Egg Yolk Pigmenting Properties of a Marigold Extract and Paprika Oleoresin in a Practical Type Diet

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1 Egg Yolk Pigmenting Properties of a Marigold Extract and Paprika Oleoresin in a Practical Type Diet D. L. FLETCHER Department of Poultry Science, University of Georgia, Athens, Georgia 2 H. R. HALLO RAN Halloran Research Farm, Inc., 524 Roscoe Road, Modesto, California 551 (Received for publication November 1, 182) ABSTRACT The egg yolk pigmenting properties of a marigold concentrate and a paprika oleoresin were evaluated when used in combination with a practical yellow corn basal diet. A total of 21 laying hens was divided at random into 42 five-bird pens assigned one of 21 test diets. The diets were formulated to contain,,,, or mg/kg added xanthophyll from a marigold concentrate or,,,, or mg/kg of added xanthophyll from a paprika oleoresin in a yellow corn basal diet with 1.5 mg/kg naturally occurring xanthophylls. Egg production, feed consumption, and egg mass were monitored. After 21 days on the test diets eggs were collected for color analysis on three separate days. Color was determined using the Association of Official Analytical Chemists method, Roche color fan, and reflectance colorimetry. Results indicated that addition of either a marigold concentrate or a paprika oleoresin separately or in combination can greatly enhance egg yolk pigmentation values. Additions of small quantities of the paprika oleoresin to the yellow corn basal diet resulted in a greater relative response than that provided by larger quantities of marigold concentrate. Thus, small quantities of a paprika oleoresin could be used to greatly enhance yolk color as opposed to much larger quantities of marigold concentrate. (Key words: pigmentation, xanthophyll, yolk color, marigold, paprika) INTRODUCTION A number of studies have been conducted to compare the egg yolk pigmenting properties of a number of xanthophyll containing feedstuffs. In most cases these studies involve a comparison of single xanthophyll sources against other sources. A limited number of studies have been conducted that compare the relative pigmenting properties of multiple sources of xanthophyll when fed in combination. An interesting concept presented by Nelson and Baptist (18) was the feeding of combined sources of red and yellow pigments to layers. They reported that the addition of red xanthophyll to the diet could produce egg yolk colors equivalent to those produced from much higher levels of just yellow xanthophyll sources. The use or potential use of red sources of xanthophyll were illustrated as early as 7 by Morgan and Woodroof, who evaluated the egg 1 The mention of specific products, trade names, or equipment does not imply endorsement or approval by the authors or the University of Georgia over other similar products or equipment that may be available. 18 Poultry Science 2:5-1 yolk pigmenting properties of waste pimiento pepper (Capsicum annuum). Brown (18) found that the predominant pimiento pigment responsible for coloring egg yolk was capsanthin. Mackay et al. (1) reported that paprika (Capsicum annuum) extract enhanced egg yolk pigmenting properties and that the improved color would not adversely affect sponge cake color. Although naturally occurring red xanthophylls have been shown to be effective egg yolk pigmenting agents, they have not found much acceptance by the industry. The concept of blending controlled levels of xanthophylls to produce desired yolk colors using synthetic xanthophyll sources was illustrated by Couch and Farr (171) and Fletcher et al. (178) among others. Controlled blending of the synthetic xanthophylls, beta-apo-8 -carotenal and canthaxanthin, was used by the previous authors to demonstrate the concept. Synthetic xanthophylls, however, are not approved for use in the United States at this time. In a previous paper (Fletcher and Halloran, 181) the use of a marigold concentrate and a paprika oleoresin as natural sources of red and Downloaded from at Penn State University (Paterno Lib) on September 18, 21 5

2 FLETCHER AND HALLORAN yellow pigments was evaluated. It was found that the two sources could be blended to produce a wide range of yolk colors at lower total xanthophyll levels than would be possible otherwise. These tests, however, were conducted using a white corn (xanthophyll-free) basal diet. Therefore, the purpose of this study was to examine and evaluate the egg yolk pigmenting properties of a marigold concentrate and paprika oleoresin when used in conjunction with a practical yellow corn based diet. EXPERIMENTAL PROCEDURE A total of 21 laying hens (Shaver 1 ), 55 weeks of age, was divided at random into 42 pens of 5 birds each and fed one of 21 test diets. The test diets were produced by adding various levels of a marigold concentrate and a paprika oleoresin to the basal diet shown in Table 1. The marigold (Tagetes erecta) con- TABLE 1. Composition of yellow corn basal diet Ingredient Yellow corn Soybean meal (48.5% protein) Alfalfa (17% protein) Poultry fat Limestone Defluorinated phosphate (Ca 2%, Salt DL-Methionine, % Trace mineral mix 1 Vitamin premix 2 Calculated analysis Metabolizable energy, kcal/kg Protein, % Lysine, % Methionine, % Methionine + cystine, % Calcium, % Available phosphorus, % Xanthophyll, mg/kg P 18%) 'Trace mineral mix provides (ppm of diet): Mn, ; Zn, 5; Fe, ; Cu, 5; I, 1.5; Ca, 75 (min.) and (max.). 2 Vitamin premix provides (per kg/diet): vitamin A, 11, IU; vitamin D, 1,1 ICU; vitamin E, 11 IU; riboflavin, 4.4 mg; Ca pantothenate, mg; nicotinic acid, 44 mg; choline CI, 22 mg; vitamin B, 2,. Mg; vitamin. B, 2.2 mg; menadione, 1.1 mg (as MSBC); folic acid,.55 mg; d-biotin,.11 mg; thiamine, 2.2 mg (as thiamine mononitrate); ethoxyquin, 5 mg. TABLE 2. Dietary treatments of added xanthophyll and total xanthophyll from marigold concentrate (MC) and paprika oleoresin (PO) Added xanthophyll Jotgl ^ Diet MC PO thophyll (mg/kg) centrate and the paprika (Capsicum annuum) oleoresin were obtained from Desy International, Inc., Dallastown, PA. 1 The products were analyzed for total xanthophyll using the procedure of the Association of Offical Analytical Chemists (AOAC, 175), and they were found to contain 4 and 1 mg/g total xanthophyll for the marigold concentrate and the paprika oleoresin, respectively. The 21 test diets, the blending schedule with added xanthophyll, and the total xanthophyll are shown in Table 2. Yellow corn and alfalfa sources were analyzed prior to formulation and determined to have 17.5 and 8.28 mg xanthophyll/kg, respectively. Following mixing, all diets were stored at 4.5 C until fed. All diets were fed for 21 days prior to a 14-day test period from which all eggs for subsequent color analysis were taken. The 21-day period prior to the test period was to allow for the hens to void their ovaries of any rapidly maturing ova that started development prior to being placed on the test diets. Egg production, egg mass, and feed consumption values were recorded by pen, throughout the 5-day period, for subsequent analysis of feed intake and feed efficiency. Downloaded from at Penn State University (Paterno Lib) on September 18, 21

3 EGG YOLK PIGMENTATION 7 Egg yolk sample preparation for subsequent color analysis was identical to procedures reported by Fletcher (18). Yolk color was determined on composite samples from all the eggs from an individual pen ( to 5 eggs) in three replicate groups (replicates consisted of separate days on which eggs were collected during the last 14-day period). Color was determined using visual comparisons with the Roche color fan, the AOAC (175) procedure of beta-carotene equivalents, and reflectance colorimetry. Colorimetric results are expressed in the International Commission on Illumination (CIE -Commission Internationale de L Eclairage) system values of dominant wavelength (nm), excitation purity (%) and luminosity (%), the CIELAB system values of lightness (), redness (), yellowness (), and a ratio of redness:yellowness (/). Results of feed conversion values and color data were analyzed using analysis of variance and Duncan's multiple range test as described by Steel and Torrie (1). RESULTS AND DISCUSSION Results for beta-carotene equivalents and visual comparisons with the Roche color fan are shown in Table. As levels of either the marigold concentrate or the paprika oleoresin increased, both the beta-carotene equivalents and the Roche color fan values increased (P<.5). There were no significant source (marigold X paprika) interactions. Relatively small quantities of the paprika oleoresin resulted in much greater increases in Roche color fan values, indicating the shift to a more red color. The basal diet with only mg/kg added xanthophyll from paprika resulted in the same Roche color fan score of as did the basal diet with mg/kg added xanthophyll from marigold. Thus, the visual response of a small quantity of a red xanthophyll source can be greater than that of a much larger quantity of yellow xanthophyll, which will not be apparent using a chemical-spectrophotometric method as the AOAC beta-carotene equivalents. The greater relative increase in the beta-carotene equivalents with increases in the marigold concentrate as opposed to the greater relative increase in Roche color fan values with increases in paprika oleoresin illustrates the relative weakness in the beta-carotene equivalents (AOAC, 175) method of yolk color analysis (Fletcher, 18). TABLE. Beta-carotene equivalents () and Roche Color Fan values () of egg yolks from hens fed various added levels of marigold concentrate (MC) and paprika oleoresin (PO) Marigold concentrate xanthophyll Main effects S 48.8 h e U. f 8. cd 11.* 5.5 ab. ef MC»«* * «* / n \ (mg/kg) 42.4 hi. ef ef 7.1 de. efd 85.5 C 1. cde 5. ab.7cde PO ** Paprika oleoresin xanthophyll.8".7cde 5.5 f g ls.o ^ 72.8 de 1. bcd 11.2 a 1.7 abc 5.2 ab 1.7 abc MCX PO f 14. ab 8. cd 14. ab 8.5 bc 14. ab 85.8 C 14. ab 74.7 d e 14.7 a 5.1 ab 14.7 a Downloaded from at Penn State University (Paterno Lib) on September 18, 21...»... ' Means within each color value with different superscripts are significantly different (P<.5). "Significantly different (P-C1). 1, Not significant.

4 8 FLETCHER AND HALLORAN The colorimetric analysis in the CIE system (Table 4) can be used to explain further the visual and chemical analyses. As levels of either of the two sources increased, dominant wavelength increased, excitation purity remained constant, and luminosity decreased (P<.5). The increased dominant wavelength indicates a shift to the more red end of the spectrum. Again, this shift was much more pronounced for additions of red pigment as opposed to yellow. The decrease in luminosity indicated that as pigment concentration increased, the samples became darker. The lack of significant (P>.5) change in EP values with increasing xanthophyll levels is in contradiction to results reported previously comparing the same two sources when used in conjunction with a white corn basal diet (Fletcher and Halloran, 181). It appears that the basal diet xanthophyll level of 1.5 mg/kg is sufficient to cause the excitation purity values to plateau and thus be ineffective at higher levels to distinguish between deeply pigmented yolk (Fry et al., 174). The CIELAB values of lightness (), redness (), yellowness (), and redness to yellowness ratio (/) are presented in Table 5. The lightness values show the same basic trend as does luminosity because the two are different mathematical versions of the same parameter. The magnitude of change of the and values is what would be expected for changes in dietary levels of yellow and red pigment sources. The values increased dramatically with increases in paprika oleoresin while values decreased. As marigold concentrate was increased, the increase in was greater than that of (with values actually decreasing with increased levels of marigold concentrate). The increase in values (and concomitant decrease in ) with increased levels of marigold extract is due to the fact that "deep" yellow has a red component as well as a yellow component (highly concentrated forms TABLE 4. Dominant wavelength (DWL), excitation purity (EP), and luminosity (hum) of egg yolks from hens fed various levels of marigold concentrate (MC) and paprika oleoresin (PO) Marigold xanthophyll Main effects DWL EP Lum 58. k 7.8 a 28. a 581.2) 75. a 27.4 a a 2.2 b 582. h 75.7 a 25.2 b a 2. cd MC / /i \ - (mg/kg) 582. h 7.7 a 25.5 b h 75. a 25.5 b a 24. l c ef 74.5a 2.2 cd 584. e 75.a 2.2 cd PO Paprika oleoresin xanthophyll a 24. C f 74. a 24.2 C d 74.7a 2.2 cd 58.2 b 7.7 a 2.88 b 585. bc 74 a 21.4% MCX PO 585. C 7. a 22.8 de 58.2 b 77.4a 21.4% 58.2 b 7.8 a 21.7% 58.2 b 74.ia 22. ef 587.ia 7. a a 7.2 a 2." Downloaded from at Penn State University (Paterno Lib) on September 18, 21 n rv /* fi ^ p or rl 11 k.,» > > >gi > >J> Means within each color value with different superscripts are significantly different (P<.5). Significantly different (P<.1). 1, Not significant.

5 EGG YOLK PIGMENTATION of yellow pigment appear red; however, dilute solutions of red do not appear yellow). For this reason, the / ratio seems to be a very effective single number expression of yolk color (much easier to interpret on a practical basis). Feed conversion values (kg of feed consumed/kg of eggs produced) ranged from 1.84 to 2.28 with a mean of 2.5. Egg production averaged 7.4% (eggs/bird/day) for all birds throughout the entire trial. There were no significant differences (P>.5) between any of the treatments for feed conversion. This would indicate that differences in feed consumption or efficiency of utilization could not account for the treatment differences noted. These results indicate that the addition of a marigold concentrate or a paprika oleoresin, or both to a commercial type diet can be used to increase dietary xanthophyll levels and potentially enhance resulting yolk color. Relatively small quantities of a paprika oleoresin resulted in a much greater color response than did the marigold concentrate when used alone. Thus, deeply colored yolks could be produced by feeding low quantities of the paprika oleoresin in place of high quantities of marigold extract or other sources of yellow xanthophylls. ACKNOWLEDGMENTS This study was supported in part by state and Hatch funds allocated to the Georgia Agricultural Experiment Station. The authors are also indebted to David Claghorn of Desy TABLE 5. Lightness (), redness (), yellowness (), and redness: yellowness ratio (/) of egg yolks from bens fed various levels of marigold concentrate (MC) and paprika oleoresin (PO) Marigold concentrate xanthophyll Main effects / a»/ a'/ / a'/ av 5.2 a 7.22 m 5.2 a a abc.1 k b 11.8 k 58. ab.2) 57.4 b.47j k 55 17abcd.2 ; J 55.8 cd bcdefg.27 h MC ** (mg/kg) 57.5 b 1.21J 5.4 abc b 1.1J 54 2 abcde.2v 5.21 c IS^O 1 " 5.7 bcdef.288" cd cdef g. ^ cd cdef g.2 ef PO %** Paprika oleoresin xanthophyll 5.7 c 14.85* 5. bcdef 8.28S h 5.28 c.8 h bcdef 8.% 55.4 cd f 52. cdef g.4 de 55.71g h 1. cd 52. cdef g.8 bc 5.4 f e 4.44 f 8.8 bc MC X PO ' 54.8 de 1.2 de 5 2bcdefg. cd 5.g h 2.14 c 5.4 bcdef g.8 bc 5.7g h 1.1 cd 4.72 ef g. b 54. ef 1.8 cd 5.28 def 8. b b 48 21g 44 a h 8 a def g 4 a Downloaded from at Penn State University (Paterno Lib) on September 18, 21,, > >,,8,, >J>,, Means within each color value with different superscripts are significantly different (P<.5). " Significantly different (P<.1). 1, Not significant.

6 1 FLETCHER AND HALLORAN International, Inc., for supplying the xanthophyll products as well as for partial support of this project. REFERENCES Association of Official Analytical Chemists, 175. Official Methods of Analysis. th ed. AOAC, Washington, DC. Brown, W. L., 18. The influence of pimiento pigments on the color of the egg yolk of fowls. J. Biol. Chem. 2:55-5. Couch, J. R., and F. M. Farr, 171. The effect of adding canthaxanthin and beta-apo-8'-carotenal to laying diets containing yellow corn and alfalfa on egg yolk pigmentation. Br. Poult. Sci. : Fletcher, D. L., 18. An evaluation of the AOAC method of yolk color analysis. Poultry Sci. 5: -1. Fletcher, D. L., and H. R. Halloran, 181. An evaluation of a commercially available marigold concentrate and paprika oleoresin on yolk pigmentation. Poultry Sci. : Fletcher, D. L., R. H. Harms, and D. M. Janky, 178. Yolk color characteristics, xanthophyll availability, and a model system for predicting egg yolk color using beta-apo-8'-carotenal and canthaxanthin. Poultry Sci. 57:24-2. Fry, J. L., C. F. Hinton, and R. H. Harms, 174. Reflectance colorimetric evaluation of egg yolk pigmentation. J. Food Sci. : Mackay, E., G. J. Mountney, and E. C. Naber, 1. Yolk color resulting from different levels of paprika extract in the ration. Poultry Sci. 42: 2-7. Morgan, W. A., and J. G. Woodroof, 7. Waste pimiento pepper for coloring egg yolks. Georgia Exp. Sta. Bull Nelson, T. S., and J. N. Baptist, 18. Feed pigments. 2. The influence of feeding single and combined sources of red and yellow pigments on egg yolk color. Poultry Sci. 47:24-1. Steel, R.G.D., and J. H. Torrie, 1. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York, NY. Downloaded from at Penn State University (Paterno Lib) on September 18, 21