of formula (VII) 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 (VIII) 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)/(21)/(22)/(23)/(24)/(25)/(26)/(27)” encompasses all possible isomers having any configuration at said centers.

The most preferred fat-soluble antioxidant is α-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 α-tocopherol and sodium ascorbate, whereby a weight ratio of α-tocopherol to sodium ascorbate ranging from 5:1 to 1:5 is especially preferred, a weight ratio of α-tocopherol to sodium ascorbate ranging from 3:1 to 1:3 is more preferred, a weight ratio of α-tocopherol to sodium ascorbate ranging from 2.5:1 to 1:1 is even more preferred, a weight ratio of α-tocopherol to sodium ascorbate ranging from 2:1 to 1:1 is especially more preferred, and a weight ratio of α-tocopherol to sodium ascorbate of 2:1 is most preferred.

Feed additives according to the present invention

The feed additive may be in liquid (= dispersion) or solid form.

The composition of such a feed additive 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 1:10, preferably ranging from 1:1 to 1:7, more preferably ranging from 1:1.5 to 1:6, most preferably ranging from 1:2 to 1:5.5. 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.

Preferred embodiments of the feed additives according to the present invention

The compositions of preferred solid feed additives according to the present invention, which comprise an absorbent, are listed in the following Tables 2-5. The total amounts of all ingredients are based on the total weight of the feed additive and sum up to 100 weight-%. It is understood that each single preferred amount of one ingredient may be combined with each preferred single amount of any other ingredient. The feed additives according to Table 4 are especially preferred.

Table 2

Instead of dried glucose syrups, also glucose syrups can be used. The amount is then calculated accordingly. In further preferred embodiments of the present invention the weight ratio of the dried glucose syrup to the lignosulfonate(s) b) is ranging from 2:1 to 1:10, preferably ranging from 1:1 to 1:7, more preferably ranging from 1:1.5 to 1:6, most preferably ranging from 1:2 to 1:5.5. Table 3

In further preferred embodiments of the present invention the weight ratio of the dextrin to the lignosulfonate(s) b) is ranging from 2:1 to 1:10, preferably ranging from 1:1 to 1:7, more preferably ranging from 1:1.5 to 1:6, most preferably ranging from 1:2 to 1:5.5.

Table 4

Instead of mixtures of dextrins with OSA-starches, also mixtures of glucose syrups or dried glucose syrups with OSA-starches can be used. In further preferred embodiments of the present invention the weight ratio of the dextrin and the OSA-starch to the lignosulfonate(s) b) is ranging from 2:1 to 1:10, preferably ranging from 1:1 to 1:7, more preferably ranging from 1:1.5 to 1:6, most preferably ranging from 1:2 to 1:5.5.

Table 5

In further preferred embodiments of the present invention the weight ratio of the hexose-dimer to the lignosulfonate(s) b) is ranging from 2:1 to 1:10, preferably ranging from 1:1 to 1:7, more preferably ranging from 1:1.5 to 1:6, most preferably ranging from 1:2 to 1:5.5..

Further preferred feed additives according to Tables 2-5 are feed additives, 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 feed additive excluding the absorbent.

If the feed additives are liquid, the amounts of their ingredients a) to d) have to be adjusted accordingly to obtain feed additives with the same relative amounts of the single ingredients a) to d) of the feed additives (see also Tables 1-5). The amount of water in said liquid feed additives is preferably ranging from 30 to 60 weight-%, more preferably ranging from 35 to 55 weight-%, even more preferably ranging from 40 to 50 weight-%, most preferably ranging from 45 to 50 weight-%, based on the total weight of the liquid 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) and the compound c) and optionally a water-soluble antioxidant in water; ii) Providing an active phase by dissolving the carotenoid and optionally the fat-soluble antioxidant in an organic solvent; iii) Mixing the matrix obtained in step i) and the active phase obtained in step ii) to obtain an emulsion; iv) Comminuting the droplets of the active phase in the emulsion obtained in step iii) and removing the organic solvent to obtain a dispersion; v) Drying said dispersion obtained in step iv) in presence of an absorbent to obtain the solid feed additive.

The feed additive may be used in liquid or in solid form. The liquid form (= dispersion) is obtained after step iv), the solid form after step v). Preferably the feed additive of the present invention is used in solid form.

The single steps of the process of the manufacture of the feed additive and its precursor, i.e. the dispersion obtained after having performed step iv), 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 liquid or solid feed additive after having performed steps i) to iv) and 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 40°C to 62°C, most preferably at a temperature ranging from 50° C to 60° C.

Step ii)

The amounts of the carotenoid a) and the fat-soluble antioxidant d), if present, are chosen so that the final amounts of these compounds in the resulting liquid or solid feed additive after having performed steps i) to iv) and i) to v), respectively, is as described above. The amount of the organic solvent and the dissolution temperature are chosen so as to dissolve the carotenoid a) and the fat-soluble antioxidant d), if present, completely. Usually it is necessary to heat up the suspension obtained when mixing all compounds present in this step to get a solution.

Examples of suitable organic solvents are methanol, ethanol, n-propanol, iso- propanol, 1-methoxy-2-butanol, 1-propoxy-2-propanol, tetrahydrofuran, acetone, dichloromethane, chloroform, tetrachloromethane, dimethyl carbonate, diethyl carbonate, propylene carbonate, ethyl formate, methyl acetate, ethyl acetate, iso- propyl acetate and methyl tert-butyl ether.

The temperature to which the suspension is heated up is e.g. ranging from 40 to 90° C, more preferably the temperature is ranging from 50 to 80°C.

Step iii)

Preferably this step is performed at a mixing temperature ranging from 30 to 70°C, more preferably at a mixing temperature ranging from 35 to 65°C, even more preferably at a mixing temperature ranging from 40°C to 60°C to obtain an emulsion.

Step iv)

The comminution 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.

The organic solvent may e.g. be removed by using a rotary evaporator or a thin film evaporator cascade. Other methods known to the person skilled in the art are also applicable. Step v)

The drying step is preferably carried out. Thereby the liquid form is converted into a solid form.

The conversion to the solid form can 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 fluidised bed granulation.

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 particle size ranging from 100 to 300 nm, preferably ranging from 120 to 250 nm, more preferably ranging from 130 to 230 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 narrow, meaning that the inner phase of the solid feed additive after step v), when re-dispersed in deionized water, has a D (10%) ranging from 60 to 190 nm, preferably ranging from 70 to 180 nm, more preferably ranging from 80 to 170 nm, even more preferably ranging from 90 to 160 nm, most preferably ranging from 100 to 150 nm, and/or a D (50%) ranging from 130 to 270 nm, preferably ranging from 140 to 260 nm, more preferably ranging from 150 to 250 nm, even more preferably ranging from 160 to 240 nm, most preferably ranging from 170 to 230 nm, and/or a D (90%) ranging from 200 to 500 nm, preferably ranging from 230 to 390 nm, more preferably ranging from 240 to 380 nm, even more preferably ranging from 250 to 370 nm, especially more preferably ranging from 260 to 360 nm, most preferably ranging from 270 to 350 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 60 to 190 nm and a D_v(50) ranging from 130 to 270 nm and a D_v(90) ranging from 200 to 500 nm. The most preferred feed additive has a D_v(10) ranging from 90 to 160 nm and a D_v(50) ranging from 170 to 230 nm and a D_v(90) ranging from 270 to 350 nm.

Dispersions according to the present invention The present invention is also directed to the dispersion as obtained after having performed step iv) as well as to the following dispersion comprising:

- at least a carotenoid in an amount ranging from 0.5 to 25 weight-%;

- at least a lignosulfonate in an amount ranging from 35 to 80 weight-%;

- 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 in an amount ranging from 5 to 30 weight-%, whereby further optionally at least one hexose may be present;

- at least an antioxidant in an amount ranging from 0 to 20 weight-%; wherein the amount of ethoxyquin in said dispersion is ≤ 0.5 weight-%; and wherein the amount of butylated hydroxytoluene in said dispersion is ≤ 0.5 weight-%; all amounts based on the total weight of the dry matter of said dispersion.

The amount of water in the dispersion is chosen in such a way so that its final amount in the dispersion is preferably ranging from 30 to 60 weight-%, more preferably its final amount is ranging from 35 to 55 weight-%, most preferably its final amount is ranging from 40 to 50 weight-%, based on the total weight of the dispersion.

The amount of the carotenoid in the dispersion is chosen in such a way so that its final amount in the dispersion is preferably ranging from 1.0 to 20 weight-%, more preferably its final amount is ranging from 2.0 to 20 weight-%, most preferably its final amount is ranging from 9 to 16 weight-%, based on the total weight of the dry matter of the dispersion.

The amount of the lignin derivative is chosen in such a way so that its final amount in the dispersion is preferably ranging from 35 to 80 weight-%, more preferably its final amount is ranging from 40 to 75 weight-%, even more preferably its final amount is ranging from 42 to 68 weight-%, most preferably its final amount is ranging from 44 to 66 weight-%, based on the total weight of the dry matter of the dispersion. The amount of the compound c) is chosen in such a way so that its final amount in the dispersion is preferably ranging from 10 to 30 weight-%, more preferably its final amount is ranging from 12 to 28 weight-%, based on the total weight of the dry matter of the dispersion.

The total amount of the antioxidant is chosen in such a way so that its final amount in the dispersion is preferably ranging from 1.0 to 12 weight-%, more preferably its final amount is ranging from 2.0 to 11 weight-%, most preferably its final amount is ranging from 5 to 11 weight-%, based on the total weight of the dry matter of the dispersion.

The particle sizes of the dispersion are the same as given above for the solid/dried feed additive.

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 14. 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 50 to 70 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 as shown in Tables 7, 8, 10-12 and 15.

Usually fish feed is stored for a maximum of 4-6 weeks. As shown in Table 15 the maximum loss of the carotenoid is 10% of the initial concentration after 12 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 ( Sporus 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 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 solar 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 50 to 70 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 1-7: Preparation of the feed additive according to the present invention

Examples 1.2.4 and 6: Method A

The used ingredients and their amounts are given in Table 6. As compound c) sucrose, dried glucose syrups such as Glucidex 21 (as commercially available from Roquette) or Glucidex IT 47 (as commercially available from Roquette), dextrins such as Dextrin Yellow (as commercially available from Roquette) and modified food starch such as Capsul and Capsul HS (as commercially available from Ingredion) are used.

The lignosulfonate, the compound c) and the water-soluble antioxidant are dissolved in deionized water¹⁾ to obtain the so-called "matrix”.

¹⁾ Method A a) whereby the pH is not changed;

Method A b) including an adjustment to pH 7.0 using aqueous sodium hydroxide (32% weight/weight).

An aliquot of 800-900 g is transferred to a reactor vessel and stirred at 36° C. Astaxanthin, the fat-soluble antioxidant (D/L-α-tocopherol) and an organic solvent is heated above the boiling point of said solvent until complete dissolution of astaxanthin and the fat-soluble antioxidant (so-called "active phase”). Subsequently, the active phase is pre-emulsified into the matrix at 5000 rpm ("rotations per minute”) using a rotor/stator. Subsequently, the pre-emulsion is further homogenized by two passages at 36°C using high-pressure homogenization. The organic solvent is evaporated under vacuum. A liquid feed additive according to the present invention is obtained.

Subsequently, the emulsion is sprayed into fluidized corn starch. The obtained beadlets (solid feed additive according to the present invention) remains in the corn starch for 45 to 60 minutes. The dried beadlets are sieved and the fraction with a particle size ranging from 160 to 630 pm used.

To determine the particle size, the dried beadlets are-dispersed in deionized water and measured using a particle analyzer Delsa™ Nano S (Beckmann Coulter, New Castle, DE, USA) via dynamic light scattering. The measured particle sizes are given in Table 6.

Examples 3 and 5: Method B

The lignosulfonate, the compound c) and the water-soluble antioxidant are dissolved in deionized water, including an adjustment to pH 7.2 using aqueous sodium hydroxide (20% weight /weight) (so-called "matrix”). An aliquot of 341 g is transferred to a reactor vessel and stirred for 20 minutes at 52°C and 1500 rpm using a rotor /stator device. Astaxanthin, the fat-soluble antioxidant (D/L-α-tocopherol) and an organic solvent is heated at the boiling point of said solvent until complete dissolution of astaxanthin and the fat-soluble antioxidant (so-called "active phase”). Subsequently, the active phase is emulsified into the matrix for 30 min at 53°C and 10 000 rpm. Subsequently, the pre-emulsion is homogenized at 150 bar. The organic solvent is evaporated. A liquid feed additive according to the present invention is obtained. Subsequently, the emulsion (including optionally a viscosity adjustment via addition of water) is sprayed into fluidized corn starch. The obtained beadlets (solid feed additive according to the present invention) remain in the corn starch for 45 to 60 minutes. The dried beadlets are sieved (160-630 pm). To determine the particle size, the dried beadlets are-dispersed in deionized water and measured using a particle analyzer Delsa™ Nano S (Beckmann Coulter, New Castle, DE, USA) via dynamic light scattering. The measured particle sizes are given in Table 6.

Table 6: All amounts are given in weight-%, based on the total weight of the solid feed additive.

Astaxanthin stability during stress testing exposing prototypes to ambient environment at 25°C / 60% r.h. and 40°C / 75% r.h.(feed additives according to examples 1-7) Aliquots of 120 mg are weighed into perforated capped plastic tubes. Thus, the samples are exposed to the environment during storage for 4 and 8 weeks in climatized rooms at 25°C / 60% relative humidity (r. h.) as well as 40°C / 75% r. h., respectively. The total astaxanthin concentration is determined by HPLC (high performance liquid chromatography) at the respective storage time. In brief, the aforementioned aliquot of 120 mg is transferred to a flask containing 200 mg BHT (butylated hydroxytyrosol). BHT prevents further degradation of the astaxanthin during the analytical procedure. Subsequently, the sample is re-dispersed in 10 mL deionized water at 50°C. After adding 100 mL ethanol and 80 ml dichloromethane, the mixture is kept for 30 min at room temperature. Afterwards, the sample is made up to 200 mL with dichloromethane and an aliquot of 10 mL is centrifuged for 5 min at 4000 rpm. The supernatant of the centrifuged stock solution is filled into amber glass vials for HPLC analysis. Astaxanthin retentions are calculated based on the initial concentration. Each test is performed two times. The average of the results is shown in Table 7. The accuracy is ± 5%. The starting value is 100%. The results show that astaxanthin is physically stable under both conditions for at least 8 weeks.

Table 7: The accuracy is ± 5%. The starting value is 100%.

Astaxanthin stability during per-se stability tests bv storing prototypes in sealed aluminum pouches at 25°C / 60% r. h. and 40°C / 75% r. h.(feed additives according to examples 1-7) Aliquots of 1.5 g are weighed into aluminum pouches and sealed. Samples are stored in climatized rooms at 25°C / 60% r. h as well as 40°C / 75% r. h. for 4 and 8 weeks. The total astaxanthin concentration is determined by HPLC at the respective storage time. In brief, 200 mg BHT and an aliquot of 120 mg sample are weighed into a volumetric flask (200 ml) and re-dispersed in 10 mL deionized water at 50°C. BHT prevents further degradation of the astaxanthin during the analytical procedure. After adding 100 mL ethanol and 80 ml dichloromethane, the mixture is kept for 30 min at room temperature. Subsequently, the sample is made up to 200 mL with dichloromethane and an aliquot of 10 mL is centrifuged for 5 min at 4000 rpm. The supernatant of the centrifuged stock solution is filled into amber vials for HPLC analysis. Astaxanthin retentions are calculated based on the initial concentration. Each test is performed two times. The average of the results is shown in Table 8. The accuracy is ± 5%. The starting value is 100%. The results show that astaxanthin is physically stable under both conditions for at least 8 weeks.

Table 8: The accuracy is ± 5%. The starting value is 100%.

Example 8a-c

Example 8 has been carried out three times on large scale. The composition is given in Table 6. The particle size distribution of the obtained solid feed additives, measured via dynamic light scattering using especially the particle analyzer Delsa™ Nano S (Beckmann Coulter, New Castle, DE, USA), are as follows:

Table 9 Furthermore, the stability of these feed additives has been evaluated at 15°C, at 30°C and a relative humidity of 75%, as well as at 40°C and a relative humidity of 75%. The results as well as the filtration residues are shown in the following Tables 10-13: Table 10

Table 11

Table 12 Table 13: Filtration Residues

Example 9: Fish trials using a feed additive according to example 6 a) Production of the feed

Pellets having a particle size of 7 and 9 mm are produced with the following composition:

Table 14

Thus, the feed additive according to the present invention is added pre-extrusion with the other micro ingredients. The 7 mm pellets are fed to fish with a weight ranging from 0.6-1.2 kg, the 9 mm pellets are fed to fish with a weight ranging from 1.2-3.0 kg.

The storage stability of the feed additive according to example 6 in the 9 mm pellets has also been determined. The astaxanthin ("AXN”)content is measured 2 weeks, 4 weeks, 8 weeks and 12 weeks after storage at 23° C. Usually fish feed is stored for a maximum of 4-6 weeks. As shown in Table 15 the maximum loss is 10% after 12 weeks which fulfills the requirements of the market:

Table 15 b) Fish trial As fish salmon is used. 345 fish are randomly distributed into 3 cages (5 x 5 m steel cages) with 115 fish per cage. All fish are individually weighed and counted into the trial. The average body weight at the start of the trial is 805 g (minimum: 709 g; maximum: 880 g; coefficient of variation ("CV") = 0.53%). Sampled fish are euthanized by a percussive blow to the head. The sampled fish is bled, gutted including the removal of the kidney, and weighed for dress-out-loss calculation. Photofish is used to assess content of fat and color in the Norwegian quality cut (so called "NQC”; see Fig. 1).

The trial started at October 17, 2019. The fish was fed to satiation by two daily meals from start to 31st of October, and one daily meal from 1st of November to 28th of February, and two daily meals from 1st of March. The daily ration was based on satiation feeding by hand, using distinctive meals. The feeding rate is adjusted according to the feeding response of the fish in the cage.

On December the 6th the fish were bulk weighed in connection with change in pellet size from 7 to 9 mm. The average weight at this point was 1438 g. The final sampling was done in week 27 (30th of June- 1st of July, 2020), the final fish weight being ca. 3.5 kg.

The color evaluation of the NQC is done by Photofish (see Fig.2). Photofish by AKVAGROUP (https://www.akvagroup.com/software-/photofish) is based on automated photo analysis offish samples. Samples are cut from the NQC cutlet and placed on the test tray ready for photography, and the measurement board is passed through the Photofish box and photographed. The actual measurement process is automated and controlled by the software. The software calulates the color given as SaloFan score and the concentration of astaxanthin [mg/kg] and fat [%].

The CIE 1976 L*a*b* color space (also referred to as CIELAB) is one of the most popular color spaces for measuring object colors. It was defined by CIE in 1976 for color communication and is widely adopted today in many industries for color control and management. In the L*a*b* color space, L* indicates lightness and a* and b* are chromaticity coordinates, a* and b* are color directions: +a* is the red axis, -a' is the green axis, +b* is the yellow axis and -b* is the blue axis. The L* and +a* and +b* were measured by a hand-held Minolta (type) on the positions indicated in Fig.3. The muscle retention is calculated as follows:

Muscle retention of astaxanthi whereby "0.6" represents the assumption that the white muscle constitutes 60% of the whole body in salmon (Wathne et al.„ Aquaculture 1998, 159, pages 217-231: "Pigmentation of Atlantic salmon (Salmo salar) fed astaxanthin in all meals or in alternating meals.”; Einen et al., Aquaculture 1999, 178, pages 149-169; "BW” = body weight and "Ax" = astaxanthin concentration in mg per kg. c) Results

The astaxanthin content in NQC as measured by photofish is 5.7 mg/kg (standard deviation: 0.2). The lightness L* is 37.7 (standard deviation: 1.6), the redness a* is 10.3 (standard deviation: 0.9) and the yellowness is 13.2 (standard deviation: 1.3).

The muscle retention is 7.8% (standard deviation: 0.2).

It is shown that salmon fed with a feed comprising the feed additive according to the present invention shows the desired muscle retention. Furthermore, the feed additive shows a good stability in the feed.

Examples 10-12: Fish trials using a feed additive according to examples 2b. 4b and 6. 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 (50-60 mg/kg). Astaxanthin is added to the mash pre-extrusion. The ingredients and the basal composition of the diets are presented in Table 16. Table 16

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 123.3 ± 3.8 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 approximately identified by each number in Fig.4. Readings are taken for mean redness, yellowness, lightness, hue, and chroma (see Table 17).

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.4. 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 accumulats in the fillet to exceed the industry target minimum of 7 mg/kg fillet astaxanthin.

Table 17: 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.

Examples 13-14: Preparation of the feed additive according to the present invention The used ingredients and their amounts are given in Table 18. The preparation is as described for examples 1-7 above according to Method A b).

To determine the particle size, the dried beadlets are-dispersed in deionized water and measured using a particle analyzer Delsa™ Nano S (Beckmann Coulter, New Castle, DE, USA) via dynamic light scattering. The measured particle sizes are given in Table 18.

Table 18: All amounts are given in weight-%, based on the total weight of the solid feed additive.

Examples 15-16: Fish trials using a feed additive according to examples 13 and 14. respectively a) Production of the feed

The feed is produced as described above for examples 10-12. b) Fish trial

The fish trial is performed as described above for examples 10-12. The average body weight at the start of the trial is 121.0 ± 0.1 g. 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 19: Mean ± standard deviation ("SD”) of fillet color characteristics, fillet astaxanthin and astaxanthin retention offish from day 0 to day 86.

Claims

1. A feed additive comprising a) at least a carotenoid in an amount ranging from 0.5 to 25 weight-%; b) at least a lignosulfonate in an amount ranging from 35 to 60 weight-%; 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 in an amount ranging from 5 to 25 weight-%, whereby further optionally at least one hexose may be present; d) at least an antioxidant in an amount ranging from 0 to 20 weight-%; e) at least an absorbent in an amount ranging from 1 to 20 weight-%; and residual moisture in an amount ranging from 0 to 10 weight-%; wherein the lignosulfonate b) and the compound c) form a matrix in which the carotenoid is encapsulated; wherein the amount of ethoxyquin in the feed additive is ≤ 0.5 weight-%; and wherein the amount of butylated hydroxytoluene in the feed additive is ≤ 0.5 weight-%; whereby all amounts sum up to 100 weight-% and are based on the total weight of the feed additive.

2. The feed additive according to claim 1, wherein the carotenoid is astaxanthin or a derivative thereof or canthaxanthin or a derivative thereof, preferably wherein the carotenoid is astaxanthin or a derivative thereof, more preferably wherein the carotenoid is astaxanthin or astaxanthin dimethyl succinate, most preferably wherein the carotenoid is astaxanthin.

3. The feed additive according to claim 1 and/or 2, wherein the lignosulfonate b) is sodium lignosulfonate or calcium lignosulfonate or any mixture thereof.

4. The feed additive according to any one or more of the preceding claims, wherein the inner phase of the feed additive has an average diameter of the particles ranging from 100 to 300 nm, preferably ranging from 120 to 250 nm, more preferably ranging from 130 to 230 nm, measured via dynamic light scattering.

5. The feed additive according to any one or more of the preceding claims having a D (90%) ranging from 200 to 500 nm, preferably ranging from 230 to 390 nm, more preferably ranging from 240 to 380 nm, even more preferably ranging from 250 to 370 nm, especially more preferably ranging from 260 to 360 nm, most preferably ranging from 270 to 350 nm, measured via dynamic light scattering.

6. The feed additive according to any one or more of the preceding claims, wherein 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 , even more preferably ranging from 3:1 to 6:1, most preferably ranging from 3.5:1 to 5.2:1.

7. The feed additive according to any one or more of the preceding claims, wherein the weight ratio of the compound(s) c) to the lignosulfonate(s) b) is ranging from 2:1 to 1:10, preferably ranging from 1:1 to 1:7, more preferably ranging from 1:1.5 to 1:6, most preferably ranging from 1:2 to 1:5.5.

8. The feed additive according to any one or more of the preceding claims, wherein compound c) is selected from dried glucose syrups 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, and their mixtures with modified food starches, whereby a weight ratio of dried glucose syrup to modified food starch of 1:1 is especially preferred.

9. The feed additive according to any one or more of claims 1 to 7, wherein compound c) is selected from starch hydrolysates that have a maximum amount of 10 weight% of reducing sugars, based on the total weight of the starch hydrolysate, and their mixtures with modified food starches.

10. The feed additive according to any one or more of the preceding claims, wherein the antioxidant is tocopherol, preferably α-tocopherol, more preferably DL- α-tocopherol, even more preferably a mixture of DL- α- tocopherol with sodium ascorbate.

11. The feed additive according to any one or more of the preceding claims, wherein the absorbent is starch.

12. A process for the manufacture of a feed additive according to any one or more of the preceding claims comprising the following steps: i) Providing a matrix by dissolving the lignosulfonate and the compound c) and optionally a water-soluble antioxidant in water; ii) Providing an active phase by dissolving the carotenoid and optionally the fat-soluble antioxidant in an organic solvent; iii) Mixing the matrix obtained in step i) and the active phase obtained in step ii) to obtain an emulsion; iv) Comminuting the droplets of the active phase in the emulsion obtained in step iii) and removing the organic solvent to obtain a dispersion; v) Drying the dispersion obtained in step iv) in presence of an absorbent to obtain the feed additive.

13. A dispersion as obtained after having performed step iv) of the process according to claim 12.

14. A dispersion comprising

- at least a carotenoid in an amount ranging from 0.5 to 25 weight-%;

- at least a lignosulfonate in an amount ranging from 35 to 80 weight-%;

- 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 in an amount ranging from 5 to 30 weight-%, whereby further optionally at least one hexose may be present; - at least an antioxidant in an amount ranging from 0 to 20 weight-%; wherein the amount of ethoxyquin in said dispersion is ≤ 0.5 weight-%; and wherein the amount of butylated hydroxytoluene in said dispersion is ≤ 0.5 weight-%; all amounts based on the total weight of the dry matter of said dispersion.

15. Feed comprising a feed additive according to any one or more of claims 1 to 11.

16. The feed according to claim 15, wherein the feed is preferably for aquatic animals, more preferably for crustaceae and farmed fish, even more preferably for salmonids, most preferably for salmons.

17. Use of the feed additive according to any one or more of claims 1 to 11 or the feed according to any one or more of claims 15 to 16 for pigmentation of an animal excluding humans.

18. A method of pigmenting an animal excluding humans by administering a feed additive according to any one or more of claims 1 to 11 or a feed according to any one or more of claims 15 to 16 to said animal.

19. The use according to claim 17 or the method according to claim 18, wherein the animal is an aquatic animal, preferably wherein the animal is crustaceae or farmed fish, more preferably wherein the animal is salmonids, most preferably wherein the animal is salmon.