Background of the Invention

The present invention relates to the use of 3'-Dehydrolutein or 3'-Dehydrolutein derivatives or metabolites thereof as an additive for animal feed.

Specific examples of metabolites of 3'-Dehydrolutein include £,£-Carotene-3,3'-dione and β,ε- Carotene-3'-one. More particular, this invention relates to the use of 3'-Dehydrolutein as an additive for poultry feed to provide desirable yellow shade coloration of egg yolks and poultry skins.

Furthermore, the present invention relates to a novel feed composition for poultry feed which composition comprises as active ingredients 3'-Dehydrolutein.

The term feed or feed composition means any preparation, mixture, premix or composition suitable for, or intended for intake by an animal.

The genus poultry encompasses turkeys, ducks and chickens including but not limited to broiler chicks and layers. Preferably the animal is a laying hen.

Lutein is commercially produced in large quantities from marigold flowers (Tagetes erecta) for use as a food or feed additive. For feed applications, Lutein is semi-purified by several means and is sold for formulation into chicken feed where it imparts a strong yellow coloration to the egg yolk and also to the skin. Specifically it is used widely to color chicken egg yolks.

However, while Lutein imparts a strong yellow color to the yolk, it is desirable in many markets to add red coloration so that the resulting egg yolk is more orange in color. Typically this red coloration is obtained by the addition of a second compound / carotenoid e.g. paprika extracts or Canthaxanthin to the feed. Addition of these extracts or of Canthaxanthin to the feed significantly increases the expense. There is a need for alternative carotenoid products which either have a higher pigmentation potency than Lutein and/or which can impart the desired coloration without subsequent addition of other colorants.

It is therefore the object of the present invention to modify lutein to a product which has higher pigmentation potency than Lutein itself and/or produces the desired orange-red coloration or a different coloration, so that less carotenoids or a single carotenoid can be used for

pigmentation.

Summary of the Invention

It has now been found surprisingly that 3'-Dehydrolutein of formula (I), an asymmetric carotenoid found as natural metabolite of Lutein or Zeaxanthin in human plasma, but also found in low amounts in plant extracts, can be used for the desired yellow coloration with increased pigmentation potency compared to Lutein.

Formula (I)

3'-Dehydrolutein of formula I can be in any possible isomeric form or in the form of mixtures of isomers, e.g. racemate mixtures.

Preferred Example of 3'-Dehydrolutein of the formula I is:

(SR.e'RJ-S'-dehydrolutein

3'-Dehydrolutein according to the invention can easily be prepared by a skilled person starting from Lutein using processes and methods well-known in the prior art.

For example, 3'-Dehydrolutein can be synthesized from Lutein by oxidation e.g. with Mn02 , [Molnar, Peter; Deli, Jozsef; Osz, Erzsebet; Toth, Gyula; Zsila, Ferenc; Herrero, Christian; Landrum, John T.; Preparation and spectroscopic characterization of 3'- oxolutein; Letters in Organic Chemistry (2006), 3(10), 723-734.] or by Oppenauer oxidation which are common methods for allylic oxidation. The conversion of Lutein to 3'-Dehydrolutein is shown in the following diagram:

acetone, AI(OiPr) ₃

CH ₂ CI ₂ , 40 °C

(3R,6'R)-3'-Dehydrolutein

In preferred embodiments, the starting compound Lutein is obtained by saponification of a plant extract. Preferably, the plant is marigold. In alternate preferred embodiments, Lutein is prepared synthetically.

In accordance with the present invention 3'-Dehydrolutein can be used in highly purified forms, in mixtures of 3'-Dehydrolutein and metabolites or derivatives thereof, or in the form of natural available plant extracts or extract-mixtures containing enriched 3'-Dehydrolutein concentrations.

By the expression "natural" and "natural substance" is in this context understood a substance which consists of compounds occurring in nature and obtained from natural products or through synthesis.

Embodiments of the invention are directed to the use of 3'-Dehydrolutein of formula (I) as an animal feed additive for pigmentation purposes, especially as an additive for poultry feed to provide desirable coloration of egg yolks and poultry skins.

In preferred embodiments, the 3'-Dehydrolutein is added to the final feed in an amount sufficient to produce a concentration of 3'-Dehydrolutein product of between about 1 to 160 mg/kg feed. Preferably, the 3'-Dehydrolutein is added to the feed of a laying hen in an amount sufficient to produce a concentration of 3'-Dehydrolutein product in the feed of between about 2 to 80 mg/kg feed. Most preferably, the 3'-Dehydrolutein is added to the feed of a laying hen in an amount sufficient to produce a concentration of 3'-Dehydrolutein product in the feed of between about 4 to 40 mg/kg feed.

Embodiments of the invention are also directed to animal feed compositions which composition comprises as active ingredients 3'-Dehydrolutein, wherein the term feed or feed composition means any preparation, mixture, premix or composition suitable for, or intended for intake by an animal.

Embodiments of the invention are also directed to a method of using 3'-Dehydrolutein of formula (I) as an animal feed additive for pigmentation purposes, especially as an additive for poultry feed to provide desirable coloration of egg yolks and poultry skins. Further aspects, features and advantages of this invention will become apparent from the detailed description of the preferred embodiments which follow.

Detailed Description of the Preferred Embodiment

A preferred method to produce 3'-Dehydrolutein is directed to an oxidation procedure for conversion of lutein to 3'-Dehydrolutein. By a simple oxidation procedure, lutein can be converted in good yields to 3'-Dehydrolutein which, in fact, possesses the desirable yellow coloration.

This oxidation procedure can be carried out in various ways such that only the desired side of the molecule is affected.

Preferably, the reaction is carried out at any temperature between 0° C and 70° C. More preferably, the reaction is carried out at temperatures between 10 and 50° C.

The carotenoid product may be separated from the reaction mixture by extraction into the organic phase. Optionally, the organic phase may be washed after extraction of the product.

In a preferred embodiment, 3'-Dehydrolutein may be synthesized in one chemical step by selective allylic oxidation of lutein using acetone/aluminium isopropylate in dichloromethane. As described in Example 1 , after 16 hours reaction time at 40 °C and following recrystallisation 3'-Dehydrolutein may be obtained in 60 - 90 % yield and up to 98% purity.

3'-Dehydrolutein obtained by the described method is added to animal feed to provide desirable yellow coloration in the resulting food products. In preferred embodiments, compositions including 3'-Dehydrolutein prepared by the described method are used as a chicken feed additive to achieve desirable yellow-orange coloration of chicken skin and/or egg yolks. For that purpose, 3'-Dehydrolutein and optionally metabolites and derivatives thereof may be used alone or as a mixture together with Lutein. Furthermore, the purified 3'-Dehydrolutein may be combined with other carotenoids to provide desired coloration as for example with Canthaxanthin in order to get a more orange-red coloration.

The 3'-Dehydrolutein product may also be added directly to foods, i.e. egg products as pasta, to provide the desirable coloration.

For the realization of their use as pigmenting agents for the feed of poultry 3'-Dehydrolutein may be incorporated in the animal feed by methods known per se in the art of feed formulation and processing, in principle by admixture with at least some of the components of the final feed at an appropriate stage of its manufacture. The 3'-Dehydrolutein is normally incorporated as a formulation, particularly a water-dispersible formulation. Such a formulation can be produced in principle by first dissolving 3'-Dehydrolutein in a plant or vegetable oil or fat, e.g. corn oil, or in an organic solvent, e.g. an alcohol, an aliphatic ether, a halogenated aliphatic hydrocarbon such as dichloromethane, or an aliphatic ester, or in a mixture of both a plant or vegetable oil or fat and an organic solvent. The dissolution can be effected in a broad temperature range, e.g. in the range from room temperature to about 150°C. In the case of using dichloromethane as the organic solvent for the dissolution, this can be affected at room temperature or at temperatures up to about 30°C, or at higher temperatures on application of elevated pressure. The use of relatively low temperatures when using dichloromethane or an alternative solvent is particularly advantageous for preventing 3'-Dehydrolutein from being subjected to unnecessarily high dissolution temperatures, and so represents an economical and mild processing. Moreover, dichloromethane in particular dissolves 3'-Dehydrolutein so readily that further economic savings are gained through the use of relatively low volumes of this solvent; as one result thereof, less of the solvent needs to be removed by evaporation and then disposed of or recycled, and as another, the processing of the lower solution volume can be effected more rapidly. After completion of the dissolution the solution is normally emulsified with an aqueous solution of a protective colloid, e.g. a plant or animal protein such as a gelatin, particularly fish gelatin; a carbohydrate; polysaccharide; or a ligninsulphonate. The solvent and water are then at least partially removed, thus affording the formulation as a concentrated emulsion. Before the actual incorporation in the feed the concentrated emulsion can either be directly spray-dried by conventional spray-drying techniques or spray-dried into fluidized starch or an alternative carrier, e.g. calcium silicate, again by conventional techniques. The product of such spray- drying consists of beadlets, which, apart from 3'-Dehydrolutein itself contains components from the previous processing, e.g. oil, protective colloid, starch etc., and which may contain up to about 25% by weight of said derivative; the content of any oil present is generally in the range from about 0.5% to about 50% by weight, the content of matrix material (principally protective colloid) generally in the range from about 50% to about 80% by weight, and the content of any carrier material from the spray-drying (starch, calcium silicate etc.) generally from about 10% to about 25% by weight. Apart from the aforementioned materials, the beadlets may contain relatively minor amounts of stabilizers, emulsifying agents and other conventional formulation aids.

In accordance with the present invention 3'-Dehydrolutein can also be used in highly purified forms of natural available plant extracts or extract-mixtures. The term "extract" as used herein includes compositions containing enriched 3'-Dehydrolutein concentrations obtained by solvent extraction (which are also known as "extracted oils"), steam distillation (which are also known as "essential oils") or other separation methods known to the skilled person.

Finally, the 3'-Dehydrolutein formulation, particularly a water-dispersible formulation thereof or a natural extract containing an enriched 3'-Dehydrolutein concentration, is conventionally mixed with other components of the feed, such as fish oil and fish meal.

This can be carried out by preparing a premix of the active ingredients and other suitable additives. Such a premix may comprise 2 to 10% by weight of the active mixture or natural extract, 0 to 40% by weight of other conventional additives, such as flavorings, and 50 to 98% by weight of any conventional absorbing support.

The support material may contain, for example, 40 to 50% by weight of wood fibers, 8 to 10% by weight of stearine, 4 to 5% by weight of curcuma powder, 4 to 5% by weight of rosemary powder, 22 to 28% by weight of limestone, 1 to 3% by weight of a gum, such as gumarabic, 5 to 50% by weight of sugar and/or starch and 5 to 15% by weight of water. This premix is then mixed with vitamins, mineral salts and other feed additive ingredients and then finally added to the feed in such quantities that the feed will contain 1 to 80 ppm, preferably 2 to 40 ppm of 3'-Dehydrolutein.

Further aspects of the present invention are formulations containing 3'-Dehydrolutein derivatives of the present invention for incorporation into poultry feed, and such feed containing an effective amount of 3'-Dehydrolutein as a pigmenting agent, particularly a poultry feed containing from about 1 to about 80 ppm of 3'-Dehydrolutein. The present invention is further illustrated with reference to the following Examples. It will be understood by those of skill in the art that numerous and various modifications can be made without departing from the spirit of the present invention. Therefore, it should be clearly understood that the examples are illustrative only and are not intended to limit the scope of the present invention.

Example 1 - Preparation of 3'-Dehvdrolutein from Lutein

45.41 g of lutein were dissolved in a mixture of dichlorom ethane and acetone. At room temperature a solution of 14.71 g of aluminium isopropoxide in dichloromethane was added. After reaction overnight at 40°C the brown solution was cooled to room temperature and washed with water. The organic phase was concentrated under reduced pressure resulting in 38.64 g of crude 3'-dehydrolutein which was purified by column chromatography. After crystallization from methanol / dichloromethane, 22.84 g of orange-red crystalline 3 '-dehydrolutein were obtained in 97.4% purity and 87% yield. Example 2 - Formulation of 3'-Dehvdrolutein

Crystalline material of 3'-dehydrolutein may be formulated according to a method as described above. The composition of such an exemplified formulation (5% formulation) is summarized as follows:

3'-Dehydrolutein cryst. (purity 98% HPLC)

Ethoxyquin (EMQ) 1.0 %

Ascorbylpalmitate 1.0 %

Porcin Gelatin BI.140 Lot Si 0501072 38.5 %

Dextrin yellow 15.7 %

Sugar cryst. 15.7 %

Corn starch fluidized 22.1 %

Example 3 - Pigmentation Trials

Pigmentation tests of 3'-Dehydrolutein and Lutein were performed in egg yolk and broiler pigmentation. The formulated carotenoids were applied as single compounds in doses of 5, 10, 20 and 40 ppm. Mixtures were not investigated. Crystalline 3'-Dehydrolutein has been formulated as a 5% form as described above. The composition of the formulation of Lutein (Lutein 5% CWS/S-TG of DSM Nutritional Products, CH Kaiseraugst) used in the trial is as follows: Lutein extracted from Tagetes e recta 5 %

Corn starch 23 %

Modified food Starch 52 %

DL-alpha-Tocopherol 1 %

Glucose Syrup 17 %

Sodium Ascorbate cryst. 2 %

The formulated compounds were tested in egg yolks for their pigmentation properties, wherein the test was carried out according to the following protocol

3 weeks

mash, ad libitum

Laying hens Hy line Brown), 47 weeks-old at the beginning of the trial

3'-Dehydrolutein (3DL) at 5.0, 10.0, 20.0, 40.0 mg/kg

Lutein (LUT) at 5.0, 10.0, 20.0, 40.0 mg/kg

each group replicated by 4 groups of three hens

3'-Dehydrolutein (3DL) 5%, RS8/10-22 (6.3%)

Lutein 5% CWS/S-TG, UT09080011

After treatment the content of carotenoids in feed and egg and coloration properties have been determined by High Performance Liquid Chromathography (HPLC) as follows:

Determination of 3'-Dehydrolutein in feed:

The assay comprises an enzymatic digestion of the formulation mixed into the feed, followed by extraction with ethanol and dichloromethane. After purification from polar ingredients by a silica gel SPE-column the extract is injected into an isocratic normal-phase HPLC system that is able to resolve 3'-Dehydrolutein (3DL) from other xanthophylls like Lutein, Zeaxanthin, Apoester or Canthaxanthin. For HPLC, a mobile phase of acetone in n-heptane (19:81 , v/v) is pumped with a flow rate of 1.5 ml/min through a LiChrosorb Si60 column (250 x 4 mm). The carotenoids are detected at a wavelength of 450 nm.

Determination of 3'-Dehydrolutein in egg yolk: The method comprises a liquid-liquid extraction of the carotenoids from the diluted yolk into a n- hexane phase which is directly injected into the HPLC. The isocratic normal-phase HPLC system is able to resolve 3'-Dehydrolutein (3 DL) from other xanthophylls like Lutein,

Zeaxanthin, Apoester or Canthaxanthin as well as from the metabolites of 3'-Dehydrolutein, ε,ε- carotene-3,3'-dione and β,ε-θ3Γθίβηβ-3'-οηβ (3'-keto-a-carotene) which are described in more detail below. For HPLC, a mobile phase of acetone in n-heptane (19:81 , v/v) is pumped with a flow rate of 1.5 ml/min through a LiChrosorb Si60 column (250 x 4 mm). The carotenoids are detected at a wavelength of 450 nm.

The results from the in vivo pigmentation test were:

3'-Dehvdrolutein content in feed and egg

Dose FEED Egg Egg

mg/kg 3DL 3DL Metabolites

5 4.45 6.73 ± 1 .12 1 .4 ± 0.28

10 9.37 16.28 ± 1 .58 3.5 ± 0.37

20 19.80 35.90 ± 3.41 6.9 ± 1 .25

40 37.30 65.28 ± 5.20 1 1 .4 ± 1 .19

Lutein content in feed and eaa

Dose FEED EGG YOLK

mg/kg LUT LUT

5 5.15 8.05 ± 0.40

10 9.56 15.85 ± 0.61

20 19.90 31 .38 ± 1 .26

40 42.90 63.90 ± 4.82

Colourimetry

Carotenoid Lightness L* Redness a* Yellownessb* Color Hueh*ab Chroma Cab

3'-Dehydrolutein 61 .1 ± 4.53 17.0 ± 8.34 81 .7 ± 1 1 .9 78.7 ± 4.20 83.6 ± 13.3

Lutein 63.4 ± 3.52 13.4± 6.36 87.2 ± 12.2 81 .6 ± 2.99 88.3A ± 12.9

When all the data were pooled together irrespective of the dosage and a simple statistical comparison of the means was done and it was found that a factor of 1.3 times the amount of Lutein was required to obtain a similar pigmentation as with 3'-Dehydrolutein. In terms of colour score, 3'-Dehydrolutein is more efficient for improving pigmentation of the egg yolk compared to Lutein.

In another pigmentation experiment carried out as described above, 3'-Dehydrolutein was also tested in broiler pigmentation at 2.5, 5.0 and 10 mg/kg feed. The results showed that the concentration of 3'-Dehydrolutein in fat and skin increased with increasing dietary inclusion levels. In addition, the results demonstrated that 3'-dehydrolutein gave increasing yellowness values in the skin with increasing dosages in the feed. Further analysis of carotenoids in egg yolk by HPLC when feeding 3'-Dehydrolutein showed three distinctive peaks. The main compound detected was 3'-Dehydrolutein (-82% total carotenoids). The other two compounds are considered derivatives of 3'-Dehydrolutein as a result of natural metabolism activity. The derivatives that also showed pigmentation efficacy were:

£,£-Carotene-3,3'-dione („Diketone") β,ε-ΰ3Γθίβηβ-3'-οηβ (3'-Keto-a-carotene)