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Pigmentation of Egg Yolks with Astaxanthin from the Yeast Phaffia rhodozyma
Pigmentation of Egg Yolks with Astaxanthin from the Yeast Phaffia rhodozyma ERIC A. JOHNSON,1 ' 3 MICHAEL J. LEWIS,1 ,4 and C. R. GRAU2 Department of Food Science and Technology and Department of Avian Sciences, University of California, Davis, California 95616 (Received for publication May 7, 1979)
1980 Poultry Science 59:1777-1782
INTRODUCTION Because of the economic i m p o r t a n c e of color in chicken egg yolk, m a n y investigators have studied the uses of natural and synthetic carotenoids as yolk pigmenters (Bauernfeind et al., 1971). Yolk with a high p i g m e n t c o n t e n t is in d e m a n d for bakery goods, noodles, m a y o n naise, and certain o t h e r foods. Traditionally, the pigment sources in chicken rations have been yellow corn and alfalfa, which supply t h e p r o m i n e n t egg yolk pigments, cryptoxantJhin [(3R)-(3, |3-carotene-3-ol], zeaxanthin [3R, 3 R)-(3, fi-carotene-3, 3 ' - d i o l ] , and lutein [(3R, 3'S, 6'R)-(3-e carotene-3, 3 ' - d i o l ] . F o r e c o n o m i c reasons, however, grains such as milo, wheat, and barley often replace corn and alfalfa in commercial chicken rations with c o n s e q u e n t loss in pigmentation. This has led t o identification of and e x p e r i m e n t a t i o n with alternative carotenoid sources. Because the chicken converts carotenes to colorless vitamin A in t h e intestinal mucosa, these cartenoids have little pigmenting ability. It is necessary t o feed x a n t h o p h y l l s or apo-
1
Department of Food Science and Technology. Department of Avian Sciences. Present address: Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, MA 02139. 4 Person to whom reprint requests should be addressed. 2 3
carotenoids t o obtain a d e q u a t e egg y o l k color. T h e x a n t h o p h y l l s comprise a wide variety of structures, and it is n o t presently possible to predict t h e pigmenting p o t e n c y of a particular carotenoid, especially if it is fed in a natural feedstuff. As a general rule, however, dihydroxy- and diketo-xandiophylls are better utilized for yolk pigmentation than m o n o hydroxy-, m o n o k e t o - , or epoxy-carotenoids (Braeunlich, 1978). It is also k n o w n t h a t esterification may decrease the pigmenting activity of particular x a n t h o p h y l l s (Couch, 1972). T h e natural pigment sources m o s t extensively studied have been yellow corn, marigold extract, and alfalfa which all contain lutein as their major carotenoid. Some experim e n t a t i o n has also been done with t h e reddish synthetic pigments )3-apo-8 -carotenal (8 -apo-)3carotene-8 -al) and c a n t h a x a n t h i n (|3,j3-carotene-4, 4 - d i o n e ) (Fletcher et al., 1 9 7 8 ) ; when used alone or mixed with small quantities of yellow pigments, these pigments cast an orange h u e o n t o t h e egg y o l k (Marusich et al, 1960). T h e use of red pigments alone is n o t recomm e n d e d in t h e p r o d u c t i o n of yolks for bakery or macaroni goods since t h e p r o d u c t s take on an undesirable red color. However, t h e p r o p e r mixing of red and yellow pigments m a y result in attractive and commercially useful egg yolks (Nelson and Baptist, 1969). The yeast Phaffia rhodozyma, which contains astaxanthin ( 3 , 3 -dihydroxy-/3, j3-carotene-4, 4 -dione) as its primary carotenoid, was f o u n d
1777
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ABSTRACT The red yeast Phaffia rhodozyma was tested as a dietary pigment source for egg yolks of laying hens and Japanese quail. It was found that astaxanthin from broken yeast or prepared yeast oil but not from intact yeast cells was deposited in egg yolks. The efficiency of carotenoid deposition was approximately 4%, and it was only slightly dependent of the astaxanthin concentration in the diet. Astaxanthin was probably deposited without metabolic alteration in egg yolks. When P. rhodozyma was fed to laying hens at several concentrations and in combination with marigold flower pigments or yellow corn, a wide range of colors was achieved; depending on the yeast concentration in the feed, the dominant wavelength of chicken egg yolks ranged from 571 nm to 593 nm. (Key words: egg yolk, pigmentation, astaxanthin, yeast)
1778
JOHNSON ET AL.
to be an excellent dietary pigment source to produce pink flesh in pen-reared salmonids (Johnson et al, 1977). Nelson and Baptist (1969) found that astaxanthin from lobster shells fed in combination with lutein, was 30 to 50 times more effective than lutein alone in pigmenting quail egg yolks. The present study was designed to investigate the possible use of P. rhodozyma as a pigment source in feed for poultry to enhance the color of egg yolks. MATERIALS AND METHODS
Ingredient
Chickens
Soybean meal (48%) Soybean protein Soybean oil Vitamin m i x 3 Mineral p r e m i x a Cellulose Methionine CaC03 CaHP04-2H,0 NaCl Milo Starch
32
Quail
28.4 3.75 1 1.31 5 .45 5 3 .99
3 1 1.31 0 0 5 3 .99 53.7
51.1
For composition see Roudybush (1977).
was remarkably constant during this period at 20g per bird per day. Each group of birds yielded 2 to 4 eggs/day with yolks of 1 lg fresh weight. Chicken Study: Effect of Mixed Pigment Sources on Egg Yolk Color. After feeding the low pigment diet, 28 chickens were transferred individually to the experimental diets 17 through 44, and 1 chicken was maintained on the unsupplemented diet (diet 16). The diet for each chicken was continuously available and was provided fresh each day from cold storage.
TABLE 2. Efficiency of carotenoid deposition into quail yolk C arotenoid utilization
Pigment source and diet c o n c e n t r a t i o n Diet no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
P. rho dozyma, ruptured, dried
Marigold concentrate
Corn, yellow, ground
Deposited per day
Ingested per day
ingested
(%)
(mg %)
(%)
(Mg)
(Mg)
(%)
0 0 0 0 0 0 2.5 6.5 13.0 25.4 50.8 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 5.1 12.7 25.4 50.8
.3 1.9 2.7 5.2 9.4 17.8 .88 1.6 3.2 7.1 16.2 1 2.4 4.8 9.4
48 65 130 238 476 22.5 58.6 117 228 457 23.4 58.4 116 234
3.95 4.15 4 3.95 3.74 3.91 2.73 2.48 3.11 3.54 4.27 4.10 4.10 4.02
0 .7 .96 1.9 3.5 7.0 0 0 0 0 0 0 0 0 0
Deposited
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Birds. Laying hens of stock bred for egg production and quail (Cotumix coturnix japonica) were housed in rooms of constant temperature (25 C) and photoperiod (14L: 10D). Food and water were always available. Eggs were collected each day between 0800 and 0900 hr and frozen and stored at —20 C until analysis. The quail and chickens were first partially depleted of pigments by feeding for 12 or 16 days, respectively, a low-pigment diet (diets 1 and 16: Tables 1, 2, and 4). Quail Study: Comparison of Efficiency of Pigment Sources. After the pigment-depletion period, the quail were offered, in groups of 4 birds, diets 2 to 15 (Table 2). These diets, which contained one of the pigment sources in replacement of an equal weight of starch, were fed for a period of eight days. Feed consumption
TABLE 1. Composition of chicken and quail basal diets (%)
EGG YOLK PIGMENTATION WITH PHAFFIA
RHODOZYMA
1779
TABLE 3. Effect of freezing on color determination of egg yolks'* Dominant wavelength
Luminosity
Excitation/ purity
Liquid yolks
575.2 (±.4) 575.9 (+.11)
24.5 (±1.7) 23.2 (±3.2)
75 (±9.8) 80.6 (±9.2)
Frozen yolks
575.7 (±.8) 575.6 (±.62)
35.9 (±2.5)
71 (±4.5) 62.6 (±7.1)
38.6 (±3.4)
The color of liquid and frozen yolks was measured for two lots of commercially produced eggs. Three yolks were used for each color determination.
marigold and corn diets, A m ax = 446 nm, E}% m = 2200 (Davies, 1976)] . For chromatography, the ether extracts of diets or eggs were dried over anhydrous Na2 S 0 4 and concentrated by rotary evaporation at 30 C. The extracts were chromatographed on a thin layer of silica gel (Silica Gel 60, .25 mm thickness, Brinkmann Instruments Inc.) using 10% acetone in petroleum ether as the developing solvent. In the chicken study, each diet was represented by 4 eggs. The dominant wavelength (dwl), luminosity (lum), and excitation purity (ep) were measured on 1 cm slices of the yolk of each of these eggs that had been frozen and thawed. The mean of these values is reported in Table 4. This procedure yielded yolk slices that were of the same dominant wavelength as fresh liquid yolk, but of higher luminosity and lower excitation purity. A Macbeth spectrophotometer (Model #M52045) with illuminant C and 8 mm sample aperture was used in the color determinations.
RESULTS AND DISCUSSION In a preliminary experiment, intact red yeast (P. rhodozyma) cells, mechanically fractured red yeast, and an oil of P. rhodozyma were fed for one day to individual chickens. Only the carotenoids of the fractured yeast and the oil were deposited in the yolk, which was evident by an orange-red ring in the sliced frozen yolks. These results agree with our previous findings in rainbow trout; pigments of intact yeast cells were not incorporated into the fish flesh, but pigments from broken yeast were rapidly deposited (Johnson, 1978). In all chicken and quail diets containing red yeast in this study, only mechanically fractured yeast was used. Comparison of Three Sources of Xanthophylls. When quail were fed a diet low in
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Almost all the chickens laid one egg each day. It required 10 to 12 days for the plateau of color in the eggs to be reached, whereupon four consecutive eggs from each chicken (i.e., each diet) were collected for color analysis. Diets. P. rhodozyma for the feeding experiments was grown as previously described (Johnson et al., 1978), broken in a MantonGaulin cell homogenizer (Johnson and Phaff, 1978), and freeze-dried for diet preparation. Yeast oil was prepared by extracting broken yeast with acetone, transferring the carotenoids to petroleum ether, drying over Na2 SO4, and concentrating the petroleum ether solutions of pigment in a rotary evaporator. Marigold meal was obtained from S. B. Penick and Co., New York, NY. Assay of the carotenoid content of P. rhodozyma, ground corn, and marigold concentrate (see below) revealed that they contained .34, .023, and 45.0 mg total carotenoid/g (dry weight basis), respectively. All feeds were protected against oxidation by addition of . 1 % butylated hydroxy toluene (BHT). Quail diets were stored at 4 C and chicken diets at 12 C during the experiment. Analysis of Egg Yolks. In the quail study, frozen quail yolks were separated from the albumen by rolling on a paper towel. The eggs collected each day from each diet group (2 to 4 eggs) were combined in a beaker, approximately 80 ml acetone were added, and after vigorous homogenization in a Virtis homogenizer under a stream of nitrogen, the mixture was centrifuged and the pellet was reextracted with acetone. The acetone extracts were pooled, the carotenoids transferred to petroleum ether by the addition of a dilute sodium chloride solution, and the carotenoid content of the solution estimated as astaxanthin or lutein by measuring the absorbance at A max: [yeast diets, X m a x = 478 nm, E|% m = 1600 (Andrewes et al., 1976);
1780
JOHNSON ET AL. TABLE 4. Colors of egg yolks laid by hens fed various concentrations of P. rhodozyma, marigold concentrate, or yellow com Diet concentration of pigment source
Diet no.
Color measurements
P. rhodozyma
Marigold Corn, concentrate yellow
P. rhodozyma
Marigold Corn
Dominant wavelength
(%)
(mg %)
(ppm)
(ppm)
(ppm)
(nm)
0 0 0 0 0 5 10 20 0 0 0 0 0 5 10 20 0 0 0 0 0 5 10 20 0 2 5 8 10
0 0 0 0 0 0 0 0 5 5 5 5 5 5 5 5 10 10 10 10 10 10 10 10 0 0 0 0 0
571.7 574.9 584.3 588.9 591.9 572.3 573.2 573.9 574.6 576.9 578.8 584.4 593.0 573.4 572.2 573.2 575.2 575.4 581.2 583.5 589.9 573.6 574.7 574.8 588.6 585.4 579.8 574.6 571.2
0 .3 .73 1.5 2.95
0 0 0 0 .3 .73 1.5 2.95
0 0 0 0 .3 .73 1.5 2.95
0 0 0 1.5 1.2 .75 .3 0
0 0 0 0 0 5.55 11.1 22.2
0 0 0 0 0 5.55 11.1 22.2
0 0 0 0 0 5.55 11.1 22.2
0 2.2 5.55 8.85 11.1
(%) 0 0 0 0 0 0 0 0 10.85 10.85 10.85 10.85 10.85 10.85 10.85 10.85 21.7 21.7 21.7 21.7 21.7 21.7 21.7 21.7
0 0 0 0 0
0 2.04
5 10.2 20.1
0 0 0 0 2.04
5 10.2 20.1
0 0 0 0 2.04
5 10.2 20.1
0 0 0 10.2
8.2 5.1 2 0
pigment, the total carotenoid content of their eggs decreased in 7 days from 108 /ig/yolk to 18 /!g/yolk and decreased slowly during the following five days to a minimum of 12 /^g/yolk (Fig. 1). Following this period the quail were fed diets which contained several concentrations of broken red yeast, marigold extract, or yellow corn (Table 2). The amount of feed consumed each day by the quail was remarkably constant at 20 g/bird. Pigment deposition was noticeable after 2 days feeding as a thin outer ring in sliced frozen yolks. After about 6 days of feeding, pigment content in the quail egg yolks plateaued (Fig. 2). In each case, the amount of carotenoid deposited in the yolks was a function of the amount of carotenoid ingested. Carotenoids were somewhat more efficiently deposited in the yolks of quail fed P. rhodozyma or corn (about 4% of carotenoid ingested) than those
Excitation Lumipurity nosity
20.8 16.8 18.8 22.9 25.9 30.9 33.2 37.4 33.0 30.2 27.3 32.0 25.7 31.5 34.9 41.6 36.7 33.4 39.0 33.2 39.5 33.3 50.7 52.0 19.6 19.8 26.5 28.5 33.9
33.3 28.8 23.8 19.9 18.8 26.0 28.6 28.5 27.2 24.8 24.2 22.3 16.9 24.8 23.9 27.2 27.5 24.9 20.1 22.0 15.9 24.6 25.2 22.3 22.6 20.8 22.1 25.1 26.1
fed marigold extract (about 3%, Table 2), but it is evident that the efficiency of transfer of carotenoids from these three pigment sources tested was poor. Our results with quail agree with the data of Marusich et al. (1960) who found the average pigment content of chicken yolks, sampled in the plateau region, increased linearly with the daily dose of carotenoid. However, our efficiency levels for carotenoid transfer are generally lower than those obtained by Marusich et al. (1960). Their values, for various pure carotenoids, ranged from 2.2% for violaxantJiin to 26.0% for canthaxanthin; 13.9% efficiency was found for dehydrated alfalfa leaf meal, which contains lutein as its major carotenoid. These differences may reflect different digestive abilities of quail and chickens or differences in the composition of our basal diet. It is inter-
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16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Carotenoid concentrations furnished by
EGG YOLK PIGMENTATION WITH PHAFFIA
110 100
-
80 -
90
70
y
60
\ \
50
\
40
\
30 20
x^^
10 )
1
i
i
i
2
3
4
5
6
7
8
9
10 II 12
FIG. 1. Depletion of egg yolk pigments in Japanese quail by feeding a diet low in carotenoid content (diet 1).
esting that in the present study the percentage of carotenoid deposited was rather constant regardless of the quantity of carotenoid ingested or its source. The carotenoids extracted from the diets and from eggs collected on the seventh day were chromatographed. It was found that the primary carotenoid present in the yolk of quail fed the yeast or corn diets cochromatographed with the main pigment in the respective feeds (rfs = .11 (astaxantiiin) and .14 (lutein), respectively); the astaxanthin of the yeast and lutein of the corn were deposited without modification in the egg yolk. However, the primary pigment found in the egg yolk of quail fed marigold extract (rf = .14) and in the marigold diet (rf = .92) were not the same. Lutein occurs in an esterified form in marigold (Quackenbush and Miller, 1972). This explains the high rf value of the major carotenoid in the marigold extract, because carotenoid esters are less polar chromatographically than the parent carotenoid (Davies, 1976). Either this predominant esterified carotenoid was altered by the bird during the deposition process, or another (unesterified) carotenoid was selectively absorbed. Influence of Mixed Pigmentation Sources on Chicken Egg Yolk Color. It was found that the dominant wavelength (dwl), the main measurement parameter of color that influences visual judgment (Fletcher et al, 1978) was the same in fresh or frozen-thawed egg yolks (Table
1781
3); however, luminosity and excitation purity were different in the two conditions. Our values for dwl are directly comparable to values obtained by other workers using fresh yolks, but lum and ep values are only valid for comparison within our experiments. To determine the effect on egg yolk color, 29 laying hens were fed diets which contained different proportions of xanthophylls derived from P. rhodozyma, marigold extract, and yellow corn. A considerable range of colors was obtained in the yolks as observed visually and measured by the dwl of the freeze-thawed yolks (Table 4). The dwl ranged from 571 nm to 593 nm. The dwl was dependent almost entirely upon the concentration of yeast in the diet. Thus, as the concentration of astaxanthin only (diets 16 to 20) was increased from 0 to 20.1 mg/kg, the dwl increased from 571.7 to 591.9. In contrast, as the concentration of marigold only (diets 16 and 21 to 23) was increased from 0 to 20.0 mg/kg, the dwl increased from 571.7 to 573.9; a similar small change was seen for corn only (diets 16, 24, and 32) or for marigold mixed with corn (diets 29 to 31 and 37 to 3 9). When corn at two levels was added to diets containing increasing concentrations of astaxanthin (diets 25 to 28 and 33 to 36 and control diets 17 to 20) the increase in dwl was 15 nm in each case, although there was a suggestion of a slightly lower dwl on average with corn present. Weiser and Manz (1972, unpublished data, cited in Braeunlich, 1978) found that a high level of yellow pigments suppressed the effect of red pigments on the deepening of egg yolk color; it is possible the
A.
B.
C.
r>6
J '~~* // / ,.5
0^
, 5
*-*— 10 S~* 4
a. 20
0
C^1 2
4
6
8
0
t^r ' 2 4
0
A 2
4
y ^ 14
6
Days
FIG. 2 Deposition of carotenoids in the egg yolks of Coturnix quail fed broken red yeast (diets 2 to 6), marigold flower extract (diets 7 to 11), or yellow corn (diets 12 to 15). Composition of diets as in Table 2.
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Day
RHODOZYMA
1782
JOHNSON ET AL.
amount of corn used here was insufficient to show its effect clearly. Diets 40 to 44 contained a constant concentration of carotenoid (10 mg/kg) but varied in the relative concentrations of yeast or marigold pigments. It was again observed that the dwl was higher with those diets containing more astaxanthin from P. rhodozyma. The yolks of the eggs described in Table 4 were yellow when the diet contained corn or marigold. When lower concentrations of yeast were also in the diet, the yolks were gold in color but were orange-pink at the highest concentration of yeast used.
REFERENCES Andrewes, A. G., H. J. Phaff, and M. P. Starr, 1976. Carotenoids of Phaffia rhodozyma, a red-pigmented fermenting yeast. Phytochemistry 15: 1003-1007. Bauernfeind, J. C , G. B. Brubacher, H. M. Klaui, and W. L. Marusich, 1971. Use of carotenoids. Pages 743—770 in Carotenoids. O. Isler, ed. Birkhauser Verlag, Basel, Stuttgart. Braeunlich, K.. 1978. The chemistry and action of pigmenters in poultry diets. World Poultry Congr. 15:236-240. (Abstr.)
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ACKNOWLEDGMENTS We thank T. Roudybush and K. Hirsch for assisting in the preparation of the diets and J. Buhlert for the color determinations.
Couch, J. R., 1972. Pigmentation. Feedstuffs. April 3 44(14): 21. Davies, B. H., 1976. Carotenoids. Page 38-165 in Chemistry and biochemistry of plant pigments. Vol. 2. T. W. Goodwin, ed. Academic Press, London. Fletcher, D. L., R. H. Harms, and D. M. Janky, 1978. Yolk color characteristics, xanthophyll availability, and a model system for predicting egg yolk color using /3eta-Apo-8'-carotenal and canthaxanthin. Poultry Sci. 57:624-629. Johnson, E. A., 1978. Astaxanthin production by the yeast Phaffia rhodozyma and its use as a pigment source in animal feeding. M.S. thesis, University of California, Davis, CA. Johnson, E. A., D. E. Conklin, and M. J. Lewis, 1977. The yeast Phaffia rhodozyma as a dietary pigment source for salmonids and crustaceans. J. Fish. Res. Board Can. 34:2417-2421. Johnson, E. A., and H. J. Phaff, 1978. Rhodotorula fujisanensis, a. new taxonomic combination. Current Microbiol. 1:223-225. Johnson, E. A., T. G. Villa, M. J. Lewis, and H. J. Phaff, 1978. Simple method for the isolation of astaxanthin from the basidiomycetous yeast Phaffia rhodozyma. Appl. Environ. Microbiol. 35:1155-1159. Marusich, W., E. De Ritter, and J. C. Bauernfeind, 1960. Evaluation of carotenoid pigments for coloring egg yolks. Poultry Sci. 39:1338—1345. Nelson, T. S., and J. N. Baptist, 1968. Feed pigments. 2. The influence of feeding single and combined sources of red and yellow pigments on egg yolk color. Poultry Sci. 4 7 : 9 2 4 - 9 3 1 . Quackenbush, F. W., and S. Miller, 1972. Composition and analysis of the carotenoids in marigold petals. J. Ass. Offic. Anal. Chem. 55:617. Roudybush, T. E., 1977. Fructose utilization in Coturnix coturnix japonica (Japanese quail). M.S. thesis, University of California, Davis. CA.
1980 Poultry Science 59:1777-1782
INTRODUCTION Because of the economic i m p o r t a n c e of color in chicken egg yolk, m a n y investigators have studied the uses of natural and synthetic carotenoids as yolk pigmenters (Bauernfeind et al., 1971). Yolk with a high p i g m e n t c o n t e n t is in d e m a n d for bakery goods, noodles, m a y o n naise, and certain o t h e r foods. Traditionally, the pigment sources in chicken rations have been yellow corn and alfalfa, which supply t h e p r o m i n e n t egg yolk pigments, cryptoxantJhin [(3R)-(3, |3-carotene-3-ol], zeaxanthin [3R, 3 R)-(3, fi-carotene-3, 3 ' - d i o l ] , and lutein [(3R, 3'S, 6'R)-(3-e carotene-3, 3 ' - d i o l ] . F o r e c o n o m i c reasons, however, grains such as milo, wheat, and barley often replace corn and alfalfa in commercial chicken rations with c o n s e q u e n t loss in pigmentation. This has led t o identification of and e x p e r i m e n t a t i o n with alternative carotenoid sources. Because the chicken converts carotenes to colorless vitamin A in t h e intestinal mucosa, these cartenoids have little pigmenting ability. It is necessary t o feed x a n t h o p h y l l s or apo-
1
Department of Food Science and Technology. Department of Avian Sciences. Present address: Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, MA 02139. 4 Person to whom reprint requests should be addressed. 2 3
carotenoids t o obtain a d e q u a t e egg y o l k color. T h e x a n t h o p h y l l s comprise a wide variety of structures, and it is n o t presently possible to predict t h e pigmenting p o t e n c y of a particular carotenoid, especially if it is fed in a natural feedstuff. As a general rule, however, dihydroxy- and diketo-xandiophylls are better utilized for yolk pigmentation than m o n o hydroxy-, m o n o k e t o - , or epoxy-carotenoids (Braeunlich, 1978). It is also k n o w n t h a t esterification may decrease the pigmenting activity of particular x a n t h o p h y l l s (Couch, 1972). T h e natural pigment sources m o s t extensively studied have been yellow corn, marigold extract, and alfalfa which all contain lutein as their major carotenoid. Some experim e n t a t i o n has also been done with t h e reddish synthetic pigments )3-apo-8 -carotenal (8 -apo-)3carotene-8 -al) and c a n t h a x a n t h i n (|3,j3-carotene-4, 4 - d i o n e ) (Fletcher et al., 1 9 7 8 ) ; when used alone or mixed with small quantities of yellow pigments, these pigments cast an orange h u e o n t o t h e egg y o l k (Marusich et al, 1960). T h e use of red pigments alone is n o t recomm e n d e d in t h e p r o d u c t i o n of yolks for bakery or macaroni goods since t h e p r o d u c t s take on an undesirable red color. However, t h e p r o p e r mixing of red and yellow pigments m a y result in attractive and commercially useful egg yolks (Nelson and Baptist, 1969). The yeast Phaffia rhodozyma, which contains astaxanthin ( 3 , 3 -dihydroxy-/3, j3-carotene-4, 4 -dione) as its primary carotenoid, was f o u n d
1777
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 14, 2015
ABSTRACT The red yeast Phaffia rhodozyma was tested as a dietary pigment source for egg yolks of laying hens and Japanese quail. It was found that astaxanthin from broken yeast or prepared yeast oil but not from intact yeast cells was deposited in egg yolks. The efficiency of carotenoid deposition was approximately 4%, and it was only slightly dependent of the astaxanthin concentration in the diet. Astaxanthin was probably deposited without metabolic alteration in egg yolks. When P. rhodozyma was fed to laying hens at several concentrations and in combination with marigold flower pigments or yellow corn, a wide range of colors was achieved; depending on the yeast concentration in the feed, the dominant wavelength of chicken egg yolks ranged from 571 nm to 593 nm. (Key words: egg yolk, pigmentation, astaxanthin, yeast)
1778
JOHNSON ET AL.
to be an excellent dietary pigment source to produce pink flesh in pen-reared salmonids (Johnson et al, 1977). Nelson and Baptist (1969) found that astaxanthin from lobster shells fed in combination with lutein, was 30 to 50 times more effective than lutein alone in pigmenting quail egg yolks. The present study was designed to investigate the possible use of P. rhodozyma as a pigment source in feed for poultry to enhance the color of egg yolks. MATERIALS AND METHODS
Ingredient
Chickens
Soybean meal (48%) Soybean protein Soybean oil Vitamin m i x 3 Mineral p r e m i x a Cellulose Methionine CaC03 CaHP04-2H,0 NaCl Milo Starch
32
Quail
28.4 3.75 1 1.31 5 .45 5 3 .99
3 1 1.31 0 0 5 3 .99 53.7
51.1
For composition see Roudybush (1977).
was remarkably constant during this period at 20g per bird per day. Each group of birds yielded 2 to 4 eggs/day with yolks of 1 lg fresh weight. Chicken Study: Effect of Mixed Pigment Sources on Egg Yolk Color. After feeding the low pigment diet, 28 chickens were transferred individually to the experimental diets 17 through 44, and 1 chicken was maintained on the unsupplemented diet (diet 16). The diet for each chicken was continuously available and was provided fresh each day from cold storage.
TABLE 2. Efficiency of carotenoid deposition into quail yolk C arotenoid utilization
Pigment source and diet c o n c e n t r a t i o n Diet no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
P. rho dozyma, ruptured, dried
Marigold concentrate
Corn, yellow, ground
Deposited per day
Ingested per day
ingested
(%)
(mg %)
(%)
(Mg)
(Mg)
(%)
0 0 0 0 0 0 2.5 6.5 13.0 25.4 50.8 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 5.1 12.7 25.4 50.8
.3 1.9 2.7 5.2 9.4 17.8 .88 1.6 3.2 7.1 16.2 1 2.4 4.8 9.4
48 65 130 238 476 22.5 58.6 117 228 457 23.4 58.4 116 234
3.95 4.15 4 3.95 3.74 3.91 2.73 2.48 3.11 3.54 4.27 4.10 4.10 4.02
0 .7 .96 1.9 3.5 7.0 0 0 0 0 0 0 0 0 0
Deposited
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Birds. Laying hens of stock bred for egg production and quail (Cotumix coturnix japonica) were housed in rooms of constant temperature (25 C) and photoperiod (14L: 10D). Food and water were always available. Eggs were collected each day between 0800 and 0900 hr and frozen and stored at —20 C until analysis. The quail and chickens were first partially depleted of pigments by feeding for 12 or 16 days, respectively, a low-pigment diet (diets 1 and 16: Tables 1, 2, and 4). Quail Study: Comparison of Efficiency of Pigment Sources. After the pigment-depletion period, the quail were offered, in groups of 4 birds, diets 2 to 15 (Table 2). These diets, which contained one of the pigment sources in replacement of an equal weight of starch, were fed for a period of eight days. Feed consumption
TABLE 1. Composition of chicken and quail basal diets (%)
EGG YOLK PIGMENTATION WITH PHAFFIA
RHODOZYMA
1779
TABLE 3. Effect of freezing on color determination of egg yolks'* Dominant wavelength
Luminosity
Excitation/ purity
Liquid yolks
575.2 (±.4) 575.9 (+.11)
24.5 (±1.7) 23.2 (±3.2)
75 (±9.8) 80.6 (±9.2)
Frozen yolks
575.7 (±.8) 575.6 (±.62)
35.9 (±2.5)
71 (±4.5) 62.6 (±7.1)
38.6 (±3.4)
The color of liquid and frozen yolks was measured for two lots of commercially produced eggs. Three yolks were used for each color determination.
marigold and corn diets, A m ax = 446 nm, E}% m = 2200 (Davies, 1976)] . For chromatography, the ether extracts of diets or eggs were dried over anhydrous Na2 S 0 4 and concentrated by rotary evaporation at 30 C. The extracts were chromatographed on a thin layer of silica gel (Silica Gel 60, .25 mm thickness, Brinkmann Instruments Inc.) using 10% acetone in petroleum ether as the developing solvent. In the chicken study, each diet was represented by 4 eggs. The dominant wavelength (dwl), luminosity (lum), and excitation purity (ep) were measured on 1 cm slices of the yolk of each of these eggs that had been frozen and thawed. The mean of these values is reported in Table 4. This procedure yielded yolk slices that were of the same dominant wavelength as fresh liquid yolk, but of higher luminosity and lower excitation purity. A Macbeth spectrophotometer (Model #M52045) with illuminant C and 8 mm sample aperture was used in the color determinations.
RESULTS AND DISCUSSION In a preliminary experiment, intact red yeast (P. rhodozyma) cells, mechanically fractured red yeast, and an oil of P. rhodozyma were fed for one day to individual chickens. Only the carotenoids of the fractured yeast and the oil were deposited in the yolk, which was evident by an orange-red ring in the sliced frozen yolks. These results agree with our previous findings in rainbow trout; pigments of intact yeast cells were not incorporated into the fish flesh, but pigments from broken yeast were rapidly deposited (Johnson, 1978). In all chicken and quail diets containing red yeast in this study, only mechanically fractured yeast was used. Comparison of Three Sources of Xanthophylls. When quail were fed a diet low in
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Almost all the chickens laid one egg each day. It required 10 to 12 days for the plateau of color in the eggs to be reached, whereupon four consecutive eggs from each chicken (i.e., each diet) were collected for color analysis. Diets. P. rhodozyma for the feeding experiments was grown as previously described (Johnson et al., 1978), broken in a MantonGaulin cell homogenizer (Johnson and Phaff, 1978), and freeze-dried for diet preparation. Yeast oil was prepared by extracting broken yeast with acetone, transferring the carotenoids to petroleum ether, drying over Na2 SO4, and concentrating the petroleum ether solutions of pigment in a rotary evaporator. Marigold meal was obtained from S. B. Penick and Co., New York, NY. Assay of the carotenoid content of P. rhodozyma, ground corn, and marigold concentrate (see below) revealed that they contained .34, .023, and 45.0 mg total carotenoid/g (dry weight basis), respectively. All feeds were protected against oxidation by addition of . 1 % butylated hydroxy toluene (BHT). Quail diets were stored at 4 C and chicken diets at 12 C during the experiment. Analysis of Egg Yolks. In the quail study, frozen quail yolks were separated from the albumen by rolling on a paper towel. The eggs collected each day from each diet group (2 to 4 eggs) were combined in a beaker, approximately 80 ml acetone were added, and after vigorous homogenization in a Virtis homogenizer under a stream of nitrogen, the mixture was centrifuged and the pellet was reextracted with acetone. The acetone extracts were pooled, the carotenoids transferred to petroleum ether by the addition of a dilute sodium chloride solution, and the carotenoid content of the solution estimated as astaxanthin or lutein by measuring the absorbance at A max: [yeast diets, X m a x = 478 nm, E|% m = 1600 (Andrewes et al., 1976);
1780
JOHNSON ET AL. TABLE 4. Colors of egg yolks laid by hens fed various concentrations of P. rhodozyma, marigold concentrate, or yellow com Diet concentration of pigment source
Diet no.
Color measurements
P. rhodozyma
Marigold Corn, concentrate yellow
P. rhodozyma
Marigold Corn
Dominant wavelength
(%)
(mg %)
(ppm)
(ppm)
(ppm)
(nm)
0 0 0 0 0 5 10 20 0 0 0 0 0 5 10 20 0 0 0 0 0 5 10 20 0 2 5 8 10
0 0 0 0 0 0 0 0 5 5 5 5 5 5 5 5 10 10 10 10 10 10 10 10 0 0 0 0 0
571.7 574.9 584.3 588.9 591.9 572.3 573.2 573.9 574.6 576.9 578.8 584.4 593.0 573.4 572.2 573.2 575.2 575.4 581.2 583.5 589.9 573.6 574.7 574.8 588.6 585.4 579.8 574.6 571.2
0 .3 .73 1.5 2.95
0 0 0 0 .3 .73 1.5 2.95
0 0 0 0 .3 .73 1.5 2.95
0 0 0 1.5 1.2 .75 .3 0
0 0 0 0 0 5.55 11.1 22.2
0 0 0 0 0 5.55 11.1 22.2
0 0 0 0 0 5.55 11.1 22.2
0 2.2 5.55 8.85 11.1
(%) 0 0 0 0 0 0 0 0 10.85 10.85 10.85 10.85 10.85 10.85 10.85 10.85 21.7 21.7 21.7 21.7 21.7 21.7 21.7 21.7
0 0 0 0 0
0 2.04
5 10.2 20.1
0 0 0 0 2.04
5 10.2 20.1
0 0 0 0 2.04
5 10.2 20.1
0 0 0 10.2
8.2 5.1 2 0
pigment, the total carotenoid content of their eggs decreased in 7 days from 108 /ig/yolk to 18 /!g/yolk and decreased slowly during the following five days to a minimum of 12 /^g/yolk (Fig. 1). Following this period the quail were fed diets which contained several concentrations of broken red yeast, marigold extract, or yellow corn (Table 2). The amount of feed consumed each day by the quail was remarkably constant at 20 g/bird. Pigment deposition was noticeable after 2 days feeding as a thin outer ring in sliced frozen yolks. After about 6 days of feeding, pigment content in the quail egg yolks plateaued (Fig. 2). In each case, the amount of carotenoid deposited in the yolks was a function of the amount of carotenoid ingested. Carotenoids were somewhat more efficiently deposited in the yolks of quail fed P. rhodozyma or corn (about 4% of carotenoid ingested) than those
Excitation Lumipurity nosity
20.8 16.8 18.8 22.9 25.9 30.9 33.2 37.4 33.0 30.2 27.3 32.0 25.7 31.5 34.9 41.6 36.7 33.4 39.0 33.2 39.5 33.3 50.7 52.0 19.6 19.8 26.5 28.5 33.9
33.3 28.8 23.8 19.9 18.8 26.0 28.6 28.5 27.2 24.8 24.2 22.3 16.9 24.8 23.9 27.2 27.5 24.9 20.1 22.0 15.9 24.6 25.2 22.3 22.6 20.8 22.1 25.1 26.1
fed marigold extract (about 3%, Table 2), but it is evident that the efficiency of transfer of carotenoids from these three pigment sources tested was poor. Our results with quail agree with the data of Marusich et al. (1960) who found the average pigment content of chicken yolks, sampled in the plateau region, increased linearly with the daily dose of carotenoid. However, our efficiency levels for carotenoid transfer are generally lower than those obtained by Marusich et al. (1960). Their values, for various pure carotenoids, ranged from 2.2% for violaxantJiin to 26.0% for canthaxanthin; 13.9% efficiency was found for dehydrated alfalfa leaf meal, which contains lutein as its major carotenoid. These differences may reflect different digestive abilities of quail and chickens or differences in the composition of our basal diet. It is inter-
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16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Carotenoid concentrations furnished by
EGG YOLK PIGMENTATION WITH PHAFFIA
110 100
-
80 -
90
70
y
60
\ \
50
\
40
\
30 20
x^^
10 )
1
i
i
i
2
3
4
5
6
7
8
9
10 II 12
FIG. 1. Depletion of egg yolk pigments in Japanese quail by feeding a diet low in carotenoid content (diet 1).
esting that in the present study the percentage of carotenoid deposited was rather constant regardless of the quantity of carotenoid ingested or its source. The carotenoids extracted from the diets and from eggs collected on the seventh day were chromatographed. It was found that the primary carotenoid present in the yolk of quail fed the yeast or corn diets cochromatographed with the main pigment in the respective feeds (rfs = .11 (astaxantiiin) and .14 (lutein), respectively); the astaxanthin of the yeast and lutein of the corn were deposited without modification in the egg yolk. However, the primary pigment found in the egg yolk of quail fed marigold extract (rf = .14) and in the marigold diet (rf = .92) were not the same. Lutein occurs in an esterified form in marigold (Quackenbush and Miller, 1972). This explains the high rf value of the major carotenoid in the marigold extract, because carotenoid esters are less polar chromatographically than the parent carotenoid (Davies, 1976). Either this predominant esterified carotenoid was altered by the bird during the deposition process, or another (unesterified) carotenoid was selectively absorbed. Influence of Mixed Pigmentation Sources on Chicken Egg Yolk Color. It was found that the dominant wavelength (dwl), the main measurement parameter of color that influences visual judgment (Fletcher et al, 1978) was the same in fresh or frozen-thawed egg yolks (Table
1781
3); however, luminosity and excitation purity were different in the two conditions. Our values for dwl are directly comparable to values obtained by other workers using fresh yolks, but lum and ep values are only valid for comparison within our experiments. To determine the effect on egg yolk color, 29 laying hens were fed diets which contained different proportions of xanthophylls derived from P. rhodozyma, marigold extract, and yellow corn. A considerable range of colors was obtained in the yolks as observed visually and measured by the dwl of the freeze-thawed yolks (Table 4). The dwl ranged from 571 nm to 593 nm. The dwl was dependent almost entirely upon the concentration of yeast in the diet. Thus, as the concentration of astaxanthin only (diets 16 to 20) was increased from 0 to 20.1 mg/kg, the dwl increased from 571.7 to 591.9. In contrast, as the concentration of marigold only (diets 16 and 21 to 23) was increased from 0 to 20.0 mg/kg, the dwl increased from 571.7 to 573.9; a similar small change was seen for corn only (diets 16, 24, and 32) or for marigold mixed with corn (diets 29 to 31 and 37 to 3 9). When corn at two levels was added to diets containing increasing concentrations of astaxanthin (diets 25 to 28 and 33 to 36 and control diets 17 to 20) the increase in dwl was 15 nm in each case, although there was a suggestion of a slightly lower dwl on average with corn present. Weiser and Manz (1972, unpublished data, cited in Braeunlich, 1978) found that a high level of yellow pigments suppressed the effect of red pigments on the deepening of egg yolk color; it is possible the
A.
B.
C.
r>6
J '~~* // / ,.5
0^
, 5
*-*— 10 S~* 4
a. 20
0
C^1 2
4
6
8
0
t^r ' 2 4
0
A 2
4
y ^ 14
6
Days
FIG. 2 Deposition of carotenoids in the egg yolks of Coturnix quail fed broken red yeast (diets 2 to 6), marigold flower extract (diets 7 to 11), or yellow corn (diets 12 to 15). Composition of diets as in Table 2.
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Day
RHODOZYMA
1782
JOHNSON ET AL.
amount of corn used here was insufficient to show its effect clearly. Diets 40 to 44 contained a constant concentration of carotenoid (10 mg/kg) but varied in the relative concentrations of yeast or marigold pigments. It was again observed that the dwl was higher with those diets containing more astaxanthin from P. rhodozyma. The yolks of the eggs described in Table 4 were yellow when the diet contained corn or marigold. When lower concentrations of yeast were also in the diet, the yolks were gold in color but were orange-pink at the highest concentration of yeast used.
REFERENCES Andrewes, A. G., H. J. Phaff, and M. P. Starr, 1976. Carotenoids of Phaffia rhodozyma, a red-pigmented fermenting yeast. Phytochemistry 15: 1003-1007. Bauernfeind, J. C , G. B. Brubacher, H. M. Klaui, and W. L. Marusich, 1971. Use of carotenoids. Pages 743—770 in Carotenoids. O. Isler, ed. Birkhauser Verlag, Basel, Stuttgart. Braeunlich, K.. 1978. The chemistry and action of pigmenters in poultry diets. World Poultry Congr. 15:236-240. (Abstr.)
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ACKNOWLEDGMENTS We thank T. Roudybush and K. Hirsch for assisting in the preparation of the diets and J. Buhlert for the color determinations.
Couch, J. R., 1972. Pigmentation. Feedstuffs. April 3 44(14): 21. Davies, B. H., 1976. Carotenoids. Page 38-165 in Chemistry and biochemistry of plant pigments. Vol. 2. T. W. Goodwin, ed. Academic Press, London. Fletcher, D. L., R. H. Harms, and D. M. Janky, 1978. Yolk color characteristics, xanthophyll availability, and a model system for predicting egg yolk color using /3eta-Apo-8'-carotenal and canthaxanthin. Poultry Sci. 57:624-629. Johnson, E. A., 1978. Astaxanthin production by the yeast Phaffia rhodozyma and its use as a pigment source in animal feeding. M.S. thesis, University of California, Davis, CA. Johnson, E. A., D. E. Conklin, and M. J. Lewis, 1977. The yeast Phaffia rhodozyma as a dietary pigment source for salmonids and crustaceans. J. Fish. Res. Board Can. 34:2417-2421. Johnson, E. A., and H. J. Phaff, 1978. Rhodotorula fujisanensis, a. new taxonomic combination. Current Microbiol. 1:223-225. Johnson, E. A., T. G. Villa, M. J. Lewis, and H. J. Phaff, 1978. Simple method for the isolation of astaxanthin from the basidiomycetous yeast Phaffia rhodozyma. Appl. Environ. Microbiol. 35:1155-1159. Marusich, W., E. De Ritter, and J. C. Bauernfeind, 1960. Evaluation of carotenoid pigments for coloring egg yolks. Poultry Sci. 39:1338—1345. Nelson, T. S., and J. N. Baptist, 1968. Feed pigments. 2. The influence of feeding single and combined sources of red and yellow pigments on egg yolk color. Poultry Sci. 4 7 : 9 2 4 - 9 3 1 . Quackenbush, F. W., and S. Miller, 1972. Composition and analysis of the carotenoids in marigold petals. J. Ass. Offic. Anal. Chem. 55:617. Roudybush, T. E., 1977. Fructose utilization in Coturnix coturnix japonica (Japanese quail). M.S. thesis, University of California, Davis. CA.