Egg Yolk Color as Affected by Saponification of Different Natural Pigmenting Sources

Transcription

1 2004 Poultry Science Association, Inc. Egg Yolk Color as Affected by Saponification of Different Natural Pigmenting Sources J. Galobart,* R. Sala,*,1 X. Rincón-Carruyo,* E. G. Manzanilla,* B. Vilà, and J. Gasa* *Departament de Ciència Animal i dels Aliments, Facultat de Veterinària, Universitat Autònoma de Barcelona, E Bellaterra, Spain; and ITPSA, R&D Department. Av. Roma, 157 E Barcelona, Spain Primary Audience: Nutritionists, Egg Producers, Egg Processors, Food Scientists SUMMARY Two experiments were conducted to study the influence of saponification of various yellow, red, or yellow and red xanthophylls from marigold flower and paprika fruit included at graded levels on egg yolk coloration in table and breaking eggs. Pigmentation was measured visually by the Roche yolk color fan, a subjective method, and by reflectance colorimetry, using a HunterLab MiniScan XE colorimeter, an objective method. Reflectance colorimetric a/b (redness to yellowness) ratio appeared to be a good variable to objectively measure egg yolk color on a practical basis. Results indicate that saponification of xanthophylls increased its pigmenting capacity in egg yolk. The effectiveness of saponification was more evident as a darker color of egg yolk was pursued. Both in table and in breaking eggs, the pigmenting capacity of saponified xanthophylls is at least 1.5-fold that of nonsaponified xanthophylls. Key words: egg yolk pigmentation, natural pigments, reflectance colorimetry, saponification 2004 J. Appl. Poult. Res. 13: DESCRIPTION OF PROBLEM Egg yolk color has always been regarded as an important egg quality characteristic. In fact, consumers tend to associate golden yellow to orange yolk with good health [1]. The color of egg yolk is a dietary response, and a suitable degree of pigmentation and xanthophyll content are of concern in table eggs as well as in eggs for manufacturers (breaking eggs). The yolk color depends not only on the levels of pigmenting substances, namely xanthophylls, present in the feed but also on the type and ratio of these compounds. Consumer preferences for egg yolk pigmentation vary among countries and even between regions of the same country. For example, in the US, the preferred yolk coloration ranges from 7 to 10 in the Roche yolk color fan (RYCF), whereas in some countries of Europe or Asia, the values preferred are higher (10 to 14) [1]. Yolk pigmenters can be included in poultry feed via feed ingredients (i.e., corn, corn gluten meal, alfalfa) and by addition of synthetic or natural pigments or a combination of both. For achieving an optimal yolk coloration, hens diets should be supplemented with yellow and red xanthophylls [2]. Yellow xanthophylls are needed to achieve a correct yellow base col- 1 To whom correspondence should be addressed: Roser.Sala@uab.es.

2 GALOBART ET AL.: NATURAL PIGMENTS ON YOLK COLOR 329 oration but are not allowed to reach RYCF values higher than 13 at commercial inclusion levels, because they are not economical [2, 3, 4, 5]. The addition of small amounts of red xanthophylls produces yolk colors equivalent to those obtained by higher levels of yellow xanthophylls alone [2, 3], allowing the egg producer to obtain the desired color at a lower cost. However, in eggs for the manufacture of pasta, bakery, sauces, liquors, and other items, the content of yellow xanthophylls is of great importance. Breaking eggs require a high content of yellow xanthophylls to ensure the good distribution of colors in the mixtures to which eggs are added [6]. Over recent years, there has been a marked trend toward the use of natural supplements in animal nutrition with marigold flower (Tagetes erecta) and paprika fruit (Capsicum annuum)as the main, natural sources of pigments. Marigold flower meal is one of the most concentrated sources of lutein (12 g/kg of total xanthophylls, 80 to 90% lutein) [7], and paprika meal has high levels of capsanthin (4 to 8 g/kg of total xanthophylls, 50 to 70% capsanthin) [8]. In marigold and paprika, xanthophylls are usually esterified to fatty acids, that is, attached to fatty acids as the ester. In birds, esterified carotenoids have to be split (saponified) before they are absorbed. So, a previous saponification of xanthophylls may improve its availability to the animal, as had been suggested by Hamilton et al. [9]. The objective of the present study was to compare the pigmenting efficiency of different natural products from marigold and paprika in esterified or saponified form on egg yolk color. MATERIALS AND METHODS Two experiments were planned with ISA- Brown laying hens to study the effect of saponification of paprika products, marigold products, or both and the effect of xanthophyll levels on egg yolk coloration for table eggs (experiment 1) and for breaking eggs (experiment 2). Performance parameters were checked to ensure that they could be considered normal for the experimental conditions. In both trials, hens were initially fed for 15 d a wheat-barley basal diet (white diet, Table 1) to deplete xanthophyll reserves of birds as observed in previous trials [8]. TABLE 1. Composition of the white basal diet (as fed) A Ingredient % Wheat Barley Full-fat extruded soy Soybean meal, 44% CP 6.37 Sunflower oil 4.00 Calcium carbonate 8.65 Dicalcium phosphate 1.54 Salt 0.29 Vitamin and mineral premix B 0.20 DL-Methionine 0.09 Capsozyme T/2 C 0.05 Nutrient contents Experiment 1 Experiment 2 Dry matter, % CP, % Ash, % Ether extract, % Crude fiber, % AME, D kcal/kg 2,900 2,900 Calcium, D % Available P, D % Lysine, D % Methionine, D % TSAA, E % A All diets were formulated using average nutritional contents of ingredients as found in NRC [10]. B Supplied per kilogram of total diet: vitamin A, 10,000 IU; vitamin D 3, 500 IU; vitamin E, 15 mg; vitamin K 3,2mg; thiamin, 1 mg; riboflavin, 4 mg; pyridoxin, 1 mg; cobalamin, 15 µg; nicotinic acid, 28 mg; pantothenic acid, 8 mg; choline chloride, 150 mg; Cu, 7 mg; Zn, 50 mg; Se, 200 µg; I, 1 mg; Mn, 70 mg; Co, 100 µg; Fe, 60 mg; ethoxyquin, 32 µg; propyl gallate, 4 µg; citric acid, 6 µg. C Capsozyme T/2: xylanase + β-glucanase, ITPSA (Industrial Técnica Pecuaria SA). D Calculated value. E TSAA calculated total sulfur amino acids value. Experimental diets were fed for 28 d and were formulated to meet or exceed NRC [10] requirements. Xanthophyll content in marigold flower and paprika fruit products were determined by AOAC method [11]. In the first experiment, 144 laying hens 62 wk of age were randomly distributed into 12 dietary treatments with 2 replicates of 6 hens each. Experimental treatments resulted from a 3 4 factorial arrangement having 3 products (SAP, EST-1, and EST-2) included at 4 levels to obtain 2.25, 4.50, 9.00, and mg/kg of red xanthophylls. The first product (SAP) resulted from the combination of a saponified marigold extract (33.24 g/kg of total xanthophylls) [12] with a saponified paprika extract (30.29 g/

3 330 JAPR: Research Report TABLE 2. Roche Yolk Color Fan (RYCF) and a/b (redness/yellowness) reflectance colorimetry values of egg yolk (experiment 1) Red xanthophylls (ppm) Item A RYCF SAP 7.56 a 9.71 a a a EST b 8.15 c b c EST b 9.05 b b b Product effect*** ppm effect*** Interaction*** a/b SAP a a a a EST b c b c EST b b b b Product effect*** ppm effect*** Interaction*** a c Values in each column and variable with no common superscript are significantly different (P < 0.05). A SAP, saponified marigold extract (33.24 g/kg of total xanthophylls) with saponified paprika extract (30.29 g/kg of total xanthophylls); EST-1, marigold meal (15.21 g/kg of total xanthophylls) with gas-extracted paprika oleoresin (71.89 g/kg of total xanthophylls); EST-2, marigold meal (15.21 g/kg of total xanthophylls) with paprika meal (3.81 g/kg of total xanthophylls). ***P < kg of total xanthophylls) [13]. The second product (EST-1) arose from the combination of a marigold meal (15.21 g/kg of total xanthophylls) with a gas-extracted paprika oleoresin (71.89 g/ kg of total xanthophylls). The third product (EST-2) came from the combination of the same marigold meal with a paprika meal (3.81 g/kg of total xanthophylls). In all treatments, yellow xanthophylls were adjusted to 5 ppm. In the second trial, 192 layers, 25 wk of age, were randomly distributed in 16 treatments (2 replicates of 6 hens per treatment). Experimental treatments resulted in a factorial arrangement with the factors of variation being 2 marigold oleoresins (A vs. B) 14, 15], either saponified (YSAP) or nonsaponified (YEST) and included at 4 levels to obtain 20, 40, 80, and 160 mg/kg of yellow xanthophylls. In both experiments, feed and water were provided ad libitum. Egg production was recorded daily, and feed consumption was measured for the whole period of each experiment. Although deposition of xanthophylls into egg yolk is quite a rapid process, it can take up to 3 wk to achieve a stable response to dietary pigments [3]. Therefore, samples were taken at d 19, 20, 21, 26, 27, and 28 of the feeding trial (6 to 10 eggs per treatment per day). Yolk color was determinated by using the RYCF, a subjective method, and by means of a MiniScan XE HunterLab [16] colorimeter (D65, 10 standard observer), an objective method, and expressed in the HunterLab scale (L, lightness; a, redness; and b, yellowness). Shell thickness and albumen height were measured, and Haugh units calculated in eggs collected during d 19 and 20. All production and egg quality data, as well as yolk coloration, were analyzed by ANOVA by using the general linear model procedure of SAS software [17]. When significance was reached means were separated by the least significant difference method. The response of egg yolk color variables (RYCF and a/b) was approximated by nonlineal regression to the level of dietary xanthophylls by using the GraphPad TABLE 3. Levels A (ppm) of the saponified and esterified red xanthophylls required to obtain a Roche Yolk Color Fan (RYCF) value from 10 to 14 (experiment 1) RYCF values SAP B EST EST A Estimated levels from regression equation between RYCF values and dietary levels of red xanthophylls. B SAP, saponified marigold extract (33.24 g/kg of total xanthophylls) with saponified paprika extract (30.29 g/kg of total xanthophylls); EST-1, marigold meal (15.21 g/kg of total xanthophylls) with gas-extracted paprika oleoresin (71.89 g/kg of total xanthophylls); EST-2, marigold meal (15.21 g/kg of total xanthophylls) with paprika meal (3.81 g/kg of total xanthophylls).

4 GALOBART ET AL.: NATURAL PIGMENTS ON YOLK COLOR 331 FIGURE 1. Regression between Roche yolk color fan (RYCF) values and level of red xanthophylls (RX) in the diet (experiment 1). SAP, saponified marigold extract (33.24 g/kg total xanthophylls) with saponified paprika extract (30.29 g/kg total xanthophylls); EST-1, marigold meal (15.21 g/kg of total xanthophylls) with gas-extracted paprika oleoresin (71.89 g/kg total xanthophylls); EST-2, marigold meal (15.21 g/kg of total xanthophylls) with paprika meal (3.81 g/kg total xanthophylls). K, expressed in the same units as the independent variable (ppm), represents the value of the independent variable when (maximum minimum)/2 is reached. SEE = standard error of the estimate. Prism program [18]. In all cases P 0.05 was considered significant. RESULTS AND DISCUSSION Production and egg quality parameters were considered normal for the conditions of the experiments and were not influenced by the dietary treatments (experiment 1, laying rate: 67 ± 10.4%; egg weight: 67.3 ± 1.41 g; feed conversion ratio (FCR): 2.45 ± 0.358; experiment 2, laying rate: 92 ± 6.2%; egg weight: 57.8 ± 1.51 g; FCR: 2.19 ± 0.205). Because there were no differences in feed consumption, differences in egg yolk pigmentation are considered to be treatment related. In experiment 1, RYCF values darkened as levels of red xanthophylls increased in the feed (Table 2). An interaction (P < 0.001) between a dose of red xanthophylls and product effect was observed, in the sense that the absolute differences on RYCF values between SAP and EST products were more pronounced as red xanthophyll levels increased (Figure 1). Pigmentation response followed the law of diminishing returns (Figure 1), which agrees with previous observations [4, 5, 19] The theoretical maximum egg yolk color, obtained from the regression equations between RYCF values and levels of red xanthophylls, is significantly higher for SAP product. Small differences were found between nonsaponified products, which were slightly higher for eggs from hens fed the paprika meal (EST-2) than those fed the paprika oleoresin (EST-1). Table 3 presents levels of red xanthophylls required to obtain egg yolk colors ranging from 10 to 14 in the RYCF. For moderate yolk color (i.e., RYCF = 11), 1.85 and 1.64 times more EST-1 and EST-2, respectively, are required than SAP, but when deeper colors (i.e., RYCF = 14) are pursued, this figure increases to 3.76 and 3.06, respectively.

5 332 JAPR: Research Report TABLE 4. Roche yolk color fan (RYCF) and a/b (redness/yellowness) reflectance colorimetry values of egg yolk (experiment 2) Yellow xanthophylls (ppm) Item A RYCF YSAP-A 9.80 a a a a YEST-A 8.49 c 9.52 c c c YSAP-B a b a a YEST-B 8.96 b 9.69 c b b Origin effect*** Saponification effect*** ppm effect*** Interactions*** A a/b YSAP-A C b a b b YEST-A c b d d YSAP-B a a a a YEST-B c b c c Origin effect*** Saponification effect*** ppm effect*** Interactions*** B a c Values in each column and variable with no common superscript are significantly different (P < 0.05). A All interactions but origin saponification ppm were significant. B All interactions were significant. C YSAP-A, marigold oleoresin ( g/kg of total xanthophylls) saponified (35.74 g/kg of total xanthophylls); YEST-A, marigold oleoresin ( g/kg of total xanthophylls) sprayed on inert carrier (53.17 g/kg); YSAP-B, marigold oleoresin ( g/kg of total xanthophylls) saponified product (32.97 g/kg); YEST-B, marigold oleoresin ( g/kg of total xanthophylls) sprayed on inert carrier (43.38 g/kg). ***P < Saponification also significantly improved colorimetric variables. When levels of red xanthophylls increased, a and a/b values increased, but L and b values decreased; the a/b ratio was the most sensitive parameter (Table 2). As it had been suggested by Fletcher and Halloran [4, 5] and Baiao et al. [20], this a/b ratio might be considered as a simple and objective expression of yolk color. Similarly to RYCF, SAP sources reached higher a/b values than EST products, with small differences between EST ones. From these results, gas extraction of paprika to concentrate xanthophylls did not result in an important modification of its pigmenting properties. An interaction (P < 0.001) was also observed, indicating that the effect of increasing red xanthophylls was more pronounced on those eggs from SAP product. In experiment 2, as in previous experiment, pigmentation response (RYCF and a/b values) was higher as level of xanthophylls increased in the feed (Table 4), following the law of diminishing returns (Figure 2). Although oleoresin B presented a slightly higher pigmenting efficiency than oleoresin A (Table 4), interaction between oleoresin and processing effect indicated that the improvement due to saponification was more pronounced in oleoresin A (Figure 2). Again, the differences in yolk coloration between saponified and nonsaponified pigments were more evident as the level of yellow xanthophylls increased in the feed. The RYCF achieved with 80 ppm of yellow xanthophylls was to 1.5 to 2.4 units higher in YSAP products than in YEST ones (Table 4). Similar results were reported by Huyghebaert [19] by using different saponified and nonsaponified concentrates from marigold extracts. In nonsapon- TABLE 5. Level A (ppm) of saponified and esterified yellow xanthophylls required to obtain redness/ yellowness (a/b) ratio from 0.6 to 0.9 (experiment 2) a/b ratio YSAP-A B YEST-A NA NA YSAP-B YEST-B A Estimated levels from regression equation between a/b ratio values and dietary levels of yellow xanthophylls. B YSAP-A, marigold oleoresin ( g/kg of total xanthophylls) saponified (35.74 g/kg of total xanthophylls); YEST-A, marigold oleoresin ( g/kg of total xanthophylls) sprayed on inert carrier (53.17 g/kg); YSAP- B, marigold oleoresin ( g/kg of total xanthophylls) saponified product (32.97 g/kg); YEST-B, marigold oleoresin ( g/kg of total xanthophylls) sprayed on inert carrier (43.38 g/kg). NA = not available.

6 GALOBART ET AL.: NATURAL PIGMENTS ON YOLK COLOR 333 FIGURE 2. Regression between redness and yellowness (a/b) ratio and level of yellow xanthophylls (YX) in the diet (experiment 2). YSAP-A, marigold oleoresin ( g/kg of total xanthophylls) saponified (35.74 g/kg of total xanthophylls); YEST-A, marigold oleoresin ( g/kg of total xanthophylls) sprayed on inert carrier (53.17 g/ kg); YSAP-B, marigold oleoresin ( g/kg of total xanthophylls) saponified product (32.97 g/kg); YEST-B, marigold oleoresin ( g/kg of total xanthophylls) sprayed on inert carrier (43.38 g/kg). K, expressed in the same units as the independent variable (ppm), represents the value of the independent variable when (maximum minimum)/2 is reached. SEE = standard error of the estimate. ified treatments, differences between oleoresins increased as the level of yellow xanthophylls increased. This tendency was also observed in saponified oleoresins, but differences were of lower magnitude, starting at 80 ppm for a/b (Figure 2). In Table 5 the pigmenting efficacy expressed in the a/b ratio is depicted. Saponification of xanthophylls not only increased their availability [9] but also reduced differences between oleoresins, improving uniformity. For a ratio of 0.8, obtained with 55 ppm of YSAP-B, more than twice the amount of YEST-B was needed. This value is not possible to achieve with YEST-A (theoretical maximum a/b value = 0.795). As it can be observed in both experiments, the use of saponified pigments resulted in a higher degree of coloration of egg yolk. These results are in agreement with those published by Hamilton et al. [9], Huyghebaert [19], Brufau [1], and González et al. [21] who described a higher degree of yolk coloration when saponified pigments were used. On the other hand, Henken [22] and Lai et al. [23] did not find any improvement of yolk pigmentation when saponified pigments were used. It has been established that esterified carotenoids have to be hydrolyzed before their intestinal absorption is deposited in the yolk mainly in the free alcohol form [23, 24] and independently of the dietary form. This is why saponified xanthophylls show a higher pigmenting ability. Nys [25] reported that saponification of Tagetes and Capsicum xanthophylls can improve their biological

7 334 availability by 30 to 40% and by 80%, respectively. From the results of Hamilton et al. [9], it is shown that saponification of xanthophylls results in a 2-fold deposition in egg yolk of these compounds. Results from the present work indicate that the relative efficiency of the xanthophylls JAPR: Research Report 1. Brufau, J Yolk The golden opportunity. International table egg production supplement. Int. Poult. Prod. 5: Belyavin, C. G., and A. G. Marangos Natural products for egg yolk pigmentation. Pages in Recent Advances in Animal Nutrition. W. Haresign and D. J. A. Cole, ed. Butterworths, London. 3. Adams, C. A Pigmenters and poultry feeds. Feed Compounder (Jun./Jul): Fletcher, D. L., and H. R. Halloran An evaluation of a commercially available marigold concentrate and paprika oleoresin on yolk pigmentation. Poult. Sci. 60: Fletcher, D. L., and H. R. Halloran Egg yolk pigmenting properties of a marigold extract and paprika oleoresin in a practical type diet. Poult. Sci. 62: Blanch, A., and J. M. Hernández Handling yellow carotenoids for optimal pigmentantion. Feed Mix 8: Quackenbush, F. W., and S. L. Miller Composition and analysis of the carotenoids in marigold petals. J. Assoc. Off. Agric. Chem. 55: Calafat, F. A Industrial Técnica Pecuaria S.A., Barcelona, Spain, personal communication. 9. Hamilton, P., F. Tirado, and F. García-Hernández Deposition in egg yolks of the carotenoids from saponified and unsaponified oleoresin of red pepper (Capsicum annuum) fed to laying hens. Poult. Sci. 69: National Research Council Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Sci., Washington, DC. 11. AOAC Method Official Methods of Analysis of the Association of Analytical Chemists. 15th ed. AOAC, Washington, DC. 12. Saponified extract from marigold flowers (Tagetes erecta) with g/kg of total xanthophylls (trade name: Capsantal EBS, Ind. Tecn. Pecuaria SA, ITPSA, Barcelona, Spain). 13. Saponified extract from paprika fruit (Capsicum annuum) with g/kg of total xanthophylls (trade name: Capsantal FS, Industrial Tecnica Pecuaria SA, ITPSA, Barcelona, Spain). 14. Marigold oleoresin A. ( g/kg of total xanthophylls): YEST-A sprayed on inert carrier g/kg; YSAP-A saponified proddepend on the target yolk color. In both trials, the higher efficiency of SAP xanthophylls, with respect to EST ones, was more evident at darker egg yolk color. This higher efficiency justifies the cost of saponification [8]. CONCLUSIONS AND APPLICATIONS 1. Saponification of natural pigmenting products from paprika and marigold results in an improvement of the pigmenting efficiency of such products. 2. The effectiveness of saponified xanthophylls increases as the desired yolk color intensity also increases. 3. Saponification of xanthophylls reduces the differences in pigmenting properties among oleoresins from different origins. REFERENCES AND NOTES uct (35.74 g/kg, trade name Capsantal EBS, Industrial Tecnica Pecuaria SA, ITPSA, Barcelona, Spain). 15. Marigold oleoresin B. ( g/kg of total xanthophylls): YEST-B sprayed on inert carrier g/kg; YSAP-B saponified product (32.97 g/kg, trade name Capsantal EBS, Industrial Tecnica Pecuaria SA, ITPSA, Barcelona, Spain). 16. MiniScanXE. HunterLab, Reston, VA. 17. SAS Institute SAS User s Guide. Version SAS Institute Inc., Cary, NC. 18. GraphPad Prism Version GraphPad, San Diego. 19. Huyghebaert, G The relative biological efficacy of yellow oxy-carotenoids for egg yolk pigmentation. Pages in Proc. IV Symp. Qual. Eggs Egg Products, Doorwerth, Netherlands. 20. Baiao, N. C., J. Mendez, J. Mateos, M. García, and G. G. Mateos Pigmenting efficacy of several oxicarotenoids on egg yolk. J. Appl. Poult. Res. 8: González, M., E. Castaño, E. Avila, and E. González de Mejía Effect of capsaicin from red pepper (Capsicum sp.) on the deposition of carotenoids in egg yolk. J. Sci. Food Agric. 79: Hencken, H Chemical and physiological behavior of feed carotenoids and their effects on pigmentation. Poult. Sci. 71: Lai, S. M., J. Gray, and C. Flegal Deposition of carotenoids in eggs from hens fed diets containing saponified and unsaponified oleoresin paprika. J. Sci. Food Agric. 72: Scheffer, J. L., J. K. Tyczkowski, C. R. Parkhust, and P. B. Hamilton Carotenoid composition of serum and egg yolks of hens fed diets varying in carotenoid composition. Poult. Sci. 67: Nys, Y Dietary carotenoids and egg yolk coloration. Pages 3 21 in Proc. VIII Eur. Symp. Qual. Eggs Egg Products, Bologna, Italy. Acknowledgments This work was partly supported by a EUREKA (no ) project led by ITPSA, Barcelona, Spain.