AU745973B2 - Increased bioavailability of lutein and zeaxanthin in humans and poultry using lysolecithin and lecithin - Google Patents

Description

WO 99/47001 PCT/US99/05925 1 INCREASED BIOAVAILABILITY OF LUTEIN AND ZEAXANTHIN IN HUMANS AND POULTRY USING LYSOLECITHIN AND LECITHIN Background of the Invention 1. Field of the Invention The invention relates generally to the absorption of carotenoids and, more specifically, to increasing the absorption and bioavailability oflutein and zeaxanthin in humans and poultry by the use of lysolecithin and lecithin.

2. Background of the Prior Art Carotenoids have long been used as important food coloring agents. In particular, xanthophylls are added to poultry feeds so that upon ingestion, the xanthophylls are deposited in the skin and egg yolks. Chickens specifically absorb the lipid-soluble pigment lutein 3'-dihydroxy-at-carotene) and deposit it intact in body and subcutaneous fat, keratinaceous tissues (feathers and beak), and eggs, particularly the yolk. Deposition of lutein and its structural isomer zeaxanthin impart a yellow color to the chicken and its eggs that is commercially desirable.

Carotenoids have been hypothesized to reduce the risk of certain types of cancers in humans through their action as anti-oxidants that quench singlet oxygen and other oxidizing species thereby terminating free radical chain reactions and limiting cellular oxidation damage. Of the nineteen carotenoids identified to date in human plasma, lutein is among the most common and is known to exhibit strong antioxidant capabilities. The structure of lutein is:

,OH

s.-i iY SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/US99/05925 2 The allylic hydroxyl group at the C-3' position of the E-end group of lutein is readily oxidized as a result of activation by the neighboring double bond. The non-allylic hydroxyl group at the C-3 position of the P-end group can also activate the C-4 carbon allylic to the double bond making this carbon highly susceptible to direct oxidation. While chemical processes for the synthesis of lutein are known, such processes are inefficient and expensive. Lutein extracted from marigold petals is commercially available in a variety of forms.

The structure of zeaxanthin is: 0" OH Lysolecithins (lysophosphatidylcholine, lysophosphatidylethanolamine, etc.) have been shown to have a variety of biological actions, all centered around modification of cell membrane permeability. Such effects include increased transfer of both cations and larger molecules across cell membranes in cultured cell lines (Duan, J. and M.P. Moffat (1991) "Protective effects of D.L-carnitine against arrhythmias induced by lysophosphatidylcholine or reperfusion", Eur. J.

Pharmacology, 192: 355-363; Boachie-Ansah, et al., (1991) "Effect of combined acidosis, lactate and lysophosphaticylcholine on action potentials and ionic currents in ventricular mycocytes", J. Mol. Cell Cardiol., 23 (Supp. S.80. On a macroscopic level it is presumed that this particular effect can result in increased absorption of nutrients in the animal gut (UK Patent No. 9205014.5). This has been accounted for by an increase in membrane transport efficiency induced by incorporation of lysolecithin into the membrane accompanied by improvement in micellar formation in SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/USq99/05925 3 the gut due to incorporation of lysolecithin into the micelle. These two effects are presumed to both increased the amounts of fatty acids biologically available from the gut (in micelles) and the efficiency of absorption of the fatty acids (increased membrane transport).

Structurally, lecithins are phosphatidyl digylcerides, by which is meant that one of the three hydroxyl groups of glycerol (propane-1,2,3-triol) is occupied by a phosphate group, which in turn is attached to a polar alkyl amine. The remaining two hydroxyls of glycerin are occupied by long-chain fatty acids. The structure of lecithin y o o S0O IS: is: Thus, lecithin, as a phosphatidyl diglyceride, has both a polar, charged end and a highly hydrophobic one the prime requirements of an emulsifier. Removal of the fatty acid in the center position by phospholipase A produces lysolecithins whose chemical properties differ noticeably from those of lecithins due to the greater hydrophilicity of its polar end. It has been found that the fatty acid remaining on lysolecithin is generally unsaturated (Dawson, et al., (1990) "Fatty acid composition fo the neutral lipid and phospholipid fractions of mechanically deboned chicken meat", Poultry Science, 64: 1411-1419. Increased membrane transport in the presence of lysolecithin is believed to occur via alteration in the membrane permeability due directly to the high polarity and hydrogen bonding potential of the hydrophilic end of lysolecithin (Sidik, K. and M.J. Smerdon (1990) "Bleomycin J m SUBSTITUTE SHEET (RULE 26) induced DNA damage and repair in human cells permeabilized with lysolecithin", Cancer Research USA, 50: 1613-1619; Kaminskas, E. and J.C. Li, (1989) "DNA fragmentation in permeablised cells and nuclei", Biochem. J. 261: 17-21.

Sufficient levels of deposition of lutein and zeaxanthin in the skin and egg yolks of poultry are required to provide the consumer demanded level of pigmentation. Lutein and zeaxanthin are further absorbed by humans where they are present in the blood and are deposited in the macular region of the retina as well as other parts of the body. Sufficient levels of lutein and zeaxanthin need to be absorbed by the human body to provide the desired biological levels of these compounds, and such levels may play a role in the prevention and treatment of disease, including melanomas and other cancers, as discussed previously. Lutein and zeaxanthin further are believed to be effective in the prevention and treatment of macular degeneration. Accordingly, a method of increasing the absorption and bioavailability of these compounds is desired.

Summary of the invention S is One aspect of the invention provides a method for increasing the absorption and bioavailability of carotenoids in humans and poultry, comprising the steps of: combining a surfactant with a carotenoid wherein the ratio of surfactant to carotenoid is between about 5wt% and about 30wt%, and feeding said combination to humans or poultry.

A formulation is made comprising the addition of a surfactant, including either 20 lysolecithin or lecithin or both, including either lutein or zeaxanthin or a mixture of both.

The formulation is fed as a food supplement and results in an increased absorption and bioavailability of the carotenoids.

[R:\LIBC]40199.doc:ais An object of the present invention is to provide a food supplement including a carotenoid which increases the absorption of the carotenoid by an animal or human that is fed the supplement.

Another object of the invention is to provide a method for increasing the absorption and bioavailability of carotenoids in food supplements.

Still another object of the invention is to provide a method of increasing the absorption and bioavailability of lutein and zeaxanthin extracted from marigold petals.

A further aspect of the invention is the use of surfactant and a carotenoid wherein the ratio of surfactant to carotenoid is between about 5wt% and about 30wt% for the o0 manufacture of a medicament for increasing the absorption and bioavailability of carotenoids in humans and poultry.

Another aspect of the invention is a method for increasing the deposition of carotenoids in the tissues and egg yolks of poultry, comprising the steps of: combining one or more of lecithin or lysolecithin surfactants with one or more of lutein or zeaxanthin 15s carotenoids, wherein the ratio of surfactant to carotenoid is between about 5wt% and about 30wt%; and feeding said composition to poultry.

Further the invention provides one or more of lecithin or lysolecithin surfactants and one or more of lutein or zeaxanthin carotenoids, wherein the ratio of surfactant to carotenoid is between about 5wt% and about 30wt% when used for increasing the 20 deposition of carotenoids in the tissues and egg yolks of poultry.

Further the invention provides use of one or more of lecithin or lysolecithin surfactants and one or more of lutein or zeaxanthin carotenoids, wherein the ratio of surfactant to carotenoid is between about 5wt% and about 30wt% for the manufacture of a medicament for increasing the deposition of carotenoids in the tissues and egg yolks of poultry.

Further the invention provides a method for increasing the deposition of carotenoids in the tissues, including the blood and macular region of the retina, of humans, comprising the steps of: combining one or more of lecithin or lysolecithin surfactants with one or more of lutein or zeaxanthin carotenoids, wherein the ratio of surfactant to carotenoid is between about 5wt% and about 30wt%; and feeding said composition to humans.

Further the invention provides one or more of lecithin or lysolecithin surfactants and one or more of lutein or zeaxanthin carotenoids, wherein the ratio of surfactant to carotenoid is between about 5wt% and about 30wt% when used for Si i increasing the 62' [I:\DayLib\LIBC]42281.doc:uff deposition of carotenoids in the tissues, including the blood and macular region of the retina, of humans.

Further the invention provides use of one or more of lecithin or lysolecithin surfactants and one or more of lutein or zeaxanthin carotenoids, wherein the ratio of surfactant to carotenoid is between about 5wt% and about 30wt% for the manufacture of a medicament for increasing the deposition of carotenoids in the tissues, including the blood and macular region of the retina, of humans.

Further the invention provides a feed supplement for increasing the absorption and bioavailability of carotenoids in humans and poultry, comprising: a carotenoid; and a surfactant, wherein the range of surfactant to said carotenoid is between about 5wt% and about The invention also provides a pharmaceutical composition comprising at least a combination as defined hereinbefore, with a pharmaceutical acceptable adjuvant, diluent S or carrier.

i These and other objects of the invention will be made known to a person skilled in the art upon a review and understanding of this specification, the associated drawings, and the appended claims.

Brief Description of the Drawings Figure 1 is a graphical representations of the results of NEPA analysis of egg yolks 20 to determine pigment uptake in chickens fed a variety of diets including control diets and ,diets supplemented with feed additives of the present invention.

44 Figure 2 is a graphical presentation of the L* a* b* Colour Space Figure in the Operation Manual of the Minolta Chroma Meter II.

Figure 3 is a graphical representation of the results of reflectance colorimetric 25 analysis of egg yolks from chickens fed lysolectithin-treated and untreated poultry feed.

Figure 4 and 5 are a graphical representations of the results of NEPA analysis of egg yolks to determine pigment uptake in chickens fed a variety of diets including control diets and diets supplemented with feed additives of the present invention.

Figure 6 is a graphical representation of the emulsifying properties of lysolecithin and measures the retardation of the phase separation over time.

[I:\ayLib\L ]42281.doc:ais t) [l:\ayLib\LIBC] 42281 .doc:ais WO 99/47001 PCT/US99/05925 6 Detailed Description of Preferred Embodiments Carotenoids, particularly the xanthophylls lutein and zeaxanthin, are important as colorants in animal feeds, particularly poultry feeds. Commercially available sources of the xanthophylls include the products of Kemin Industries, Inc., that are extracts of marigold (Tagetes erecta) and sold under the marks ORO GLO® Liquid and FloraGLO® Lutein (20% Liquid). ORO GLO® Liquid has 7 grams of xanthophyll activity per pound (as determined by a method condensed from the Association of Official Analytical Chemists, paragraph 43.108, and available from Kemin Industries, Inc., part no. 02205). FloraGLO® Lutein (20% Liquid) has 20% lutein and 0.86% zeaxanthin. The sources of xanthophylls have been used as additives to animal feeds to incorporate the pigments into the body tissues and products of the animals.

A method and formulation for increasing the level of absorption and bioavailability of the carotenoids has been developed. A formulation containing lutein and zeaxanthin is combined with lecithin and with lysolecithin at a concentration of between about 5% to about 30% by weight. The lutein and zeaxanthin source is mixed with the source of lecithin or lysolecithin and the mixture is added to feed for the animal. The treated feed is fed to animals for a predetermined length of time and the body tissues and animal products were assayed for xanthophyll content. The treated feed is found to increase the amount of the xanthophylls incorporated by the animal by between about 2% and about 50% over control feed that included the same amounts of xanthophylls but which had not been combined with lecithin or lysolecithin.

In particular, a quantity of a liquid source of xanthophylls was blended with a liquid source of lecithin or lysolecithin at high speed for an extended period of time to thoroughly mix the ingredients. The mixture was added to a low xanthophyll feed in SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/IIUS99/05925 7 a mixer to assure uniform distribution. A control feed was formulated using the same low xanthophyll feed to which was added the same quantity of the liquid xanthophyll source also in a mixer to assure uniform distribution. The treated feed and the control feed were fed to separate groups of chickens for approximately one-month. Eggs were collected from both groups and analyzed using the NEPA test for pigment content. The pigment content of the eggs showed an increased pigment content in chickens fed the treated diet that correspond to chickens that had been fed an untreated diet including up to 50% higher concentrations of xanthophylls. By combining the xanthophyll source with lecithin or lysolecithin prior to incorporation into an animal feed, the absorption and bioavailability of the xanthophylls was increased by up to Deposition of lutein and zeaxanthin in the yolk gives colorimetric responses that are linear with respect to added pigment. Maruisch, W.L. and J.C. Bauerfeind "Oxycarotenoids in poultry feed" in: Carotenoids as Colorants and Vitamin A Precursors, J.C. Bauernfeind, ed., 1981, Academic Press, pp. 330ff By contrast, visual observation gives results that fit a logarithmic curve in which perception of differences in color becomes very difficult at high levels of pigment content. In addition, actual mass of pigment incorporated into the yolk can be determined with the extraction-based spectroscopic method AOAC No. 17.004 (Journal of the Association of Official Analytical Chemists, JAOAC, 1958, 41: 274; 1973, 56:272), also known as the NEPA test. The NEPA test has the advantage of measuring actual pigment levels rather than measuring changes in perceived or reflected colors.

Accordingly, the increased absorption of the xanthophylls can be objectively measured.

SUBSTITUTE SHEET (RULE 26) WO 99/47001 PrT/I S9o/n0092 8 Experiment 1 A feeding trial was conducted over a 28 consecutive day period. The trial used two hundred and twenty-four white Leghorn Hyline W36 cross birds, 29 weeks of age at the start of the study. The birds were housed in battery pens of eight or nine birds. All birds were fed a low-pigment diet for 1 month immediately prior to the start of the trial. Composition of the low xanthophyll diet used in this study is shown in Table 1 Each treatment was fed to three replicate pens holding either eight or nine birds. The remaining birds received control low xanthophyll feed. Egg production was monitored throughout the test and remaining test feed for each treatment was weighed to determine feed consumption.

rlr~ SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/1US99/n05925; 9 TABLE 1: COMPOSITION OF XANTHOPHYLL-FREE POULTRY DIET Ingredient Percent Milo 25.90 Millet 5.00 Oats 12.50 Wheat 9.00 Barley 5.00 Sunflower hearts 5.00 Wheat midds 2.50 Beet pulp 2.50 47.5% Soybean meal 12.00 38% Extruded beans 5.00 Meat meal 5.00 Whole whey 2.50 Fishmeal 1.00 Calcium 5.00 18.5% Dical 0.90 Salt 0.50 Vitamins 0.35 Trace minerals 0.25 MYCO CURB' brand 0.111 Methionine 0.05 0.05 TERMOXTM brand 0.0125 Equivalent to 1 kg/ton.

2 Equal to 125 ppm.

The source of xanthophylls used in the trial was ORO GLO Liquid having a measured pigment content of 5.2 g/lb xanthophyll activity. The source of lysolecithin used was Lysoprin-brand lysolecithin purchased from Lovesgrove Research (Aberystwyth, Wales), product no. 10544. Target application rates for ORO GLO and Lysoprin are shown in Table 2. For the treatments with Lysoprin, 0.78, 1.17 and 1.56 lb of ORO GLOliquid were added to 0.23 Ib of Lysoprin. The mixture was blended at high speed in a Waring blender for 15 minutes. Each mixture was poured, during mixing, into a ribbon mixer containing 195 lbs of low xanthophyll feed which had SUBSTITUTE SHEET (RULE 26) been previously treated with 150 ppm TERMOX® dry (from Antitox, Buford, Georgia) and 1,000 ppm Myco CURB® liquid (from kemin Industries, Inc.). The diets thus produced contained the equivalent of 40, 60 and 80 grams of xanthophyll activity per ton of feed, respectively. Control feed was prepared by addition of 0.78, 1.17 and 1.56 lb of ORO GLO liquid directly to low xanthophyll feed without addition of Lysoprin. Samples of each feed were taken and analysed for pigment content using a method based on AOAC, Paragraph 43.018, without saponification and column chromatography.

Table 2: ORO GLO AND LYSOLECITHIN APPLICATION RATES IN A STUDY OF HIGH-LEVEL LYSOLECITHIN-INDUCED ABSORPTION

OF

io LUTEIN INTO EGG YOLKS ORO GLO(g Total Lysolecithin (Lb/Ton) Xanthophyll/Ton) 0 0 0 60 0 80 0 0 2.28 2.37 2.30 So*: 80 2.28 *oo At the end of the trial one dozen eggs were collected from each pen. The eggs were weighed and analysed for colour reflectance and pigment content. Three eggs were randomly selected from each dozen for yolk evaluation. The selected eggs were cracked 15 open and the yolk separated from the white. Colour reflectance was determined in accordance with procedures outlined in the Operation Manual for the Minolta Colorimeter (Chroma Meter II Reflectance Operation Manual Yolks were then composited and blended thoroughly. A 2.5 gram sample was taken from each yolk composite and analysed by a modification of AOAC method 17.004 [l:\DayLib\LIBC]4228 I.doc:ais WO 99/47001 PCT/IUS99/05925 11 (Journal of the Association of Official Analytical Chemists, JAOAC, 1958, 41: 274; 1973, 56: 272) in which total yolk pigment is extracted with acetone and determined spectroscopically. Pigment content was estimated using an El%/cm value of 2,360 which is used in AOAC methods for xanthophyll in feed, rather than by 3 -carotene equivalents.

Throughout the remainder of this disclosure, treatment levels will be referred to by the target level of total xanthophyll activity in grams of xanthophyll per ton of feed as described in the Methods and Materials section and Table 2. Egg collection data (feed/bird/day and average egg weight) and pigment content are presented in Table 3. As can be seen, addition of lysolecithin to the diet made no numerical or statistical difference in either feed consumption per bird per day or average egg weight.

TABLE 3: RAW DATA FROM OF HIGH-LEVEL LYSOLECITHIN LAYER TRIAL: FEED CONSUMED PER BIRD PER DAY, EGG WEIGHT AND FEED PIGMENT CONTENT Treatment (Grams/Ton) Xantho Lysoprin Feed/Bird/Day Average Egg Measured Feed phyll (Grams) Wt. (Grams) Pigment (Grams/Ton) 0 0 105.5 58.1 1.89 a 0 97.9 59.9 41.78 b 0 105.0 59.2 59.85

C

0 102.4 58.8 82.85 d 0 200 106.7 60.4 2.04a 200 101.8 61.0 43.66 b 200 106.8 60.3 63.45c 200 97.7 57.7 82.77d Average 103.0 59.4 Standard Deviation 5.0 1.1 a-d Entries in column with no common superscripts differ significantly (P<0.05).

SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/US99/05925 12 There are two strategies which can be used in evaluating the pigment content of egg yolks. The first is to measure the actual mass of pigment within the yolk. This can be done, for example, by the NEPA method. The term "NEPA" is an acronym for the National Egg Products Association which originally developed it. In modified form, it has been accepted as a standard method by the AOAC (Journal of the Association of Official Analytical Chemists, JAOAC, 1958, 41: 274; 1973, 56: 272).

In this method, pigment is extracted from the yolk and quantitated by visible spectroscopy in terms of .ig of pigment per gram of yolk. The second approach is to measure perceived changes in color. This can be done either by an evaluation panel or by reflectance colorimetry. In this study, the latter was used due to its greater sensitivity to changes in pigment content at high pigment levels and its lower variability versus panel evaluation (at all pigment levels). Both the NEPA method and reflectance colorimetry can be correlated with human color perception aids such as the Roche Fan (Marusich, W.L. and J. C. Bauernfeind "Oxycarotenoids in poultry feed" in: Carotenoids as Colorants and Vitamin A Precursors, J. C. Bauernfeind, ed., 1981, Academic Press, pp. 330ff).

The results of NEPA analysis are shown in Table 4 and Figure 1. As stated above, the NEPA method directly measures mass of pigment extracted from the yolk.

Hence, any increases in the amount of pigment incorporated into the yolk can be directly observed without regard to color perception. As can be seen in Table 4, treating poultry feed with lysolecithin resulted in statistically valid (P<0.05) increases in yolk pigment content versus control at every application level. Addition of lysolecithin without additional available pigment gave no statistical increase in pigment content. These results are shown graphically as Figure 1. This pigment SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/US99/05925 13 range (56.4 to 122.1 lg/g) correlates to a Roche Fan score of roughly 11 to 14, well into the range in which perceived changes in yolk color are difficult to differentiate.

TABLE 4: RESULTS OF ANALYSIS OF EGG YOLKS BY NEPA Treatment (Grams/Ton) NEPA Pigment (grams/gram Yolk) ORO GLO Lysoprin 0 0 0 200 3.7a 0 56.4 b 200 75.1c 0 77.60 200 9 7 .3 d 0 107.3e 200 12 2 .1f a-f Entries in column with no common superscripts differ significantly (P<0.05).

The increase in pigment content is most dramatic at 40 g ORO GLO/ton, where there is a 37.4% increase in yolk pigment content upon addition of lysolecithin to the diet. For pigment dosages of 60 g/ton and 80 g/ton, the increases were 28.3% and respectively. As can be seen in Table 4 and Figure 1, there is no statistical difference, and very little numerical difference in the actual pigment content of yolks from 40 g/ton pigment feed which contained lysolecithin and in yolks from chickens fed 60 g pigment/ton without lysolecithin.

Unlike direct pigment mass determination, colorimeter data is obtained and reported as the values a* and b* which correspond to the position of the reflected color in a three-dimensional system. The L* value is a measure of brightness (luminosity) and a* and b* are chromaticity coordinates along green-red and yellowblue axes, respectively. The coordinate system is shown graphically as Figure 2.

SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/US99/05925 14 While actual color changes can most accurately be presented on a three-dimensional graph, it is common practice to report the individual a* and b* values.

Colorimetric results are shown as Table 5 and Figure 3.

For the addition of an orange pigment such as lutein, it is anticipated that increasing lutein content will result in increasing a* and b* values. Similarly, as pigment content increases, total luminosity should decrease. As can be seen in Table and Figure 3, L* value does decrease as anticipated, a* values increase with increasing pigment levels and b* undergoes an initial increase in yellow value upon addition of pigment (40 g/ton), followed by little or no change at higher pigment 0o levels. This behavior closely fits the "yolk strip" region of the CIE (Commission Internationale d'Eclairage) color triangle in which increasing pigment content is perceived in terms of an increasing red color component versus a fixed or decreasing yellow contribution.

TABLE 5: RESULTS OF ANALYSIS OF EGG YOLKS BY MINOLTA

COLORIMETER

Treatment (Grams/Ton) ORO GLO Lysoprin L*t a* b* 0 0 66.81 -8.774 24.21" 0 200 67.08 -9.13a 27.79 b 0 62.24 -2.

8 9 b 52.03 d 200 61.17 -0.38c 51.46 c d 0 61.13 -0.19c 5 0 9 2 c,d 200 59.30 1.64 d 47.89c 0 60.14 2.28 d e 51.43cd 200 59.26 3.20e 49.78 c d a-f Entries in column with no common superscripts differ significantly (P<0.05) Not statistically evaluated. L* value contributes to changes in color perception, not chromaticity.

The L* a* b* system is designed to mimic human color perception. The relatively small changes in a* and b* upon addition of pigment correlates well SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCrT/i T1CO/nOZ1 with the known difficulty in perceiving color changes at the pigment levels used in this study. Considering the lack of linearity of response within the CIE triangle to yolk color changes, the high degree of linearity in the a* value response for untreated feed (Figure 3) with increasing pigment levels observed in this study should be noted.

The correlation coefficient (R 2 from untreated feed is 0.999; it is 0.973 from lysolecithin-treated feed.

More importantly, a* values obtained from yolks produced with lysolecithin-treated feed containing 40 g/ton ORO GLO and feed without lysolecithin containing 60 g/ton ORO GLO are the sole members of a statistically homologous o0 group (Table 4 and Figure This observation exactly parallels the result of the NEPA analyses above (Table Incorporation of high levels of Lysoprin brand lysolecithin into pigment-supplemented poultry diets for active layers were seen to yield statistically valid increases in the pigment content of egg yolks. Based on the above data, a pigment level of 40 g ORO GLOliquid/ton of feed, with addition of lysolecithin at the application rate used in this study, will show yolk color performance equivalent to the addition of 60 g ORO GLO liquid/ton of feed without lysolecithin.

Experiment 2 A feeding trial was for twenty-eight days. The study was conducted on 224 white Leghorn Hyline W36 cross birds, 47 weeks of age at the start of the study. All birds were fed a low-pigment diet for three weeks immediately prior to the start of the trial. Composition of the low xanthophyll diet used in this study is shown in Table 1.

Each treatment was fed to three replicate battery pens holding either eight or nine birds. The remaining birds received control low xanthophyll feed. Egg production was monitored throughout the test and remaining test feed for each treatment was SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/US99/05925 16 weighed to determine feed consumption. House temperatures ranged from 690 F to 850 F during the trial.

The ORO GLO liquid used for the trial had a pigment content of 5.32 g/lb.

Target application rates for ORO GLO, Lysoprin brand lysolecithin and bulk soy lecithin are shown in Table 6. For the treatments with Lysoprin and lecithin, 55, 110 or 165 grams of either Lysoprin or lecithin were blended with 550 grams of ORO GLO liquid. The mixture was blended at high speed in a Waring blender for minutes and at low speed for 5 minutes. The ORO GLO used in the control treatment was not blended. Treatment was affected by pouring each of the above pigment mixtures onto feed in a ribbon mixer containing 210 lbs of low xanthophyll feed which had been previously treated with 150 ppm TERMOX dry and 1,000 ppm MYCO CURB liquid. Control feed was prepared by addition of appropriate amounts of ORO GLO liquid, as outlined in Table 2, directly to low xanthophyll feed without addition of Lysoprin or lecithin. Each treated feed was run through a hammer mill and mill screen to aid in pigment distribution, then weighed in lots of 35 lbs into polylined Kraft bags. Samples of each feed were taken and analyzed for pigment content using a method based on AOAC, Paragraph 43.018, without saponification and column chromatography.

I

SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/US99/05925 17 TABLE 6: ORO GLO, LECITHIN AND LYSOLECITHIN APPLICATION RATES IN A STUDY OF SURFACTANTS IN LAYER DIETS ORO GLO (g Total Treatment (Lb/Ton) Xanthophyll/Ton) 0 1.15 Leci 1 1.15 Prin 2 2.31 Leci 2.31 Prin 3.46 Leci 3.46 Prin Leci bulk soy lecithin.

2 Prin Lysoprin brand lysolecithin.

At the end of the trial one dozen eggs were collected from each pen along with excess treated feed and production data. The eggs were weighed and analyzed pigment content. Three eggs were randomly selected from each dozen for yolk evaluation. Eggs were cracked open and the yolk separated from the white. Yolks were then composited and blended thoroughly. A 2.5 gram sample was taken from each yolk composite and analyzed by a modification of AOAC method 17.004 in which total yolk pigment is extracted with acetone and determined spectroscopically.

Pigment content was determined by comparing extractant absorbance at 450 nm to a 3-carotene standard curve. Results were converted to and reported as P-carotene equivalents (tg BCE/g yolk).

Egg collection data (feed/bird/day) and pigment content are presented in Table 7. As can be seen, addition of lysolecithin to the diet made no numerical or statistical difference in feed consumption per bird per day. However, all of the feeds treated with surfactant showed statistically lower (P<0.01) final pigment content when compared to the non-surfactant-treated control.

SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/US99/05925 18 TABLE 7: RAW DATA FROM LECITHIN VERSUS LYSOLECITHIN

LAYER

TRIAL: FEED CONSUMED PER BIRD PER DAY, INITIAL AND FINAL FEED PIGMENT CONTENT Xanthophyll Treatment Feed/Bird/Day Measured Measured (g/Ton) (Lbs/Ton) Initial Final Pigment Pigment (g/Ton) (g/Ton) 0 103.5 61.10 51.1L 1.15 Leci 107.8 60.40 44.9 ab 1.15 Prin 103.3 65.60 4 5 .2ab 2.31 Leci 103.3 59.30 4 5 .9 abc 2.31 Prin 109.1 67.60 47.9c 3.46 Leci 106.4 59.80 4 6 9 bc 3.46 Prin 108.8 59.40 44.3a Average 106.03 61.89 46.6 Standard deviation 2.64 3.33 2.33 a-d Entries in column with no common superscripts differ significantly (P<0.05).

SLeci bulk soy lecithin.

2 Prin Lysoprin brand lysolecithin.

The results of NEPA analysis are shown in Table 8 and Figure 4. As stated above, the NEPA method directly measures mass of pigment extracted from the yolk.

Thus, any increases in the amount of pigment incorporated into the yolk can be directly observed without regard to color perception. As can be seen in Table 8, treating poultry feed supplemented with 60 g/ton xanthophylls with 2.31 Ibs/ton of either Lysoprin brand liquid lysolecithin supplement or bulk soy lysolecithin resulted in statistically valid (P<0.001) increases in yolk pigment content versus control. In addition, the feed treated with 2.31 lb/ton of Lysoprin showed a statistically higher pigment content than feed treated with lecithin at that level. By contrast, treatment at either 1.15 lb/ton or 3.46 lb/ton with either Lysoprin or lecithin showed no increase in yolk pigment content versus either each other or control. Notably, at the highest surfactant treatment level, yolk pigment deposition was lower than at the 2.31 lb/ton SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/US99/05925 19 treatment level. Further, though the control feed showed the highest pigment stability (see Table it gave the lowest pigment deposition level (Table These results are shown graphically as Figure 4.

TABLE 8: RESULTS OF ANALYSIS OF EGG YOLKS BY NEPA Treatment NEPA Pigment g Change in BCE'/g Yolk) Pigmentation vs. Control) ORO GLO Lysoprin (Lb/Ton) (g/Ton) 0 84.6' 0 1.15 Leci 2 8 9 .6ab 5.91 1.15 Prin 3 86.8a 2.60 2.31 Leci 96.1c 13.59 2.31 Prin 10 9 .8d 29.79 3.46 Leci 86.6a 2.36 3.46 Prin 94.6 b 11.82 SBCE 3 Carotene Equivalents a-d Entries in column with no common superscripts differ significantly (P<0.05).

2 Leci bulk soy lecithin.

3 Prin Lysoprin brand lysolecithin.

Addition of lysolecithin to poultry diet results in statistical improvements in pigment incorporation into egg yolk. The largest increase, 29.79%, was seen at a Lysoprin treatment level of 2.31 lb/ton. In addition, this study shows that a similar, albeit smaller, effect can be observed using soy lecithin in place of lysolecithin.

Addition of 2.31 lb/ton of lecithin gave a 13.59% increase in yolk pigment. Statistical improvements in pigment deposition occur in spite of statistically lower pigment stability in the surfactant-treated feeds.

Experiment 3 A feeding trial was done for 28 days. Two hundred and twenty-four white Leghorn Hyline W36 cross birds were used, 47 weeks of age at the start of the study.

SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/US99/05925 All birds were fed a low-pigment diet for 3 weeks immediately prior to the start of the trial. Composition of the low xanthophyll diet used in this study is shown in Table 1.

Each treatment was fed to three replicate battery pens holding either eight or nine birds. The remaining birds received control low xanthophyll feed. Egg production was monitored throughout the test and remaining test feed for each treatment was weighed to determine feed consumption. House temperatures ranged from 580 to 720 F during the trial.

The ORO GLO liquid used for the trial had a pigment content of 5.4 g/lb.

Target application rates for ORO GLO, LYSOFORTE and Lysoprin lysolecithin supplements are shown in Table 9. Since LYSOFORTE is a 10% suspension of Lysoprin on an inert carrier, application rates for LYSOFORTE were set at 10 times those for Lysoprin. Thus, a 2.2 lb/ton treatment of LYSOFORTE is designed to parallel a 0.22 lb/ton application of Lysoprin.

For the treatments with Lysoprin, 10.5, 42 and 105 grams of Lysoprin were blended with 352 grams of ORO GLO liquid. The mixture was blended at high speed in a Waring blender for 15 minutes. Treatment was affected by pouring each of the above pigment mixtures onto feed in a ribbon mixer containing 210 lbs of low xanthophyll feed which had been previously treated with 150 ppm TERMOX dry and 1,000 ppm MYCO CURB liquid. LYSOFORTE treatments were prepared by adding 105 and 420 grams of LYSOFORTE (10% Lysoprin) to the mixer after adding pigment. Control feed was prepared by addition of appropriate amounts of ORO GLO liquid, as outlined in Table 2, directly to low xanthophyll feed without addition of Lysoprin or LYSOFORTE. Samples of each feed were taken and analyzed for pigment content using a method based on AOAC, Paragraph 43.018, without saponification and column chromatography.

SUBSTITUTE SHEET (RULE 26) WO 99/47001 PrT/ 21 TABLE 9: ORO GLO AND LYSOLECITHTN APPLICATION RATES IN A STUDY OF HIGH-LEVEL LYSOLECITHIN IN LAYER DIETS ORO GLO (g Total Treatment (Lb/Ton) Xanthophyll/Ton) 0 0 0 0 0.22 Lysoprin 0.88 Lysoprin 2.2 Lysoprin 2.2 LYSOFORTE 8.8 LYSOFORTE At the end of the trial, one dozen eggs were collected from each pen. The eggs were weighed and analyzed for color reflectance and pigment content. Three eggs were randomly selected from each dozen for yolk evaluation. Eggs were cracked open and the yolk separated from the white. Yolks were then composited and blended thoroughly. A 2.5 gram sample was taken from each yolk composite and analyzed by a modification of AOAC method 17.004 in which total yolk pigment is extracted with acetone and determined spectroscopically. Pigment content was determined by comparing extractant absorbance at 450 nm to a p-carotene standard curve. Results were converted to and reported as p-carotene equivalents (jg BCE/g yolk).

Egg collection data (feed/bird/day and average egg weight) and pigment content are presented in Table 10. As can be seen, addition of lysolecithin to the diet made no numerical or statistical difference in either feed consumption per bird per day or average egg weight.

SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT[US99/05925 22 TABLE 10: RAW DATA FROM HIGH-LEVEL LYSOLECITHIN LAYER TRIAL: FEED CONSUMED PER BIRD PER DAY, EGG WEIGHT AND FEED PIGMENT

CONTENT

Xanthoph Treatment Feed/Bird/Day Average Egg Measured Initial yll (Lb/Ton) Wt. Pigment (g/Ton) (g/Ton) 0 0 106.7 65.19 5.30a 0 108.6 64.89 44.92 b 0 103.7 66.14 67.36c 0.22 Lysoprin 105.2 64.16 42.73 b 0.88 Lysoprin 106.9 64.97 45.49 b 2.2 Lysoprin 109.4 66.30 42.02 b 2.2 LYSOFORTE 105.1 64.70 39.72 b 8.8 LYSOFORTE 106.2 62.43 39.42 b Average 106.48 64.85 Standard deviation 1.87 1.21 a-c Entries in column with no common superscripts differ significantly (P<0.05).

The results of NEPA analysis are shown in Table 11 and Figure 5. As stated above, the NEPA method directly measures mass of pigment extracted from the yolk.

Hence, any increases in the amount of pigment incorporated into the yolk can be directly observed without regard to color perception. As can be seen in Table 11, treating poultry feed supplemented with 40 g/ton xanthophylls with Lysoprin liquid lysolecithin supplement resulted in statistically valid (P<0.05) increases in yolk pigment content versus control at every Lysoprin treatment level. By contrast, addition of LYSOFORTE dry lysolecithin supplement did not yield statistically valid increases in yolk pigment content. For a 40 g/ton xanthophyll supplement, at the 2.2 lb/ton treatment level, there was a statistically valid decrease in yolk pigment content versus feed that had not been treated with LYSOFORTE. These results are shown graphically as Figure SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/IUS99/05925 23 TABLE 11: RESULTS OF ANALYSIS OF EGG YOLKS BY NEPA Treatment (Grams/Ton) NEPA Pigment (gg Percent BCE/g Yolk) Change vs. g/ton ORO

GLO

ORO GLO Lysoprin 0 0 2.24 0 52.85c 0 0 7 6 3 0 g 44.37086 0.22 Lysoprin 55 3 7 d 4.768212 0.88 Lysoprin 57.69e 9.157994 2.2 Lysoprin 60.36' 14.21003 2.2 LYSOFORTE 4 9 0 3 b -7.228 8.8 LYSOFORTE 53.93C 2.043519 a- Entries in column with no common superscripts differ significantly (P<0.05).

Incorporation of Lysoprin lysolecithin into pigment-supplemented poultry diets for active layers yields statistically valid increases in the pigment content of egg yolks. Based on the above data, a pigment level of 40 g ORO GLO liquid/ton of feed, with addition of lysolecithin at the application rates used in this study, will show yolk color performance between that of 40 g/ton and 60 g/ton untreated feeds. Conversely, addition of LYSOFORTE dry lysolecithin supplement did not improve pigment incorporation.

SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/US99/05925 24 Experiment 4 One-hundred and twenty white Leghorn Hyline W36 cross birds housed in battery pens were used in the layer trial. The hens were 84 weeks old at the start of the test. Prior to the collection of the eggs, the birds were given the experimental diets and water ad libitum for 28 days. Each battery pen, containing eight birds, was used as an experimental unit. Each experimental diet was fed to three pens randomly placed in the layer facility. In conjunction with the layer trial, an experimental assay testing the quality of Lysoprin lots was developed. This assay is based on the water-in-oil emulsifying properties of lysolecithin versus lecithin. A close correlation of the results of this assay and the trial results, high quality product as determined in the assay leads to enhanced yolk pigmentation, would allow this assay to be used as a quality control assay for incoming lots of lysolecithin.

The experimental diets were prepared in the following manner. Five samples of 210 lbs of the low-xanthophyll diet of Table I were supplemented with one of the following: 2.2 lbs/ton of Lysoprin an alternative commercial product (Blendmax 322D liquid), 11 lbs/ton LYSOFORTE, and an untreated control. In addition each sample was treated with 8 lbs/ton ORO GLO® brand liquid and 2 lbs/ton MYCO CURB® brand liquid. The liquid lysolecithin products were mixed with the ORO GLO liquid in a Waring Blender for one minute before treating the feed. The lysolecithin and/or pigment products were added to the feed at the indicated levels by slowly pouring the additive onto feed during mixing in a ribbon mixer. After running through a hammer mill with a mill screen, 35 lbs of each treatment was weighed into poly-lined Kraft bags.

SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/I US99/5925 The quality assurance test were carried out in the following manner. 1.6 g of liquid Lysoprin and 8 g of dry LYSOFORTE are added to separate 100 ml samples of canola oil. These solutions were then mixed thoroughly, after which the dry LYSOFORTE was filtered to remove all carrier material. At the same time a NaCI solution was prepared. Fifty ml of the lysolecithin solutions and 50 ml of the NaCI solution were added to a 150 ml beaker. The materials were mixed with a 10,000 rpm bio-homogenizer for 15 seconds. The emulsion was immediately transferred to a 100 ml graduated cylinder. The volume of the lower phase (the free NaCI solution) of the emulsion was observed at time 0 and then every five minutes over a 60 minute period. A blank of canola oil and the 15% NaCI was also run for comparison.

For the analysis of the pigments six eggs per treatment, two from each of the three replicates, were randomly selected. The yolks were separated from the white and further cleaned with a paper towel. The egg yolks were combined, weighed and homogenized with a bio-homogenizer.

The yolk fan scores were conducted first. Ten grams of the composite egg yolk material from each treatment was placed in a plastic 60 x 15 mm Falcon 1007 petri dish. Six individuals in the research facility were then asked to determine the color fan score of each yolk composite. The fan scores were then analyzed at the confidence level using Duncan's multiple comparison procedure (Statgraphics Plus, Manugistics, Inc., 1995).

The intensity red and yellow pigmentation was measured using a CR-300 Minolta Colorimeter. Again, 10 g of the composites egg yolk from each treatment was placed in a plastic 60 x 15 mm Falcon 1007 petri dish. To measure the L*a*b of each treatment the sample was placed on the top of the measuring head with

I

SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/US99/05925 26 an open orifice for color head. Measurements of L*a*b were replicated twice for each sample. The L, a and b scores were then analyzed at the 95% confidence level using Duncan's multiple comparison procedure.

The p-carotene equivalents were conducted according to the AOAC method 17.002 through 17.004. The measured egg pigment or p-carotene equivalents were then analyzed at the 95% confidence level using Duncan's multiple comparison procedure.

The results of the quick assay of the two lots of liquid Lysoprin, the dry LYSOFORTE and the competitive product (Blendmax 322D) used in the trial can be seen in Figure 6. The assay is based on the emulsifying properties of lysolecithin and measures the retardation of the phase separation over time when a sodium chloride/water solution and an oil/lysolecithin solution are mixed. All three liquid products showed a significant retardation of the phase separation. Given these results, the best emulsifier of the group appears to be the Blendmax 322D product, followed by the two lots of Lysoprin. No effect was seen for the dry LYSOFORTE.

Significant differences were observed between the fan scores of the treated yolks with Lysoprin lot 200795 performing the best. As seen in Table 12, the mean fan score from the yolks treated with Lysoprin lot 200795 was significantly higher than the control and the other mean fan scores, while the dry LYSOFORTE treatment showed the lowest fan score.

SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/I SO/05o925 27 TABLE 12: MEAN FAN SCORES OF YOLKS TREATED WITH DIFFERENT LYSOLECITHIN PRODUCTS Treatment Mean Fan Score Control Lysoprin (lot 200795) 9 58 b Lysoprin (lot 250795) 8.42a LYSOFORTE (lot 5053086) Competitive product (Blendmax 322D) a,b Different superscripts indicate significant differences (P<0.05).

The results of the Minolta meter measurements are found in Table 13. When compared to the control and the other treatments the dry LYSOFORTE exhibited the lowest L score reflecting less pigmentation while Lysoprin lot 200795 resulted in a significantly stronger red hew which likely accounts for the higher scoring on the fan.

TABLE 13: L, a AND b MEASUREMENT OF YOLKS TREATED WITH DIFFERENT LYSOLECITHIN PRODUCTS Pigment Measurement Treatment L a b Control 52.15 b 45.75' Lysoprin (lot 200795) 52.5c 0 .9h 4 7 .0k Lysoprin (lot 250795) 51.9" -0.05 S 46.15i LYSOFORTE (lot 5053086) 54.0 d -1.0 49.0' Competitive product (Blendmax 322D) 52.5c 0.05" 46.85 k Different superscripts within each column indicate significant differences (P<0.05).

The results of the beta-carotene equivalency method (Table 14) confirm what was observed using the fan score method. Lysoprin lot 200795 improves the pigmentation by 15% while the other two liquid lysolecithin treatments had a lesser effect. Egg yolks treated with the dry LYSOFORTE, however, showed a significant reduction in pigmentation.

I

SUBSTITUTE SHEET (RULE 26) WO 99/47001 PrT/U S9/n<0Q2 28 TABLE 14: P-CAROTENE EQUIVALENTS OF YOLKS TREATED WITH DIFFERENT LYSOLECITHIN

PRODUCTS

P-Carotene Equivalents (pg/g) Treatment Rep. 1 Rep. 2 Mean Change Control 68.89 70.44 69.67 b Lysoprin (lot 200795) 80.61 80.48 80.54e 15.6 Lysoprin (lot 250795) 74.06 73.12 73.59 5.6 LYSOFORTE (lot 5053086) 63.05 63.33 63.19 a -9.3 Competitive product (Blendmax 76.69 79.15 77.92 11.8 322D) -e Different superscripts indicate significant differences (P<0.05).

The results of the layer trial conclusively show what was observed in a previous trial, that liquid Lysoprin when included in a layer feed at a level of 0.11% can enhance the pigmentation of egg yolks by up to 15%, while dry LYSOFORTE actually decreases the efficacy of pigment uptake and/or deposition. As far as efficacy is concerned, substantial variations are observed among different liquid lysolecithin lots. In this trial lot 200795 was superior over a product from Central Soya, Blendmax 322D.

The results of this trial also indicate that there is a positive, although limited, correlation between the results of the quick assay and the results of the layer trial. The three liquid Lysoprin lots that performed well in the layer trial also performed well in the quick assay while the dry LYSOFORTE product failed in both, the quick assay as well as the layer trial. The quick assay, however, failed to predict the order of performance that was seen in the layer trial Blendmax 322D performed best in the quick assay but was inferior in the trial). Secondly, the emulsifying properties of the lysolecithin products are consistently seen only in canola oil, not however in corn oil or mineral oil. The Blendmax 322D product, however, showed a good effect also in mineral oil.

J/rJ SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/US99/05925 29 Experiment The study was conducted over 28 days. Two hundred twenty-four white leghorn Hyline W36 cross birds housed in battery pens of eight or nine birds were used. The hens were 21 weeks old at the start of the test. For three weeks prior to the trial the birds were fed a low xanthophyll diet as detailed in Table 1.

Temperature in the house during the trial ranged from 200 C to 320 C. The experimental ORO GLO samples were prepared in the following manner. To equal quantities of Kemin Yellow the surfactant as indicated in Table 15 was blended until the mixture was homogenous. The blend was then added to the dry carrier. These experimental formulas were applied to the low xanthophyll, low fat poultry mash feed. A mixture of 1:1 soybean oil and poultry fat was prepared and applied to half of each treatment. The treatments used in this trial are listed below: TABLE 15: TREATMENTS OF LAYER FEED Xanthophylls Surfactant Added Fat Treatment (Theoretical) kg/ton kg/ton Control ORO GLO dry 30 g/ton OGD Corn oil 30 g/ton 0.916 kg/ton Corn Oil OGD Lysoprin 30 g/ton 0.916 kg/ton Lysoprin OGD Lecithin 30 g/ton 0.916 kg/ton Lecithin OGD; High fat diet 30 g/ton 36.6 OGD Corn oil; high fat 30 g/ton 0.916 kg /ton Corn Oil 36.6 diet OGD Lysoprin; high fat 30 g/ton 0.916 kg /ton Lysoprin 36.6 diet OGD Lecithin; high fat 30 g/ton 0.916 kg/ton Lecithin 36.6 diet MYCO CURB® brand liquid was also added to each treatment at 0.916 kg/ton. Mixing of the feed samples was done in a ribbon mixer at low speed. Each treatment was weighed into poly-lined Kraft bags, after running through a hammer SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/IIS99/05925 mill with a mill screen. Samples were taken and analyzed for pigment content using QC method QPM-10 without saponification or chromatography.

Four randomly selected pens were given each of the nine treatments. Feed and water were given ad libitum for the duration of the trial. Eggs from each pen were collected and counted each day. On the last day of the trial eggs from each pen were collected for analysis. Samples of eggs from each pen were weighed to determine average egg weight and five eggs from each dozen were randomly selected and pooled with the appropriate treatment. These eggs were cracked and the yolks were separated from the whites, composites were made and blended thoroughly, from which a 2.5 g sample was analyzed for p-carotene equivalence by AOAC method 958.05, and by Roche fan scores.

Pigment conversion was calculated according to the following formula: Pigment conversion 100% x (BCE) x (volk weight) x (eggs/dav/hen) (Pigment) x (feed/hen/day) For the statistical analysis an analysis of variance with a Duncan multiple range test was conducted.

Tables 16 displays the results of the quantitative determination of the pigments and pigment conversion in the egg yolks. Addition of ORO GLO to the low pigment feed increased the pigmentation from 7.49 to 52.00 p-carotene equivalents. Inclusion of 4% or 36.6 kg/ton soybean oil/poultry fat 1:1 enhanced pigmentation from 52.00 to 65.2 p-carotene equivalents. The addition of 36.6 kg/ton soybean oil/poultry fat 1:1 to the diet resulted in a 35.0% better pigment conversion as compared to the low pigment diet. When corn oil, lecithin or Lysoprin up to a final concentration of 0.916 kg/ton feed were added with the ORO GLO dry to the low fat or the high fat diet, SUBSTITUTE SHEET (RULE 26) WO 99/47001 PCT/US99/05925 31 lecithin showed the greatest increase in pigmentation and pigment conversion (22.2% over the ORO GLO control) for the low fat diet, while Lysoprin resulted in the best increase for the high fat diet (23.3% over the ORO GLO/high fat control). However, surfactant treatments of the low fat diet were not statistically different among each other.

The egg productivity of the laying hens was also monitored for the duration of the trial. The results are displayed in Table 17. No significant differences in egg productivity were seen for the various treatments.

TABLE 16: PIGMENTATION OF EGG YOLKS AS DETERMINED BY THE P- CAROTENE EQUIVALENCY AND FAN SCORE METHODS Treatment Control ORO GLO dry OGD Corn oil OGD Lysoprin OGD Lecithin OGD; High fat diet OGD Corn oil; high fat diet OGD Lysoprin; high fat diet OGD Lecithin; high fat diet Feed Pigment (g/ton) n/a 27.5 26.5 28.9 26.9 24.4 22.7 23.2 24.2 P-carotene equivalents (4g/g) 7.49

A

52.00

B

53.18

B

53.81B 62.31 c 65.20 c 71.22

D

75.41D 72.30

D

Fan Score 2.1 7.7c 7.2 7.1 7.5B,c 8.

C,D

8.0 C D 8.5D 8.1C,D Pigment Conversion n/a 16.14a 17.49 ,b 18.10 a b 19.77 a, 21.79a,b,c 21.48a,b,c 26.88 c 23.40 b,c Different superscripts indicate significant differences (P<0.05) TABLE 17: PRODUCTIVITY AS EGGS/HEN/DAY Treatment Eggs/hen/day Yolk Wt. Egg Wt. (g) Control 0.804 13.5 55.5 ORO GLO dry 0.837 13.7 53.8 OGD Corn oil 0.886 13.3 54.9 OGD Lysoprin 0.908 13.7 55.7 OGD Lecithin 0.857 13.7 55.7 OGD; High fat diet 0.839 13.6 54.6 OGD Corn oil; high fat diet 0.754 13.4 54.4 OGD Lysoprin; high fat diet 0.895 13.7 55.4 OGD Lecithin; high fat diet 0.815 13.3 56.4 SUBSTITUTE SHEET (RULE 26) WO 99/47001 PrT/i 4COo/nC'A 32 A A high fat diet was observed to lead to higher pigment utilization and pigment conversion as compared to a low or no fat diet. It further appears that while lecithin performs better in a low fat diet, Lysoprin performs better in a high fat diet as measured by pigment conversion. The first observation can be explained reasonably by the fact that uptake by the bird and deposition of the hydrophobic carotenoid molecules in the egg yolk is more effective when dispersed in fat in the animal's gut.

The second observation, however, seems to reflect a more specific effect with much higher efficacy for the lecithin and Lysoprin as compared to the soybean oil/poultry fat blend. While the 4% fat was added to the feed, corn oil, lecithin and Lysoprin were mixed into the ORO GLO product at only 0.1% relative to the final feed. This effect may be even more pronounced than is reflected in the results of this study.

Broilers eat according to their energy requirement. They consume less feed and, thus, less pigment when a high energy diet is fed. Results of this study were not corrected for the reduction in feed intake when the high fat feed was consumed. It can be speculated that the close proximity between lipids and carotenoids in the ORO GLO product contributes to the observed efficacy. As has been investigated at several occasions lysolecithin as compared to lecithin contains an additional 1-2% lysophophatidyl lipids which seems to be responsible for the enhanced emulsifying properties of Lysoprin. It appears that Lysoprin can display full efficacy as an emulsifying agent only in a diet supplemented with a certain amount of fat.

The results of this and previous studies suggest the possibility of including Lysoprin or lecithin in a commercial product for better pigmenting efficacy. With lecithin or Lysoprin included in a pigmenter product a desired pigmentation could be achieved with less pigment and/or lower fat. Also, the supplementation of just yellow pigment could lead to higher pigmentation scores.

SUBSTITUTE SHEET (RULE 26)

Claims (30)

1. A method for increasing the absorption and bioavailability of carotenoids in humans and poultry, comprising the steps of: combining a surfactant with a carotenoid wherein the ratio of surfactant to carotenoid is between about 5wt% and about and feeding said combination to humans or poultry.

2. A method as defined in claim 1, wherein said surfactant is lecithin or lysolecithin.

3. A method as defined in claim 1 or claim 2, wherein said carotenoid is a xanthophyll.

4. A method as defined in claim 1 or claim 2, wherein said carotenoid is lutein or zeaxanthin. A method as defined in claim 1, wherein said surfactant is one or more of lecithin or lysolecithin and said carotenoid is one or more of lutein or zeaxanthin.

6. A method as defined in claim 1 or claim 2, wherein said carotenoids are S 15 obtained from petals of Tagetes erecta.

7. A method for increasing the absorption and bioavailability of carotenoids in poultry said, method being substantially as hereinbefore described with reference to any one of the examples but excluding the comparative examples.

8. A surfactant and a carotenoid wherein the ratio of surfactant to carotenoid is 20 between about 5wt% and about 30wt% when used for increasing the absorption and bioavailability of carotenoids in humans and poultry.

9. A combination as defined in claim 8, wherein said surfactant is lecithin or lysolecithin.

10. A combination as defined in claim 8 or claim 9, wherein said carotenoid is a xanthophyll.

11. A combination as defined in claim 8 or claim 9, wherein said carotenoid is lutein or zeaxanthin.

12. A combination as defined in claim 8, wherein said surfactant is one or more of lecithin or lysolecithin and said carotenoid is one or more of lutein or zeaxanthin.

13. A combination as defined in claim 8 or claim 9, wherein said carotenoids are obtained from petals of Tagetes erecta.

14. A surfactant and a carotenoid, said combination being substantially as hereinbefore described with reference to any one of the examples but excluding the [I:\DayLib\LIBC]0833 I.doc:ais comparative examples, when used for increasing the absorption and bioavailability of carotenoids in poultry. Use of surfactant and a carotenoid wherein the ratio of surfactant to carotenoid is between about 5wt% and about 30wt% for the manufacture of a medicament for increasing the absorption and bioavailability of carotenoids in humans and poultry.

16. Use as defined in claim 15, wherein said surfactant is lecithin or lysolecithin.

17. Use as defined in claim 15 or claim 16, wherein said carotenoid is a xanthophyll.

18. Use as defined in claim 15 or claim 16, wherein said carotenoid is lutein or 0t zeaxanthin.

19. Use as defined in claim 15, wherein said surfactant is one or more of lecithin or lysolecithin and said carotenoid is one or more of lutein or zeaxanthin.

20. Use as defined in claim 15 or claim 16, wherein said carotenoids are obtained from petals of Tagetes erecta. 15 21. Use of surfactant and a carotenoid, said combination being substantially as 0 hereinbefore described with reference to any one of the examples but excluding the comparative examples, for the manufacture of a medicament for increasing the absorption and bioavailability of carotenoids in poultry. 2 222. A method for increasing the deposition of carotenoids in the tissues and egg 20 yolks of poultry, comprising the steps of: combining one or more of lecithin or 0 e lysolecithin surfactants with one or more of lutein or zeaxanthin carotenoids, wherein the ratio of surfactant to carotenoid is between about 5wt% and about 30wt%; and feeding said composition to poultry.

23. A method for increasing the deposition of carotenoids in the tissues and egg yolks of poultry, said method being substantially as hereinbefore described with reference to any one of the examples but excluding the comparative examples.

24. One or more of lecithin or lysolecithin surfactants and one or more of lutein or zeaxanthin carotenoids, wherein the ratio of surfactant to carotenoid is between about and about 30wt% when used for increasing the deposition of carotenoids in the tissues and egg yolks of poultry. One or more of lecithin or lysolecithin surfactants and one or more lutein or zeaxanthin carotenoids, said composition being substantially as hereinbefore described with reference to any one of the examples but excluding the comparative examples, when used for increasing the deposition of carotenoids in the tissues and egg yolks of poultry. [I:\DayLib\LIBC]0833 1.doc:ais

26. Use of one or more of lecithin or lysolecithin surfactants and one or more of lutein or zeaxanthin carotenoids, wherein the ratio of surfactant to carotenoid is between about 5wt% and about 30wt% for the manufacture of a medicament for increasing the deposition of carotenoids in the tissues and egg yolks of poultry.

27. Use of one or more of lecithin or lysolecithin surfactants and one or more of lutein or zeaxanthin carotenoids, said composition being substantially as hereinbefore described with reference to any one of the examples but excluding the comparative examples, for manufacture of a medicament for increasing the deposition of carotenoids and the tissue and egg yolks of poultry.

28. A method for increasing the deposition of carotenoids in the tissues, including the blood and macular region of the retina, of humans, comprising the steps of: combining one or more of lecithin or lysolecithin surfactants with one or more of lutein or zeaxanthin carotenoids, wherein the ratio of surfactant to carotenoid is between about 5wt% and about 30wt%; and feeding said composition to humans. 1. 15 29. One or more of lecithin or lysolecithin surfactants and one or more of lutein or zeaxanthin carotenoids, wherein the ratio of surfactant to carotenoid is between about and about 30wt% when used for increasing the deposition of carotenoids in the tissues, including the blood and macular region of the retina, of humans.

30. Use of one or more of lecithin or lysolecithin surfactants and one or more of 20 lutein or zeaxanthin carotenoids, wherein the ratio of surfactant to carotenoid is between about 5wt% and about 30wt% for the manufacture of a medicament for increasing the deposition of carotenoids in the tissues, including the blood and macular region of the retina, of humans.

31. A feed supplement for increasing the absorption and bioavailability of carotenoids in humans and poultry, comprising: a carotenoid; and a surfactant, wherein the range of surfactant to said carotenoid is between about 5wt% and about

32. A feed supplement as defined in claim 31, wherein said carotenoid is a xanthophyll.

33. A feed supplement as defined in claim 31, wherein said carotenoid is lutein or zeaxanthin.

34. A feed supplement as defined in claim 31, wherein said carotenoids are obtained from petals of Tagetes erecta. A feed supplement as defined in any one of claims 31 to 34, wherein said Ssurfactant is lecithin or lysolecithin. O F C 4 [I:\DayLib\LIBC]08331.doc:ais

36. A feed supplement, substantially as hereinbefore described with reference to any one of the examples, but excluding the comparative examples.

37. A pharmaceutical composition comprising at least a combination as defined in any one of claims 7 to 13, together with a pharmaceutical acceptable adjuvant, diluent or carrier. Dated 21 January 2002 KEMIN INDUSTRIES, INC. Patent Attorneys for the Applicant/Nominated Person oa SPRUSON FERGUSON *9 -Z [I:\DayLib\LBC]0833 I.doc:ais