Carotenoid ketones and esters

Abstract: New carotenoids of the formula , wherein R and n have the significances given herein, are useful for pigmenting the egg yolk, integuments and/or subcutaneous fat of poultry and the meat and/or integuments of fish and crustacea when included in the feed of the poultry, fish or crustacea. As well as such carotenoids and method of pigmenting the invention further embraces the pertinent carotenoid-enriched feed for poultry, fish or crustacea, the premixes for incorporation in such feeds and beadlets containing one or more of these carotenoids for incorporation in such premixes and the use of the carotenoids in the pigmenting method. The process of manufacturing such carotenoids, which more specifically may be designated as carotenoid ketones, monoesters and diesters, also represents an aspect of the invention.

Description: It is well known that carotenoids are natural pigments which occur abundantly in the plant and animal kingdoms and which in some cases have also been produced by synthetic means. Further synthetically produced carotenoids do not appear to occur in nature. Many important carotenoids are employed as pigments in the food and feedstuff industries, e.g. for colouring egg yolk, poultry, fish and crustacea, notably in the cases of ethyl β-apo-8'-carotenoate, citranaxanthin, canthaxanthin and astaxanthin. For this purpose the carotenoid pigments are added to the animals' rations as a method of imparting an enhanced and aesthetically more acceptable visual impression of colour, be it in the animal integuments, such as the skin, shanks and beaks of poultry and the skin, scales and shells of fish and crustacea, as appropriate, subcutaneous fat of poultry and the meat of fish and crustacea, or in such animal products as eggs (yolk). The enhancement of pigmentation depends on the particular light-absorbing conjugated double bond system of the carotenoid concerned, the degree of ease with which the carotenoid is taken up into the animal body following consumption of the carotenoid-enriched feed (deposition rate) and the concentration of the carotenoid or any metabolites in the target animal body tissue or product, amongst many other factors. However, from a knowledge of the structure of the selected carotenoid it cannot be predicted how effectively it functions as a pigment in this area of application. A further factor involved is the stability of the carotenoid, e.g. towards atmospheric oxidation, light, temperature and dampness, in an animal feedstuff when stored under the normal conditions to which such a feedstuff is subjected. With respect to poultry an acceptable level and quality of pigmentation is desired for the integuments of the birds destined for consumption and for egg yolk. The use of materials to enhance yolk colour, for example, is generally promoted because consumers prefer deeply (particularly rich golden yellow) pigmented yolks. The visual appearance is indeed an important factor in the assessment of quality. Broilers and ornamental birds, for example, are in many parts of the world more aesthetically acceptable if their integuments, particularly skin, shanks and beaks, in the case of broilers also their subcutaneous fat, and in the case of ornamental birds also their plumage, satisfy certain criteria of pigmentation. The need for supplementary pigmentation is especially prevalent today in view of the reduction of grass consumption with the modern methods of intensive poultry rearing, which involve the use of low fibre, high energy feeds, rendering difficult the production of well-pigmented poultry and egg yolks. The pigmentation of fish meat and integuments, especially of various species of trout and salmon, and the meat and integuments of crustacea, e.g. crabs, lobsters and shrimps, is also well known to be achieved by feeding the fish and crustacea with carotenoid-enriched feed preparations with a view to rendering the edible products more attractive to consumers. Moreover, alimentary products such as dairy products, e.g. butter and cheese, and such cosmetic products as lipsticks are known to be pigmentable by incorporation of carotenoids at a suitable stage of the pertinent production processes. It has now been found that certain new carotenoids, as will be specified hereinafter, are effective as pigments in the above-indicated applications, or surprisingly are even more effective as such, than the carotenoids known to have been used hitherto for such purposes. According to the present invention there are provided new carotenoids of the general formula wherein R signifies a group (b) R 2 signifies C 1-15 -alkyl, and n signifies zero or an integer 1 to 4. The above and the hereinafter presented structural formulae are represented in the abbreviated manner, using simple lines, which is usual in carotenoid chemistry. In the scope of the present invention the term "C 1-6 -alkyl group" or "C 2-6 -alkenyl group" embraces straight-chain and branched groups such as, for example, methyl, ethyl, isopropyl and tert. butyl or, respectively, vinyl and 4-methyl-3-pentenyl. Similarly, the term "C 1-15 -alkyl" also embraces straight-chain and branched groups, and may be exemplified by methyl and pentadecyl. The above formula I (and each later appearing formula) embraces isomeric forms, e.g. optically active and cis/trans or E/Z isomers, as well as mixture thereof, unless an isomeric form is specifically indicated. With respect to E/Z isomerism, the all-E isomers are in general preferred. Specific examples of the new compounds of formula I are: The present invention further provides a method of pigmenting the egg yolk, integuments and/or subcutaneous fat of poultry, and the meat and/or integuments of fish and crustacea by including one or more carotenoids in the feed of such poultry, fish or crustacea, characterized in that said carotenoids are those of the general formula I given above. As a still further aspect the present invention provides a carotenoid-enriched feed for poultry, fish or crustacea and intended for the pigmentation of egg yolk, integuments and/or subcutaneous fat of the poultry and the meat and/or integuments of the fish and crustacea, characterized in that it contains as the carotenoid(s) one or more of the carotenoids of the general formula I given above in an effective amount. Those carotenoids of the formula I in which R signifies a group (b) [which are collectively embraced by the general formula and may conveniently be designated as carotenoid monoesters] can be produced by reacting the corresponding carotenoid alcohol, of the general formula with the appropriate alkanoic acid of the general formula R 2 COOH or with a reactive derivative thereof. The process of production of the carotenoids of the present invention hereinabove represents a further aspect of the present invention. As a reactive derivative of the alkanoic acid of formula VII useful in the process there may be used the acid chloride or anhydride. If the alkanoic acid itself is used, it is convenient to conduct the esterification in the presence of a dehydrating agent such as N,N'-dicyclohexylcarbodiimide. In either case it is furthermore convenient to conduct the esterification under basic conditions, i.e. in the presence of an added base, e.g. pyridine (which also simultaneously acts as solvent) or 4-dimethylaminopyridine, and indeed such esterification procedures are essentially well known to persons skilled in the art. A particularly preferred method of producing the carotenoid monoacetates (i.e. the carotenoid esters of formula Ib, in which R 2 signifies methyl), involves using acetic anhydride as the reactive derivative of acetic acid and conducting the esterification in a slightly basic organic solvent, especially pyridine, at temperatures in the range from about 50°C to about 75°C. The exclusion of air, conveniently by conducting the reaction under an atmosphere of inert gas, e.g. argon, is recommended. Isolation and purification of the product is conveniently effected by hydrolysing the post-reaction mixture with an ice/water mixture, extracting with diethyl ether, washing the organic phase with dilute sulphuric acid and water, drying it, e.g. over anhydrous sodium sulphate, filtering, and evaporating off the solvent, after which the so-isolated product can be purified by chromatography and recrystallization. A particularly preferred method of producing those carotenoid monoesters of formulae Ib, in which R 2 signifies a C 2-15 alkyl group, i.e. ethyl to pentadecyl, involves reacting the pertinent alcohol of formula V with an excess of the appropriate alkanoic acid in an aprotic solvent, especially toluene, in the presence of 4-dimethylaminopyridine as a base and of N,N'-dicyclohexylcarbodiimide as a dehydrating agent, at temperatures in the range from about 50°C to about 75°C. As in the case of the acetate, the reaction is advisably effected under an inert gas, e.g. argon, and the isolation and purification is also conveniently effected analogously, whereby in this case extra toluene rather than diethyl ether is preferably added to the post-reaction mixture and to extract the product. In general, an excess amount of the alkanoic acid of formula VII relative to the amount of carotenoid alcohol of formula V, is used in both methods, whereby an excess of preferably 4-5 mol of the carotenoid monoesters of formula Ib is required. The starting materials used in this process, viz of formulae V, VII, are either known compounds or can be produced by methods known per se, i.e. analogous to the methods for producing the known compounds. Amongst the known compounds of formulae V are 2'-dehydroplectaniaxanthin. For realizing the method of the present invention the carotenoid is applied via the poultry, fish or crustacea feed, as appropriate, and in these circumstances the carotenoid responsible for the enhanced pigmentation is ingested by the pertinent animal in a natural manner. The pertinent feed may also contain other carotenoids which themselves contribute to the normal pigmentation of egg yolk, integuments and/or subcutaneous fat of poultry and the meat and/or integuments of fish and crustacea. The content of added carotenoid(s) of the formula I in the feed is generally in the range 0.1 ppm to 150 ppm (mg/kg or 0.00001 to 0.015% by weight) based on the total weight of the carotenoid-enriched feed. In poultry feed, in particular for laying hens and broilers, the content of added carotenoid(s) is preferably in the range 0.25 ppm to 20 ppm, and in the case of feed for fish or crustacea this content is preferably in the range 2.5 ppm to 150 ppm. The ingredients of the poultry, fish or crustacea feed according to the present invention can otherwise be conventional, and the feed can also be produced by conventional methods, involving physical admixture, pelleting, extrusion, microencapsulation, spraying etc., whereby at some stage during the production process one or more of the carotenoids of the formula I is/are incorporated. The conventional ingredients of poultry feed include, for example, wheat, maize, barley, sorghum, oats, rice and/or soybean meal, usually in ground or broken form, as appropriate, in major proportions (at least about 10 percent by weight in each case). Further ingredients in minor amounts (up to about 5 percent by weight, or in certain cases less than 1 percent by weight) include, for example, fish, meat and/or bone meal, wheat bran, straw, yeast, hydrolysed fat, tallow, lard, limestone, salt, methionine premix, mineral premix, vitamin premix and/or anticaking agent. Any poultry feed can be enriched with one or more of the carotenoids of formula I to afford a poultry feed according to the present invention. Typical fish or crustacea feeds of the present invention include, apart from the added carotenoid(s), fish meal, as the major source of proteins, wheat and bone meal, soybean meal, wheat flour, cooked starch, yeast, fish oil, soybean oil, soya lecithin, methionin, vitamins and minerals. The protein, lipids and carbohydrate content of such a feed is approximately 40-50%, 15-30% and 10% by weight, respectively. The carotenoids or carotenoid mixtures from those for incorporation in the poultry, fish or crustacea feed can be mixed in the form of beadlets into a so-called premix, which in turn is added to the feed. The beadlets themselves, which represent a further aspect of the present invention, conveniently contain, apart from the carotenoid(s), a starch-coated matrix of gelatin and carbohydrate, and one or more anti-oxidants, e.g. ethoxyquin and/or ascorbyl palmitate. The beadlets generally contain from 1 to 20 percent by weight of one or more of the carotenoids of formula I as the active ingredient, whereas the premix generally contains from 0.001 to 15% by weight of the active ingredient. The present invention further provides the use of one or more of the carotenoids of formula I for pigmenting the egg yolk, integuments and/or subcutaneous fat of poultry, and the meat and/or integuments of fish and crustacea. This use preferably involves the pigmenting of egg yolk of laying hens. The invention is illustrated by the following Examples: Example 1 Preparation of 2'-dehydroplectaniaxanthin acetate A mixture of 556 mg (1.0 mmol) of 2'-dehydroplectaniaxanthin, 20 ml (25.7 mmol) of pyridine and 10 ml (10.5 mmol) of acetic anhydride are heated with an oil bath at 75°C with stirring for 17 hours and then cooled to room temperature. To the mixture are then added 50 ml of absolute diethyl ether and in portions thereafter 20 g of ice, and the whole is stirred at room temperature for 40 minutes. To isolate and purify the product the aqueous ethereal mixture is extracted with further diethyl ether and the ether phase washed in turn with five 50 ml portions of cold 3N sulphuric acid, 100 ml of ice water, a further 100 ml of ice water containing 5 ml of saturated sodium bicarbonate solution and two 100 ml portions of ice water. The aqueous phase is neutral at this stage. Then the ethereal phase is dried over anhydrous sodium sulphate, filtered and the solvent evaporated off at 35°C under reduced pressure (water jet vacuum). Finally the product is dried under high vacuum. In this way there are obtained 641 mg of very dark red crystals. A chromatographic purification through a silica gel column affords 560 mg of product. On submitting this to a recrystallization from a methylene chloride/n-hexane mixture and to stirring over about 16 hours at 40°C there are finally obtained 141 mg of 2'-dehydroplectaniaxanthin acetate of 99.7% purity according to HPLC as dark red crystals, m.p. 177-178°C. UV/VIS(n-hexane + 1% methylene chloride + 1% ethanol): λ max = 493 nm; A 1 1 = 2476; ε =150500. Example 2 Preparation of 2'-dehydroplectaniaxanthin palmitate A solution of 566 mg (1.0 mmol) of 2'-dehydroplectaniaxanthin in 50 ml of absolute toluene is treated with a whole portion of 1126 mg (4.4 mmol) of palmitic acid and 390 mg (3.2 mmol) of 4-dimethylaminopyridine, whereafter the reaction mixture is warmed to an internal temperature of 60-62°C. 906 mg (4.4 mmol) of N,N'-dicyclohexylcarbodiimide dissolved in 10 ml of absolute toluene are then added, and the mixture in then maintained at 60-62°C for 44 hours. After cooling the mixture to 5°C it is extracted with toluene, and the toluene phase is washed in turn with five 50 ml portions of ice water, 50 ml of ice water containing 1 ml of 1N sulphuric acid and two 50 ml portions of ice water. Then the organic phase is dried over anhydrous sodium sulphate and the solvent removed by evaporation at 35°C under a water jet vacuum. The resulting very dark red oil is chromatographed with n-hexan/diethyl-ether (9:1) through a silica gel column, and the collected fractions are evaporated to dryness under reduced pressure (water jet vacuum) at 35°C . The residue is recrystallized from methylene chloride/ethanol. After being stirred at 4°C for about 16 hours the crystals are dried under high vacuum at 30°C, affording 285 mg of 2'-dihydroplectaniaxanthin palmitate as very dark red crystals, m.p. 124-126°C. According to HPLC the purity is 95.6%. UV/VIS(n-hexane + 2% methylene chloride): λ max = 493 nm; A 1 1 = 1686; ε = 135700. Example 3 Carotenoid - containing beadlet composition Constituent Percent by weight Carotenoid of the invention 6.0 Ethoxyquin 1.0 Ascorbyl palmitate 1.0 Gelatin, Bloom No. 140 38.5 Dextrin yellow 15.7 Crystalline sugar 15.7 Fluidized corn starch approx. 22.1 Total 100.0 Example 4 Utility of the carotenoids as egg yolk pigmenters One of the new carotenoids, was evaluated at a level of 5 ppm in combination with 80% its weight (4 ppm) of ethyl-β-apo-8'-carotenoate ("apo-ester") for its egg yolk pigmenting efficacy in a primary screen with laying hens, and compared with the known pigmenting carotenoid canthaxanthin (the "positive control") under the same conditions, i.e. inter alia at a level of 5 ppm in combination with 4 ppm of apo-ester. Then the combinations carotenoid +apo-ester and canthaxanthin +apo-ester and apo-ester alone were incorporated into low-carotenoid basal diets to the extents (inclusion levels) given above (5 ppm + 4 ppm in each combination case and 4ppm, respectively). The supplemented basal diets contained in addition 1.10 ppm of lutein and 0.30 ppm of zeaxanthin (a total of 1.40 ppm of xanthophylls) as well as in each case a normal level of supplemental vitamins. The composition of the low-carotenoid basal diet is presented in the following Table 1: Composition of low carotenoid basal diet Ingredients Content in weight percent Wheat, ground 37.00 Oats, ground 11.00 Rice, broken 13.00 Soybean meal 10.00 Fish meal [70 % crude protein (CP)] 5.00 Meat meal, full fat (82 % CP) 1.30 Wheat bran 3.50 Straw meal(NaOH treated) 2.40 Yeast 1.00 Hydrolysed fat 3.60 Ground limestone 9.55 Salt 0.20 Methionine premix (25 %) 0.50 Anticaking agent (Diamol®) 1.00 Mineral premix (trace elements) 0.20 Vitamin premix 0.75 Calculated content (weight percent): Crude protein (%) 16.80 Metabolizable energy (MJ/kg) 11.39 Crude fibre (%) 3.78 Crude fat (%) 5.77 Calcium (%) 4.22 Phosphorus (%) 0.43 The incorporated carotenoids were checked for content in the feed by HPLC. The laying hens used in the test were kept in individual cages in a 3-floor battery. 48 "Isa Brown" hens of age 62 weeks were involved, whereby four replicate groups, each of three hens were assigned to each dietary treatment with the combination of carotenoids, with apo-ester alone at an inclusion level of 4 ppm and without a carotenoid (negative control, i.e. basal diet without added test carotenoid). Before the actual trial the hens were fed the above-indicated low carotenoid basal diet in order to reduce the existing egg yolk pigmentation. Then, during the 3 weeks trial, the hens were fed the same low carotenoid basal diets supplemented with the test carotenoid or containing no such carotenoid (negative control). The feed, in mash form, and water were provided "ad libitum". The laying performance of the hens was monitored once weekly, and the last two eggs per hen were collected and pooled for the laboratory assays (in total, 6 eggs per group). The pigmenting efficacy was assessed by means of visual scoring (Roche Colour Fan) and reflectance colorimetry (CIE Lab system and CIE-Yxy system), as well as by measurement of carotenoid deposition in the egg yolk. The reflectance measurements were effected using the Xenocolor LS 100 (CIE Lab System) and the Gardner XL colorimeter, and the determined tristimulus values were converted into the CIE system of values of dominant wavelength and spectral saturation [ see J.P. Vuilleumier, The "Roche Yolk Colour Fan" - An Instrument for Measuring Yolk Colour, Poultry Science 48, 767-779 (1969)]. The carotenoid deposition measurements, which gave the levels of the test carotenoids in pooled egg yolks, were determined by the same HPLC methods as used for the determination of the carotenoid levels in the diets before feeding, and involved the quantification of the carotenoid originally incorporated in the feed. The results (average values) are presented in the following tables, viz. Tables 2-4: Analytical data in feed and deposition of carotenoids in egg yolks of hens Carotenoid (combination); Amount (of each) in ppm Analytical Content in Feed (mg/kg) Analytical Content in Egg Yolk (mg/kg) Apo-ester / Other carotenoid -(negative control) Apo-ester/ - 4/ - 4.20 / - 16.48 ± 2.12 / - Apo-ester/Carotenoid A 4/5 3.90/4.80 16.20 ± 1.41 / 9.13 ± 1.21 Apo-ester/Canth. 4/5 4.40/5.00 15.15 ± 0.83 / 13.70 ± 0.71 Values of reflectance colorimetry (CIE-Lab system) for egg yolks of hens supplemented with carotenoids Carotenoid (combination) Amount (of each) in ppm Lightness L* Redness a* Yellowness b* Hue h*ab Chroma C*ab -(negative control) 64.97±1.15 -2.09±0.19 38.19±0.56 93.14±0.32 38.25±0.54 Apo-ester/-4/- 61.85±1.37 5.37±1.26 55.88±1.11 84.53±1.21 56.15±1.18 Apo-ester/Carotenoid A 4/5 46.31±1.46 31.25±1.01 34.38±1.66 47.71±2.27 46.49±0.58 Apo-ester/Canth. 4/5 55.81±0.79 21.07±0.51 50.15±0.47 67.22±0.54 54.40±0.47 Values of reflectance colorimetry (CIE-Xxy system) for egg yolks of hens supplemented with carotenoids Carotenoid (combination); Amount (of each) in ppm Visual scoring (Roche Yolk Colour Fan) Dominant wavelength (nm) Spectral saturation -(negative control) 4.0 578.1±0.19 0.59±0.02 Apo-ester/-4/- 9.0 581.8±0.69 0.86±0.03 Apo-ester/Carotenoid A 4/5 >15.0 597.9±1.51 0.85±0.00 Apo-ester/Canth. 4/5 13.0 587.9±0.38 0.89±0.00 The results show that Carotenoid A [5,9,13,18,22-pentamethyl-24-(2,6,6-trimethyl-1-cyclohex-1-yl)-3,5,7,9,11,13,15,17,19,21,23-tetracosaundecaen-2-one] was deposited in considerable quantity in the egg yolk (Table 2: 67% of the canthaxanthin concentration), as a result of which this carotenoid improved very significantly the redness of the egg yolks (Table 3). Accordingly the respective colour hue (Table 3) was more favourable than that of canthaxanthin. Due to a relatively low portion of yellowness with carotenoid A, chroma of canthaxanthin was superior to that of carotenoid A. These findings were entirely confirmed by the values for dominant wavelength and colour fan (Table 4), indicating that carotenoid A has a much higher pigmenting potency than canthaxanthin and is an excellent red pigmenting agent for egg yolks. Example 5 Utility of the carotenoids as egg yolk pigmenters A further new carotenoid, namely 5,9,13,17,22,26-hexamethyl-28-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3,5,7,9,11,13,15,17,19,21,23,25,27-octacosatridecaen-2-one [or (16'-torulenylidene)acetone; hereinafter referred to as carotenoid B] was evaluated at levels of 5 ppm, 2.5 ppm, 1.5 ppm and 0.75 ppm in combination with 4 ppm of ethyl β-apo-8'-carotenoate ("apoester") for its egg yolk pigmenting efficacy in a primary screen with laying hens, and compared with the known pigmenting carotenoids canthaxanthin (canth.) and 2'-dehydroplectaniaxanthin (2'-DHP) at a dose level of 5 ppm and 0.85 ppm, respectively, in combination with 4 ppm of apo-ester under the same conditions. Control treatments involved diets with no carotenoid supplementation and with 4 ppm of apo-ester as the only carotenoid. Carotenoid B was formulated as beadlets of the composition given hereinabove in Example 3. Then the combinations carotenoid B + apo-ester, canthaxanthin + apo-ester and 2'-dehydroplectaniaxanthin + apo-ester, and apo-ester as the sole supplementing carotenoid, were incorporated into low-carotenoid basal diets to the extents (levels) given above (5/2.5/1.5/0.75 ppm + 4 ppm; 5 ppm + 4 ppm; 0.85 ppm + 4 ppm; and 4 ppm, respectively). The composition of the low-carotenoid basal diet was essentially the same as that presented in Table 1 in Example 4, but the vitamin premix (also of 0.75 weight percent content) was of somewhat different constitution, as follows: Per kg feed: Vitamin A 10000 IU, vitamin D 3 2000 IU, vitamin E 71 mg, vitamin K3 2.6 mg, vitamin C 200 mg, vitamin B 1 4 mg, vitamin B 2 12.1 mg, vitamin B 6 8 mg, vitamin B 12 0.04 mg, biotin 0.06 mg, Ca-pantothenate 48 mg, nicotinic acid 104 mg, folic acid 2 mg, choline 1719 mg. Furthermore, the supplemental basal diets contained in addition 1.00 mg/kg of lutein and 0.40 mg/kg of zeaxanthin (a total of 1.40 mg/kg of xanthophylls) (first trial) or 0.30 mg/kg of lutein and < 0.10 mg/kg of zeaxanthin (a total of 0.30-0.40 mg/kg of xanthophylls) (second trial). The incorporated carotenoids were checked for content in the feed by HPLC. The laying hens used in the two trials (1 and 2) which were conducted were kept in individual cages in a 3-floor battery. "Isa Brown" hens of age 35 weeks (trial 1) or 40 weeks (trial 2) were involved, whereby four replicate groups, each of three hens, were assigned to each dietary treatment with the combination of two carotenoids, with apo-ester alone at an inclusion level of 4 ppm and without a carotenoid (negative control, i.e. basal diet without added test carotenoid). The two trials were conducted, and the pigmenting efficacies assessed, in the same way as described for the trial and assessment reported in Example 4 hereinbefore, and the results (average values) are presented in the following tables, viz. Tables 5.1 to 8.2: Analytical data in feed and deposition of carotenoids in egg yolks of hens (Trial 1) Carotenoid (combination); Amount (of each) in ppm Analytical Content in Feed (mg/kg) Analytical Content in Egg Yolk (mg/kg) -(negative control) - - Apo-ester/ -4/ - 4.10 / - 11.93 ± 0.68 / - Apo-ester/Carotenoid B 4/5.00 3.90/5.60 13.10 ± 0.55 / 3.48 ± 0.15 Apo-ester/Carotenoid B 4/2.50 4.00/2.90 13.73 ± 0.39 / 2.33 ± 0.05 Apo-ester/Canth. 4/5.00 3.80/6.70 12.13 ± 0.59 / 11.65 ± 0.71 Apo-ester/2'-DHP 4/0.85 4.50/1.00 11.98 ± 0.34 / 1.93± 0.19 Analytical data in feed and deposition of carotenoids in egg yolks of hens (Trial 2) Carotenoid (combination); Amount (of each) in ppm Analytical Content in Feed (mg/kg) Analytical Content in Egg Yolk (mg/kg) -(negative control) - - Apo-ester/ -4/ - 4.00 / - 13.80 ± 1.78 / - Apo-ester/Carotenoid B 4/1.50 3.60/1.50 12.98 ± 0.25 / 1.38 ± 0.13 Apo-ester/Carotenoid B 4/0.75 4.00/0.85 12.88 ± 0.81 / 0.70 ± 0.08 Apo-ester/Canth. 4/5.00 3.90/5.20 14.45 ± 0.42 / 12.90 ± 0.54 Apo-ester/2'-DHP 4/0.85 4.70/0.70 15.80 ± 1.09 / 2.38 ± 0.13 Values of reflectance colorimetry (CIE-Lab system) for egg yolks of hens supplemented with carotenoids (Trial 1) Carotenoid (combination); Amount (of each) in ppm Lightness L* Redness a* Yellowness b* Colour hue h*ab Chroma C*ab -(negative control) 66.44±0.84 -4.31±0.12 35.21±0.91 96.98±6.36 35.47±0.90 Apo-ester/-4/- 62.30±0.53 3.65±0.46 55.67±1.02 86.26±0.41 55.79±1.04 Apo-ester/Carotenoid B 4/5.00 45.90±0.56 26.76±0.54 31.74±0.34 49.86±0.58 41.52±0.48 Apo-ester/Carotenoid B 4/2.50 48.80±0.50 22.84±0.14 36.05±0.47 57.65±0.43 42.67±0.37 Apo-ester/Canth. 4/5.00 55.12±0.96 21.02±0.74 49.55±0.95 67.01±0.99 53.83±0.78 Apo-ester/2'-DHP 4/0.85 54.82±0.54 16.14±0.86 43.92±1.02 69.83±0.87 46.80±1.13 Values of reflectance colorimetry (CIE-Lab system) for egg yolks of hens supplemented with carotenoids (Trial 2) Carotenoid (combination); Amount (of each) in ppm Lightness L* Redness a* Yellowness b* Colour hue h*ab Chroma C*ab - (negative control) 67.20±0.83 -4.01±0.16 30.51±1.39 97.50±0.42 30.78±1.38 Apo-ester/-4/- 62.95±0.71 3.72±0.55 54.22±0.44 86.08±0.55 54.35±0.47 Apo-ester/Carotenoid B 4/1.50 54.25±0.66 17.25±0.16 41.65±1.40 67.49±0.68 45.08±1.30 Apo-ester/Carotenoid B 4/0.75 57.13±0.61 12.22±0.60 45.98±0.80 75.12±0.74 47.58±0.79 Apo-ester/Canth. 4/5.00 56.56±0.56 20.03±0.53 49.82±0.89 68.10±0.30 53.70±0.99 Apo-ester/2'-DHP 4/0.85 56.01±0.80 16.10±0.39 44.29±1.03 70.03±0.33 47.12±1.06 Values of reflectance colorimetry (CIE-Xxy system) for egg yolks of hens supplemented with carotenoids (Trial 1) Carotenoid (combination); Amount (of each) in ppm Visual scoring (Roche Yolk Colour Fan) Dominant wavelength (nm) Spectral saturation - (negative control) 3.0±0 578.3±0.1 0.53±0.01 Apo-ester/-4/- 8.0±0 582.0±0.2 0.83±0.01 Apo-ester/Carotenoid B 4/5.00 15.0±0 594.3±0.3 0.78±0.01 Apo-ester/Carotenoid B 4/2.50 15.0±0 591.2±0.1 0.80±0.01 Apo-ester/Canth. 4/5.00 13.0±0 588.8±0.4 0.90±0.01 Apo-ester/2'-DHP 4/0.85 13.0±0 587.2±0.4 0.84±0.01 Values of reflectance colorimetry (CIE-Xxy system) for egg yolks of hens supplemented with carotenoids (Trial 2) Carotenoid (combination); Amount (of each) in ppm Visual scoring (Roche Yolk Colour Fan) Dominant wavelength (nm) Spectral saturation - (negative control) 2.0±0 578.0±0.2 0.47±0.02 Apo-ester/-4/- 8.0±0 582.0±0.2 0.82±0.01 Apo-ester/Carotenoid B 4/1.50 13.0±0 587.8±0.2 0.82±0.01 Apo-ester/Carotenoid B 4/0.75 12.0±0 585.4±0.3 0.83±0.01 Apo-ester/Canth. 4/5.00 13.0±0 588.2±0.2 0.89±0.01 Apo-ester/2'-DHP 4/0.85 13.0±0 587.1±0.2 0.84±0.01 Production data of hens supplemented with carotenoids (Trial 1) Carotenoid (combination); Amount (of each) in ppm No of eggs/group : total/intact Egg production (%) Feed intake /group (g) Feed intake /egg (g) Mean egg weight (g) Egg mass (g) Feed conversion - (negative control) 61/60 96.0 6285 104 58 3512 1.79 Apo-ester/-4/- 61/61 97.2 6696 109 59 3621 1.85 Apo-ester/Carotenoid B 4/5.00 62/60 97.6 6870 112 61 3722 1.85 Apo-ester/Carotenoid B 4/2.50 58/57 92.5 6624 114 60 3490 1.90 Apo-ester/Canth. 4/5.00 56/55 88.5 6644 122 59 3307 2.06 Apo-ester/2'-2'-DHP 4/0.85 59/57 94.1 6791 115 61 3589 1.90 Production data of hens supplemented with carotenoids (Trial 2) Carotenoid (combination); Amount (of each) in ppm No of eggs/group group total/intact Egg production (%) 6) Feed intake /group (g) Feed intake /egg (g) 6) Mean egg weight (g) 7) Egg mass (g) 8) Feed conversion - (negative control) 59/59 93.3 6137 104 59 3484 1.76 Apo-ester/-4/- 57/56 90.5 6481 114 61 3494 1.85 Apo-ester/Carotenoid B 4/1.50 59/57 93.3 6718 115 62 3618 1.86 Apo-ester/Carotenoid B 4/0.75 51/50 81.4 6503 129 63 3211 2.05 Apo-ester/Canth. 4/5.00 59/54 93.7 6966 119 64 3783 1.85 Apo-ester/2'-DHP 4/0.85 59/57 94.1 6702 113 61 3603 1.86 The results show inter alia that the analytical concentrations of the carotenoids in the feeds were in an acceptable range for the target levels, whereby a tendency for slightly higher values was observed, particularly for canthaxanthin in trial 1 (see Table 5.1). The concentrations of apo-ester in the egg yolks ranged between about 12 and about 14 mg/kg (trial 1) or between about 13 and about 16 mg/kg (trial 2) and were not influenced by the other carotenoid present in the combinations. Carotenoid B was deposited in considerable quantity in the egg yolk (Tables 5.1 and 5.2). As regards the reflectance colorimetry results (Tables 6 and 7), the higher dose levels of carotenoid B tested (5.00 and 2.50 ppm in trial 1) caused a strong red pigmentation, so that this carotenoid was also tested, in trial 2, at the lower dose levels 1.50 and 0.75 ppm. Carotenoid B exhibited at the dose level 1.50 ppm a pigmentation level comparable with that of 2'-DHP and a slightly lower colour hue (Tables 6.2 and 7.2), whereas the pigmentation obtained with the lower dose 0.75 ppm was weaker. In comparison to canthaxanthin in the pertinent trial 2 the values for redness and colour saturation (chroma) were lower. From Tables 8.1 and 8.2 it can be seen that although variations occurred in the production data of the laying hens the egg production was not influenced by the various carotenoid treatments. In conclusion, carotenoid B [5,9,13,17,22,26-hexamethyl-28-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3,5,7,9,11,13,15,17,19,21,23,25,27-octacosatridecaen-2-one, or (16'-torulenylidene)acetone] is a carotenoid with a good deposition in egg yolk and a high red pigmenting potential. Example 6 Utility of the carotenoids as egg yolk pigmenters Two further new carotenoids, namely 1',2'-dihydro-17'-nor-beta-χ-caroten-2'-one (hereinafter referred to as carotenoid C) and (all E)-2,9,13,17,22,26-hexamethyl-28-(2,6,6-trimethyl-cyclohex-1-enyl)-octacosa-2,7,9,11,13,15,17,19,21,23,25,27-dodecaen-6-one [hereafter referred to as carotenoid D) were evaluated at levels of 5.0 ppm and 2.5 ppm (carotenoid C) and 5.0 ppm 2.0 ppm and 0.8 ppm (carotenoid D) in combination with 4 ppm of ethyl β-apo-8'-carotenoate ("apo-ester") for its egg yolk pigmenting efficacy in a primary screen with laying hens, and compared with the known pigmenting carotenoids canthaxanthin and 2'-dehydroplectaniaxanthin (2'-DHP) at dose levels of 5.0 ppm and 2.5 ppm (canthaxanthin) and 0.85 ppm (2'-DHP) in combination with 4 ppm of apo-ester under the same conditions. Control treatments involved diets with no carotenoid supplementation and with 4 ppm of apo-ester as the only carotenoid. Both carotenoids C and D were formulated as beadlets of the composition given hereinabove in Example 3. Then the combinations carotenoid C + apo-ester, carotenoid D + apo-ester, canthaxanthin + apo-ester and 2'-dehydroplectaniaxanthin + apo-ester, and apo-ester as the sole supplementing carotenoid, were incorporated into low-carotenoid basal diets to the extents (levels) given above (5/2.5 ppm + 4 ppm; 5/2/0.8 ppm + 4 ppm; 5/2.5 ppm + 4 ppm; 0.85 ppm + 4 ppm; and 4 ppm, respectively). The composition of the low-carotenoid basal diet was essentially the same as that presented in Table 1 in Example 4, but the vitamin premix (also of 0.75 weight percent content) was of somewhat different constitution, as follows: Per kg feed: Vitamin A 10000 IU, vitamin D 3 2000 IU, vitamin E 71 mg, vitamin K 3 2.6 mg, vitamin C 200 mg, vitamin B 1 4 mg, vitamin B 2 12.1 mg, vitamin B 6 8 mg, vitamin B 12 0.04 mg, biotin 0.06 mg, Ca-pantothenate 48 mg, nicotinic acid 104 mg, folic acid 2 mg, choline 1719 mg. Furthermore, the supplemental basal diets contained in addition 0.60 mg/kg of lutein and 0.10 mg/kg of zeaxanthin (a total of 0.70 mg/kg of xanthophylls). The incorporated carotenoids were checked for content in the feed by HPLC. The laying hens used in the trial which was conducted were kept in individual cages in a 3-floor battery. "Isa Brown" hens of age 49 weeks were involved, whereby four replicate groups, each of three hens were assigned to each dietary treatment with the combination of two carotenoids, with apo-ester alone at an inclusion level of 4 ppm and without a carotenoid (negative control, i.e. basal diet without added test carotenoid). The trial was conducted, and the pigmenting efficacies assessed, in the same way as described for the trial and assessment reported in Example 4 hereinbefore, and the results (average values) are presented in the following tables, viz. Tables 9 - 12: Analytical data in feed and deposition of carotenoids in egg yolks of hens Carotenoid (combination); Amount (of each) in ppm Analytical Content in Feed (mg/kg) Analytical Content in Egg Yolk (mg/kg) -(negative control) - - Apo-ester/ 4/ - 4.60 / - 15.75 ± 0.64 / - Apo-ester/Carotenoid C 4/5.00 4.20/5.20 17.60 ± 1.37/ 8.53 ± 0.72 Apo-ester/Carotenoid C 4/2.50 4.20/2.60 16.73 ± 1.42 / 4.75 ± 0.47 Apo-ester/Carotenoid D 4/5.00 4.10/5.00 15.13 ± 0.87 / 1.20 ± 0.12 Apo-ester/Carotenoid D 4/2.00 4.40/2.20 15.88 ± 0.21 / 1.05 ± 0.17 Apo-ester/Carotenoid D 4/0.80 4.00/1.00 15.58 ± 0.93 / 0.78 ± 0.13 Apo-ester/Canth. 4/5.00 4.30/5.70 15.90 ± 0.68 / 15.15 ± 0.78 Apo-ester/Canth. 4/2.50 3.90/2.70 15.05 ± 0.62 / 7.20 ± 0.37 Apo-ester/2'-DHP 4/0.85 4.60/0.80 15.08 ± 1.38 / 2.05± 0.34 Values of reflectance colorimetry (CIE-Lab system) for egg yolks of hens supplemented with carotenoids Carotenoid (combination); Amount (of each) in ppm Lightness L* Redness a* Yellowness b* Colour hue h*ab Chroma C*ab - (negative control) 66.26±0.54 -4.04±0.25 35.24±1.11 96.54±0.46 35.47±1.10 Apo-ester/ 4/- 62.28±0.79 4.45±0.32 56.01±0.87 85.46±0.37 56.19±0.85 Apo-ester/ Carotenoid C 4/5.00 48.33±1.22 31.53±0.87 37.27±1.83 49.74±1.84 48.83±1.28 Apo-ester/ Carotenoid C 4/2.50 51.04±0.94 26.21±0.50 40.34±0.69 56.98±0.80 48.11±0.53 Apo-ester/ Carotenoid D 4/5.00 57.70±0.18 12.46±1.06 48.30±0.89 75.55±1.09 49.89±0.99 Apo-ester/ Carotenoid D 4/2.00 59.72±1.12 11.06±1.31 49.89±1.24 77.50±1.46 51.11±1.26 Apo-ester/ Carotenoid D 4/0.80 59.77±0.35 9.57±1.07 51.05±0.49 79.39±1.17 51.95±0.53 Apo-ester/ Canth. 4/5.00 54.86±1.15 21.44±0.60 49.11+1.46 66.41±0.74 53.58±1.42 Apo-ester/ Canth. 4/2.50 57.96±0.92 15.42±0.36 51.73±0.48 73.39±0.50 53.98±0.37 Apo-ester/ 2'-DHP 4/0.85 54.61±1.03 17.54±0.70 44.64±0.72 68.55±1.10 47.97±0.41 Values of reflectance colorimetry (CIE-Xxy system) for egg yolks of hens supplemented with carotenoids Carotenoid (combination); Amount (of each) in ppm Visual scoring (Roche Yolk Colour Fan) Dominant wavelength (nm) Spectral saturation - (negative control) 2.0±0 578.4±0.2 0.53±0.01 Apo-ester/ 4/- 8.0±0 582.1±0.1 0.84±0.01 Apo-ester/ Carotenoid C 4/5.00 >15.0 595.3±0.8 >0.84 Apo-ester/ Carotenoid C 4/2.50 15.0±0 592.0±0.4 >0.84 Apo-ester/ Carotenoid D 4/5.00 12.0±0 585.4±0.5 0.85±0.01 Apo-ester/ Carotenoid D 4/2.00 11.0±0 584.6±0.6 0.86±0.02 Apo-ester/ Carotenoid D 4/0.80 11.0±0 584.1±0.5 0.85±0.01 Apo-ester/Canth. 4/5.00 13.0±0 589.9±0.3 0.90±0.01 Apo-ester/Canth. 4/2.50 11.0±0 586.4±0.2 0.89±0.00 Apo-ester/2'-DHP 4/0.85 14.0±0 587.7±0.5 0.84±0.00 Production data of hens supplemented with carotenoids Carotenoid (combination); Amount (of each) in ppm No of eggs/ group : total/ intact Egg production (%) Feed intake /group (g) Feed intake /egg (g) Mean egg weight (g) Egg mass (g) Feed conversion - (negative control) 54/54 83.3 6151 115 61 3291 1.87 Apo-ester/ 4/- 55/54 83.3 6368 116 60 3270 1.95 Apo-ester/ Carotenoid C 4/5.00 47/47 75.0 5989 130 61 2865 2.14 Apo-ester/ Carotenoid C 4/2.50 52/50 82.9 6415 124 62 3219 2.01 Apo-ester/ Carotenoid D 4/5.00 55/52 86.9 6475 119 61 3360 1.94 Apo-ester/ Carotenoid D 4/2.00 54/54 85.7 6321 117 62 3328 1.90 Apo-ester/ Carotenoid D 4/0.80 52/50 81.7 6202 121 63 3265 1.90 Apo-ester/ Canth. 4/5.00 53/51 84.5 6662 125 63 3363 1.99 Apo-ester/ Canth. 4/2.50 57/56 89.7 6331 112 60 3409 1.86 Apo-ester/ 2'-DHP 4/0.85 51/50 81.0 6184 121 62 3163 1.96 The results show that the analytical concentrations of the carotenoids in the feeds were in an acceptable range for the target levels. The test and comparison (canthaxanthin and 2'-DHP) carotenoids were deposited in the egg yolk, and it was observed that the concentration increased with increasing dose levels. Compared with canthaxanthin the concentrations of carotenoids C and D in the egg yolk were lower (decreasing values from carotenoid C to carotenoid D). According to the results presented in Tables 10 and 11 carotenoid C showed high red pigmenting properties reflected in high values for redness and low values for colour hue. The red pigmentation was stronger than that of canthaxanthin. Carotinoid C had lower values for lightness, yellowness and chroma than canthaxanthin, attributable to the stronger pigmentation. Carotenoid D also caused a significant red pigmentation of the egg yolk, with increasing redness and decreasing colour hue as the dose was increased, but the pigmentation efficacy was lower than for canthaxanthin and 2'-DHP. In respect of yellowness, carotenoid D had similar values to canthaxanthin and higher values than those of 2'-DHP, and the chroma values were between those of these comparison carotenoids. From Table 12 it can be seen that although variations occurred in the production data of the laying hens the egg production was not influenced by the various carotenoid treatments. In conclusion, carotenoid C (1',2'-dihydro-17'-nor-beta-χ-caroten-2'-one) has a high potential for red pigmentation of egg yolk, and carotenoid D [(all E)-2,9,13,17,22,26-hexamethyl-28-(2,6,6-trimethyl-cyclohex-1-enyl)-octacosa-2,7,9,11,13,15,17,19,21,23,25,27-dodecaen-6-one] also causes considerable pigmentation, albeit somewhat lower than that caused by canthaxanthin and 2'-DHP.

Claims: Carotenoids of the general formula wherein R signifies a group (b) in each of which R 2 signifies C 1-15 -alkyl, and n signifies zero or an integer 1 to 4. The carotenoids of claim 1: 2'-dehydroplectaniaxanthin acetate and 2'-dehydroplectaniaxanthin palmitate. A carotenoid-enriched feed for poultry, fish or crustacea and intended for the pigmentation of egg yolk, meat, integuments and/or subcutaneous fat of the poultry and the meat and/or integuments of the fish and crustacea, characterized in that it contains as the carotenoid(s) one or more of the carotenoids according to claim 1 or claim 2 in an effective amount. A carotenoid-enriched feed according to claim 3, characterized in that the content of the carotenoid(s) used for the pigmentation is in the range 0.1 ppm to 150 ppm based on the total weight of the feed. A carotenoid-enriched feed according to claim 3 or 4, characterized in that it is for poultry. Beadlets containing one or more carotenoids for incorporation in a premix or feed for poultry, fish or crustacea, characterized in that they contain as the carotenoid(s) one or more of the carotenoids according to claim 1 or claim 2 in an amount from 1 to 20 percent by weight. A premix containing one or more carotenoids for incorporation in a feed for poultry, fish or crustacea, characterized in that it contains as the carotenoid(s) one or more of the carotenoids according to claim 1 or claim 2 in an amount from 0.001 to 15% by weight. A process for manufacturing carotenoids of the general formula wherein R signifies a group (b) in each of which R 2 signifies C 1-15 -alkyl, and n signifies zero or an integer 1 to 4, characterized by (iii) reacting a carotenoid alcohol of the general formula with the appropriate alkanoic acid of the general formula R 2 COOH or with a reactive derivative thereof. A method of pigmenting the egg yolk, integuments and/or subcutaneous fat of poultry, and the meat and/or integuments of fish and crustacea by including one or more carotenoids in the feed of such poultry, fish or crustacea, characterized in that said carotenoids are one or more of the carotenoids according to claim 1 or claim 2. A method according to claim 9, characterized in that the content of carotenoid(s) in the feed is in the range 0.1 ppm to 150 ppm based on the total weight of the feed. A method according to claim 9 or claim 10, characterized in that it is for pigmenting the egg yolk of laying hens. The use of one or more of the carotenoids claimed in claim 1 or claim 2 for pigmenting the egg yolk, integuments and/or subcutaneous fat of poultry, and the meat and/or integuments of fish and crustacea.

Inventors: GIGER ALFRED, SIMON WERNER, GIGER, ALFRED, SIMON, WERNER
Applicants: HOFFMANN LA ROCHE, F. HOFFMANN-LA ROCHE AG
Application year: 1995
Main classification: C07C403
IPC codes: A23K1, C07C403, A23L1