Enhanced lycopene fortification in eggs using poultry feed containing a carotenoid-oil preparation derived from metabolically engineered baker’s yeast

Abstract

Carotenoids such as lycopene are important additives for food and animal feed due to their health-promoting effects as well as their industrial utility as color agents. However, the low bioavailability of lycopene from common sources limits its effective utilization as an additive in food and feed. Fortification of eggs is a promising strategy to increase dietary uptake of lycopene as well as enhance the pigmentation of egg yolk. Baker’s yeast, Saccharomyces cerevisiae, engineered to produce lycopene and beta-carotene, was examined as a supplement in poultry feed to replace synthetic dyes and fortify eggs with carotenoids. Improved bioavailability of lycopene was achieved using an oil-based formulation of carotenoids extracted from yeast. Feed supplemented with the carotenoid extract oil enhanced yolk color by as much as 4.5 points on the DSM color fan scale, compared to feed without added dyes, giving a color score of 10, equivalent to commercial feed. Feed containing 56 mg lycopene/kg resulted in the accumulation of 32.5 mg lycopene/g egg yolk, a value eight-fold higher than previous reports. These results demonstrate the utility of extracts from engineered yeast as a carotenoid supplement with high bioavailability for poultry feed, both as a color agent and to promote lycopene fortification.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-38825-9.

Introduction

Functional foods are attracting increasing attention due to their health-promoting properties. One class of functional foods is those fortified with vitamins, minerals, or other ingredients that exhibit health benefits¹. Food fortification with bioactive pigments has increased as natural pigments are preferred by consumers over artificial colorants². Carotenoids are naturally occurring pigments with yellow to red coloration that are often utilized in functional food preparation^3,4. Several epidemiologic studies have demonstrated a relationship between carotenoid consumption and decreased incidence of chronic diseases, including blindness, cancers, cardiovascular disease, and diabetes^5–8.

Lycopene, a member of the carotenoid family⁹, is a valuable food colorant due to its strong color and low toxicity¹⁰. In addition to its function as a color agent, lycopene is a powerful antioxidant and has the potential to protect lipids, proteins, and DNA from oxidative damage^11–13. A lycopene-rich diet appears to be associated with a lower incidence of some cancers^13,14 and prevents or lessens the risk of cardiovascular disease by regulating cholesterol levels¹¹. This carotenoid also has important applications in the production of cosmetics and nutraceutical products^13,15. However, the bioavailability of lycopene in commonly consumed foods, the fraction that is available for absorption, is variable and affected by the food matrix^16,17. For example, the bioavailability of lycopene from tomato juice is 5%¹⁸.

Fortification of commonly consumed foods can enhance the dietary intake of carotenoids. Eggs are an interesting choice for carotenoid fortification as poultry feed enriched with lycopene and beta-carotene, can promote their accumulation in egg yolk^19,20. In addition, carotenoids from fortified egg yolk have greater bioavailability in human diets²¹. Egg yolk color can be a determining factor in consumer’s preference for purchasing specific brands of eggs²². The egg yolk color preference varies depending on location with orange yolk color preferred in North America and more orange to red color desired in Europe and some Asian countries. Overall eggs with yolk color scores between 9 and 12 on the yolk DSM color fan are preferred by consumers compared to eggs that have more pale yolk color. It is often the perception that a vivid yolk color is an indicator of a more nutritious egg and good health of the laying hen²³. The desire of consumers for highly colored egg yolks has motivated many egg producers to utilize synthetic pigments in poultry feed to enhance yolk color²⁰. Replacement of these synthetic color agents with natural carotenoids may have economic benefits as well as allow the production of eggs with yolk color from natural pigments.

Several challenges are associated with the carotenoid fortification of animal feed and human food. Major sources of carotenoids in poultry feed are corn, alfalfa, and grass meals²⁴. However, the carotenoid content of these ingredients is unpredictable²⁵. The preparation of carotenoid supplements is a labor-intensive and expensive extraction process. The use of organic solvents, such as hexane and ethyl acetate, are commonly utilized for carotenoid extraction from plant sources²⁶. Multiple rounds of extraction are typically required resulting in the use of large amounts of organic solvents that are expensive, toxic, and require disposal¹².

A strategy to supply low-cost lycopene and beta-carotene is to utilize extracts from microorganisms that have been metabolically engineered to over-produce these carotenoids. The baker’s yeast Saccharomyces cerevisiae is an organism that can be engineered to produce carotenoids and is also often used as a component of animal feed. S. cerevisiae is a microorganism that is generally recognized as safe for use in food and feed products. Many preparations of poultry feed utilize up to 20% yeast extract as a protein source in place of soy or fish meal²⁷. Carotenoid-producing yeast have the potential to overcome several of the challenges to feed fortification as well as allowing for yolk pigmentation with natural colorants. The objectives of this study are to identify poultry feed formulation, utilizing yeast produced carotenoids, capable of enhancing yolk pigmentation and fortification of egg yolk with lycopene. The ultimate goal of this study is to produce an industrial compatible lycopene fortified feed for use as both a color-enhancing agent for egg yolk as well as providing a health benefit to egg consumers.

Materials and methods

Yeast strains and growth conditions

S. cerevisiae strains used in this study were derived from BY4742 (Mat α, leu2∆0, lys2∆0, ura3∆0, his3∆1)²⁸. The single deletion strain ypl062w∆::kanMX4 was obtained from Open Biosystems (Lafayette, CO, USA). Yeast transformations were performed using the lithium acetate procedure²⁹. Cells were propagated at 30 ˚C either in enriched yeast extract, peptone-based medium supplemented with 2% glucose (YPD) or synthetic complete (SC) medium with 2% glucose³⁰. Transformants were selected using SC medium lacking appropriate nutrients. Cultures for carotenoid production were grown in flasks with shaking using SC medium lacking uracil at 30 ˚C for 48 h.

Chemicals

Chemical reagents including methanol, hexane, acetone, acetonitrile, and dichloromethane were purchased from Merck-Millipore (Darmstadt, Germany), all chemicals were of HPLC grade. Standards for lycopene and beta-carotene (98% pure) were purchased from ChemFaces Biochemical Co., Ltd. (Wuhan, China).

Plasmids

Expression cassettes containing Xanthophyllomyces dendrohous crtE and crtYB genes flanked by the TEF2 promoter and ADH1 terminator sequences were PCR amplified from plasmids POT2-TEF2p-ycrtE-ADH1t and POT5-TEF2p-ycrtYB-ADH1t, digested with XbaI and XhoI and ligated sequentially into plasmid POT4-TDH3p-ycrtI-ADH1t³¹, digested with SpeI and XhoI. The resulting plasmid pCS002 contained crtE, crtI, and crtYB expressed from strong constitutive promoters. A second copy of the expression cassette for crtYB was added by digesting pCS002 with SpeI and XhoI and ligating pTEF2-crtYB-ADH1t cut XbaI and XhoI generating plasmid pCS012. The truncated HMG1 (tHMG1) sequences, encoding residues 530 to 1054, were PCR from yeast genomic DNA, digested with BamHI and EcoRI, and ligated into plasmid pCS021 containing the TDH3 promoter and ADH1 terminator resulting in plasmid pCS014. The expression cassette for tHMG1 was excised with XbaI and XhoI and ligated into pCS012 cut with SpeI and XhoI resulting in plasmid pCS033.]  Plasmid pRS316 has been described previously⁸⁵.  Plasmids are listed in Supplemental Table 1.

Production of carotenoid supplements

Carotenoid-producing yeast were cultured in selective SC medium lacking uracil for 48 h at 30 ˚C with shaking. Cells were harvested by centrifugation using a Sorval Lynx4000 superspeed centrifuge (Thermo Fisher Scientific, Waltham, MA, USA) at 10,000 x g for 5 min, washed with deionized water and freeze-dried. Carotenoid-enriched extracts from yeast were prepared with a novel extraction process. The yeast cell wall was softened chemically by treatment with Tris-SO₄ pH 9.4 and 10 mM dithiothreitol (Tris-SO₄/DTT) for 30 min. After washing the cells twice with deionized water, the cell mass was then disrupted using 400 μm glass beads with simultaneous extraction of carotenoids with acetone/methanol (80:20) or in later experiments 100% acetone as inclusion of methanol did not improve carotenoid extraction. Samples were mixed by vortexing for 6 min (2 min vortexing followed by 2 min on ice). Extraction used 20 mL acetone per gram of dried yeast. Cells were removed by centrifugation at 3,000 rpm for 3 min and supernatant was collected. This procedure allowed rapid and efficient carotenoid extraction, decreasing the time of preparation. The carotenoid-containing fractions were dried using a rotary evaporator to remove organic solvent. The extracted carotenoids were then resuspended in soybean oil.

Yeast with broken cell wall were prepared with Tris-SO₄/DTT treatment for 30 min, followed by washing twice with 1.2 M Sorbitol/HEPES (pH 7.5). Samples were disrupted using sonication for 6 min (2 min sonication followed by 2 min on ice) and resuspended in soybean oil. Intact dried yeast were directly resuspended in soybean oil.

All carotenoid extraction procedures were performed under low light conditions, overhead lights were not utilized, and blinds were used to limit the amount of indirect natural light. In addition, samples were shielded from light either using amber tubes or with aluminum foil covering. Carotenoid samples were stored at -20 ˚C in the dark prior to use.

Analysis of carotenoid content and composition

The carotenoid composition was determined in yeast and yeast extracts following the procedure described by Fongsuk et al.³² with slight modification. All HPLC analyses were performed using Shimadzu LC-20AC pumps, SPD-M20A diode array detector, and an Inertsil^® C-18 column (150 × 4.6 mm i.d., 5 μm). The mobile phase was acetonitrile: dichloromethane (75:25, v/v). The mobile phase was filtered through a 0.45 μm membrane. The mobile phase flow rate was 1 mL/min. The column temperature was 25 ± 0.5 ˚C and the absorbance was detected at 475 nm. Results are reported as mg carotenoid/g dry cell weight (DCW).

The carotenoid content of egg yolk was determined following saponification using the procedure of Hong et al.³³ with modification. Yolks were freeze-dried and carotenoids were extracted with ethanol containing 0.1% (w/v) butylated hydroxytoluene. Potassium hydroxide was added to a final concentration of 5% and samples were incubated at 35 ˚C with shaking at 100 rpm for 60 min. To terminate the saponification reaction, phosphate buffer pH 2 was added to 1 M final concentration. Hexane was added to 35% and carotenoids were extracted. The hexane fraction was removed and evaporated. The resulting carotenoids were resuspended in acetone for HPLC analysis.

Poultry feed

Two types of poultry diets were used in this study, mash feed and pellet feed. Mash is a complete feed that is finely ground and mixed³⁴. Pellet feed is formed, often with the same base ingredients, through processing with added moisture, pressure and temperature³⁵. Mash feed has an advantage in formulation experiments in that the composition can be easily modified. However, pellet feeds are considered more convenient to handle. In addition, feed intake is often enhanced with pellet feed compared to mash feed preparations. A disadvantage of pellets feed is cost, with mash feeds formulated with the same ingredients often being less expensive³⁶.

Feed CP-524 (CP Feed, Thailand) is a commercial feed containing Carophyll Yellow (1.5 µg/kg) and Carophyll Red (4.5 µg/kg) as color agents. This commercial feed was used for the positive control group (M1). CL-3-801 is a mash feed without an added color agent and was used for the negative control group (M2). The CL-3-801 mash feed was supplemented with control yeast without carotenoids (M3) or yeast-producing carotenoids (M4, M5) by directly mixing freeze-dried yeast that was ground to a fine powder with the feed. The ingredients and nutrient composition of the mash feed are listed in Supplemental Table 2.

Pre-prepared pellet feeds CL-3-802, containing Carophyll Yellow (1.5 µg/kg) and Carophyll Red (4.5 µg/kg) as color agents, and CL-3-803, without added color agents, were used. During feed formulation, oil was not added to either CL-3-802 or CL-3-803 feeds. CL-3-802 with added soybean oil was used as feed for the positive control (T1). CL-3-803 feed (no added color agents) with added soybean oil was used for the negative control (T2). CL-3-802 feed prepared with carotenoid extract-oil mixture (T3), broken yeast-oil mixture (T4), and yeast-oil mixture (T5). Soybean oil or carotenoid supplements in oil were added to pellet feed using an electric oil sprayer (Buildec, Zhejiang, China) to surface coat the feed pellets. A sealed chamber, with black cover to shield against light exposure, was used to prevent loss of the sprayed oil mixture during application. The pellet feed was dried and protected from direct light and excess heat. To avoid lycopene degradation, large batches of lycopene supplemented feed were not prepared. Instead, sufficient feed was prepared each week for feeding experiments. All feeds were stored in the dark in sealed Ziplock plastic bags at 25 ˚C prior to use.

The final content of soybean oil for each feed preparation was 20 mL soybean oil/kg feed. The ingredients and nutrient composition of the pellet feed CL-3-802 and CL-3-803 are listed in Supplemental Table 3.

Laying hens

Thirty Hy-Line Brown laying hens were used in each of the three feeding trials for a total of 90 birds. All the hens and facilities were provided and supported by the Feed Research and Innovation Center (FRIC, CP Company, Chonburi, Thailand). Feeding trials were initiated when hens reached the age of 47 weeks old. Birds were kept in cage batteries for one bird each for the duration of the feeding trials. Feed and water were provided ad libitum and the feeding trial lasted three weeks for each concentration of carotenoid supplement. The lighting program consisted of a 17-hour light and 7-hour dark cycle. The temperature of the facility was maintained at 28 ˚C during the experimental period. Laying hens were arranged in the house such that members of each group were distributed in the room to limit the effects of temperature, drafts, and lighting on the feeding results. The hen arrangement for the third feeding trials is shown as an example in Figure S1.

Feeding trial with laying hens

Feeding experiments were performed in collaboration with CP staff at the Feed Research and Innovation Center (FRIC, CP Company, Chonburi, Thailand). To establish the baseline for egg weight, yolk color, and Haugh unit, hens were given feed without a color agent for three weeks before the feeding trial. Analysis of egg parameters utilized a DET 6000 digital egg tester (NABEL Co., Ltd., Japan). This study was reviewed and approved by the Institutional Animal Care and Use Committee of the Feed Research and Innovation Center, Charoen Pokphand Foods, Protocol numbers 2,408,006, 2,308,021, and 2,408,001. All experiments were performed in accordance with relevant regulations in The Animals for Scientific Purposes Act 2015³⁷. Experiments were conducted following the guidelines from “Guide for the Care and Use of Agricultural Animals in Research and Teaching, 4th edition, 2020”³⁸ and “The Ethical Principles and Guidelines for the Use of Animals for Scientific Purposes” from the National Research Council of Thailand (NRCT)³⁹.

A range of carotenoid concentrations was examined to identify the optimal content to enhance egg yolk coloration. The range utilized varied between the feed types with mash feed supplemented with 10 to 190 mg carotenoids/kg feed. In the case of the mash feed 190 mg carotenoids/kg feed was selected as highest dose as this was the content provided by addition of 10 g of yeast powder per one kg of feed. In contrast, pellet feed was supplemented with 17.5, 35, and 70 mg carotenoids/kg feed.

Feeding trial 1: The ability of dried yeast to promote carotenoid fortification of egg yolk when supplemented to mash feed was examined. Mash feed was utilized in this feeding experiment for lycopene supplementation since producing a mixture of feed with dried yeast powder would be a simple and flexible way to fortify feed with carotenoids.

In the first feeding, trial 24 hens were randomly assigned to 1 of 4 four groups, six birds in each group, with six birds in reserve in case any hens in the treatment groups did not display good egg production or had other issues that required replacement. The commercial feed CP-524 containing color agents Carophyll Yellow (1.5 mg/kg feed) and Carophyll Red (4.5 mg/kg feed) was used as a positive control. Feed CL-3-801 was fed to all hens for three weeks to establish a no color agent baseline.

In weeks 1 to 3 of the feeding trial, the groups were as follows: M1, commercial feed CP-524 (positive control); M2, mash feed CL-3-801 (negative control); M3, mash feed CL-3-801 supplemented with control yeast (no carotenoids); and M4, mash feed CL-3-801 supplemented with carotenoid yeast equivalent to 10 mg carotenoid/kg feed. During the feeding trial the M1, M2, and M3 groups were continued with the CP-524, CL-3-801, and CL-3-801 with control yeast feed, respectively. During weeks 4 to 6, hens in the M4 group were provided mash feed CL-3-801 supplemented with carotenoid yeast equivalent to 20 mg carotenoid/kg feed. The six birds in the reserve group were added to a new treatment group (M5) to allow analysis of a wider range of yeast carotenoid supplements.

During weeks 4 to 6, the M5 group was provided CL-3-801 mash feed supplemented with yeast equivalent to 45 mg carotenoid/kg feed. For weeks 7 to 9, the carotenoid supplement was increased to 90 mg carotenoid/kg feed in group M4 and 190 mg carotenoid/kg feed in group M5 in CL-3-801 mash feed. Supplementation with 190 mg carotenoid/kg feed was chosen as it was the highest concentration possible with the carotenoid yeast that had been prepared for the feeding trial. Eggs were collected daily. The experimental diagram is shown in Figure S2.

Feeding trial 2: Pellet feed was utilized in the second feeding experiments. In this trial soybean oil-based carotenoid extracts (carotenoid extract-oil) was examined. The purpose of this feeding trial was to determine if the carotenoid-oil mixture would be effective in feed fortification. Pellet feed is the standard type of poultry feed used in laying hens and the ability to fortify this type of feed would allow its use in industrial egg production.

In the second feeding trial, hens were divided into three groups, eight birds in each group, with six birds in reserve. Feed CL-3-803, basal pellet feed, was given to all hens for three weeks before the feeding trial to establish a no color agent baseline. In weeks 1 to 3 of the second feeding trial, the groups were as follows: T1, commercial pellet feed CL-3-802 containing color agents Carophyll Yellow (1.5 mg/kg feed) and Carophyll Red (4.5 mg/kg feed) as a positive control; T2, basal pellet feed CL-3-803 (negative control); T3, basal pellet feed CL-3-803 supplemented carotenoid-oil mixture extracted from yeast equivalent to 35 mg carotenoid/kg feed. The second feeding trial was ended after three weeks, egg yolk color in each group reached a plateau starting from 3rd week of the feeding period. The experimental diagram is shown in Figure S3.

Feeding trial 3: For the third feeding trial pellet feed was utilized, the same as in the second trial. In this trial the effectiveness of yolk carotenoid fortification from different sources was compared. The same carotenoid content was supplemented to feed from the carotenoid extract-oil used in feeding trial 2, broken yeast suspended in soybean oil (broken yeast-oil), and whole intact yeast in soybean oil (yeast-oil). This test was performed to examine the contribution of the soybean oil to bioavailability of carotenoids in the yeast preparations.

The number of hens used in the T1, feed CL-3-802 (positive control) and T2, feed CL-3-803 (negative control) were reduced to three birds each since data had already been collected for these treatments during the second trial. The remaining hens were placed into three groups of six birds each: T3, carotenoid-oil; T4, broken yeast-oil; and T5, yeast-oil mixtures. During weeks 1 to 3 groups T3, T4, and T5 were provided CL-3-803 feed with 17.5 mg carotenoid/kg feed of the respective carotenoid supplement. In weeks 4 to 6 the carotenoid supplement was increased to 35 mg carotenoid/kg feed. In weeks 7 to 9 the carotenoid supplement was increased to 70 mg carotenoid/kg feed. The experimental diagram is shown in Figure S4.

The duration of treatment for control conditions, both commercial feed (positive controls: CP-524 and CL-3-802) and feed without added color agents (negative controls: CL-3-801 and CL-3-803) was for the entire duration of the feeding trial. The control conditions were for 9 weeks in trial 1, 3 weeks in trial 2, and 9 weeks in trial 3. The use of the control feed for the entire feeding trail was to ensure that changes in egg parameters including yolk color, in with carotenoid fortified feed were not due to other factors such as changes to the environment in the hen house. Treatment was for three weeks for carotenoid containing feeds. Three weeks was found to be a sufficient time to allow maximum effects of the carotenoid feeds on yolk color.

Performance of hens and egg quality

The effect of supplementation of yeast-derived carotenoids on the laying hens, egg production, and parameters of the resulting eggs were monitored. Feed intake of laying hens from each group was measured weekly. Egg production (%), feed intake, and egg mass were determined using the equations shown in Supplemental Methods.

Egg quality determination included albumen height, yolk color, Haugh unit, and eggshell strength and thickness. Monitoring of these parameters was performed using a DET 6000 digital egg tester (NABEL Co., Ltd., Japan). Eggs from each group were collected daily. Egg yolk color was scored using a DSM-Firmenich YolkFan™ function available in the DET 6000 digital egg tester.

Statistics and data analysis

Experimental data are reported as the mean ± the standard deviation (SD). Significant differences between groups and no color agent diet (negative control) group are indicated with *P < 0.05, **P < 0.01, and ***P < 0.001. Significant differences between groups and commercial feed diet (positive control) group are indicated with ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001. Data were analyzed with one-way ANOVA with post-hoc Tukey test using GraphPad Prism (version 8.4.2). Power scores were determined using the Experimental Design Assistant (https://eda.nc3rs.org.uk/experimental-design-group) according to the method described by Dell et al. utilizing biological effect size and sample variability⁴⁰. Standard effect size for egg parameters were taken from Wang et al., Almeida et al., and Narinc et al.^41–43. Results of power scores and sample size per group are listed in Supplemental Tables 4 and Supplemental Table 5 for experiments utilizing mash feed and pellet feed, respectively.

Feed intake was reported for the third week of each trial from at least 30 measurements for each test group. Egg parameters were analyzed for each test group daily. Results for yolk color are reported as either the daily average for each test group, at least 6 eggs per group, or as the average to from eggs collected in the third week of each condition with at least 30 eggs used for analysis. Analysis of egg parameters (egg mass, egg production, eggshell strength, eggshell thickness, albumin height, and Haugh unit) were reported for eggs collected in the third week of each treatment with at least 30 eggs used for analysis.

Limit of detection (LOD) and limit of quantitation (LOQ) were calculated from HPLC calibration curves with the linear regression method using Microsoft Excel v. 16.103.4⁴⁴.

Results

Carotenoid production in S. cerevisiae

S. cerevisiae cannot produce lycopene or beta-carotene as it lacks several enzymes required for the production of these carotenoids. However, farnesyl diphosphate (FPP), a precursor required for beta-carotene synthesis, is readily formed in S. cerevisiae for ergosterol production. Geranylgeranyl diphosphate (GGPP) is also synthesized by S. cerevisiae but at relatively low levels. Yeast expressing crtE (GGPP synthase), crtI (phytoene desaturase), and crtYB (bifunctional phytoene synthase and lycopene cyclase) from X. dendrorhous are capable of converting FPP to lycopene and subsequently to beta-carotene^31,45.

As seen in Fig. 1A, color consistent with carotenoid production was observed in WT yeast expressing crtYB, crtE, and crtI (strain 2) compared to the vector control (strain 1). Increased color intensity was observed through the use of a yeast strain deleted for the uncharacterized gene YPL062w, which is reported to promote carotenoid production (strain 3)⁴⁶. In addition, using an additional copy of crtYB (strain 4) and tHMG1, a truncated HMG CoA reductase, lacking the regulatory domain (strain 5), further improved orange-red color formation.

Fig. 1.

Carotenoid producing yeast used in this study. (A) Color of yeast strains is shown after 3 days of incubation at 30 ˚C on SC medium lacking uracil. Yeast for HPLC analysis were grown in liquid SC medium lacking uracil for 48 h for optimal carotenoid production. (B) HPLC trace of strain 5 (ypl062w∆ 2x crtYB, crtE, crtI, tHMG1). The peaks for lycopene and beta-carotene are indicated. (C) Quantitation of lycopene and beta-carotene in each strain shown in A from HPLC analysis. The percent lycopene and beta-carotene is also reported.

Carotenoids were extracted from yeast to obtain a carotenoid-enriched fraction containing lycopene and beta-carotene. Initial extraction experiments were performed with strain 4, prior to generation of the tHMG1 expression plasmid and strain 5. A solid/liquid organic extraction method was used to isolate carotenoids from dried yeast. The cell pellet was resuspended in DMSO without heating, to limit thermal degradation of carotenoids, followed by addition of acetone/methanol (80:20) as the solvent according to established procedures for carotenoid isolation from yeast⁴⁷. However, the addition of acetone/methanol to the DMSO-treated yeast resulted in a solid pellet that was difficult to homogenize and process. Grinding with glass beads was not efficient and yeast cells retained a substantial amount of color after three rounds of grinding. Pre-wetting the yeast pellet with either deionized H₂O or sodium phosphate buffer (pH 7) did not improve the carotenoid extraction. Using this procedure carotenoid content of the extract prepared using 1 g yeast in 10 mL acetone/methanol (80:20) was determined by HPLC as 7.4 ± 1.1 mg lycopene/ g DCW (740 ± 110 µg lycopene/mL) and beta-carotene content was below the limit of detection. Limit of detection (LOD) and limit of quantitation (LOQ) by HPCL are as follows: LOD 5.0 µg lycopene/mL and LOQ 15.1 µg lycopene/mL; and beta-carotene LOD 3.4 µg beta-carotene /mL, LOQ 10.3 µg beta-carotene /mL.

As an alternative to utilizing DMSO to soften the yeast cell wall, pre-treatment with Tris-SO4 (pH 9.4) with 10 mM dithiothreitol (DTT) was performed. This procedure is used to soften yeast before generation of spheroplasts⁴⁸. Pre-treatment with Tris-SO₄/DTT resulted in a softer pellet that was easily homogenized in acetone/methanol (10 mL solvent/1 g DCW). The more complete resuspension of the yeast pellet appeared to promote cell lysis and enhance extraction of carotenoids. Carotenoids extracted using the Tris-SO₄/DTT procedure was 15.3 ± 1.2 mg lycopene/g DCW (1530 ± 110 µg lycopene/mL) and 3.6 ± 0.3 mg beta-carotene/g DCW (360 ± 27 µg beta-carotene/mL) detected. Recovery yield was not quantified as the extracted lycopene and beta-carotene content was determined not total carotenoid in the cell mass. However, the content of lycopene and beta-carotene per g DCW was reproducible across multiple extractions (Supplemental Table 6). A representative HPLC chromatogram from strain 5 is shown in Fig. 1B.

Consistent with the observed color of yeast strains analysis of carotenoid content by HPLC demonstrated increased content of lycopene and beta-carotene content in progression from strain 2 to strain 5 (Fig. 1C). Strain 5, ypl062w∆ strain expressing 2crtYB, crtE, crtI, and tHMG1 (plasmid pCS033), was utilized to produce carotenoids for feed formulations. The composition of carotenoids from our yeast system was approximately 80% lycopene and 20% beta-carotene (Fig. 1C). This composition will be referred to as “carotenoids” in subsequent sections.

Enhanced yolk color with yeast-derived carotenoids

Feeding Trial 1: Mash feed consisting of finely ground ingredients was utilized in the initial feeding trial. A major consideration was mixing of the dried yeast powder with the feed preparations. Mash feed formulations can be easily modified and seemed a good choice for combination with our yeast preparations. If successful, this technique would be a simple and flexible technique for feed supplementation.

Mash feed was supplemented with control yeast, not producing carotenoids, and a range of carotenoid presented as dried yeast powder (Fig. 2A and B). Addition of control yeast to the mash feed (M3 group) lowered the feed intake by approximately 10%. The lower feed intake in the M3 group appears to be associated with a reduction in egg production. However, inclusion of carotenoid yeast to the mash feed (M4 and M5 groups) did not cause adverse effects on egg quality or hen performance. Compared to the M2 group, feed intake, egg production, eggshell strength, egg shell thickness, and Haugh unit in the M4 and M5 groups were substantially altered. Compared to both commercial diet (M1 group) and the M2 group, the M4 and M5 groups had a small increase in egg mass, eggshell thickness, and albumen height. These parameters were reduced in the M2 group relative to commercial diet (M1 group). Addition of carotenoid yeast in the M4 and M5 groups restored the egg parameters to values seen in the M1 group (Tables 1 and 2).

Fig. 2.

Carotenoid fortification using commercial feed CP-534 and mash feed CL-3-801. (A) Yeast were freeze dried and ground to a powder. Photographs of control yeast strain 1 (WT with vector) and carotenoid producing yeast strain 5 (ypl062w∆ 2x crtYB, crtE, crtI, tHMG1) from Fig. 1. (B) Mash feed CL-3-801 with carotenoid producing yeast powder added before mixing (left panel) and after mixing (right panel). Feed mixed with the control yeast is also shown. (C) Yolk color scores for eggs from hens fed with each of the mash feed diets, as indicated, for three weeks. Results are the mean ± standard deviation for eggs produced during the third week of treatment. Statistical analysis employed one-way ANOVA with post-hoc Tukey test with differences relative to the basal feed group, ** P < 0.01.

Table 1.

Performance of hens on commercial diet and Mash feed formulations.

	Carotenoid supplement (mg/kg feed)	Feed intake (g/bird/day)	Egg Production (%)	Egg mass (g)
CP-524 Commercial diet (M1)	–	102 ± 5.5	94.3 ± 6.0	55.7 ± 1.3**
CL-3-801 Basal mash feed no color agent (M2)	–	97 ± 5.6	96.4 ± 3.8	53.8 ± 1.1##
CL-3-801 + control yeast powder (M3)	–	91 ± 18.6###	90.2 ± 9.4***	53.9 ± 1.0##
CL-3-801 + carotenoid producing yeast powder (M4, M5)	10	93 ± 5.2##	95.0 ± 3.1	57.0 ± 0.9**,#
	20	100 ± 6.7	98.5 ± 1.5	57.2 ± 1.6**, #
	45	97 ± 4.2	96.9 ± 4.4	55.6 ± 1.3*
	90	95 ± 6.0#	95.8 ± 7.2	54.5 ± 1.3#
	190	98 ± 6.4	91.7 ± 2.1	56.8 ± 1.5**, #

* P < 0.05, ** P < 0.01, *** P < 0.001 compared to Basal Feed with no color agent (M2).

^# P < 0.05, ^## P < 0.01, ^### P < 0.001 compared to Commercial Diet (M1).

Table 2.

Quality of chicken eggs from hens provided Mash feed formulations.

	Carotenoid supplement (mg/kg feed)	Eggshell strength (kgf)	Eggshell thickness (µm)	Albumen height (mm)	Haugh unit
CP-524 Commercial diet (M1)	–	4.7 ± 1.0	420 ± 30**	7.9 ± 1.8***	83.2 ± 8.2
CL-3-801 Basal feed no color agent (M2)	–	4.1 ± 1.1	370 ± 40##	5.0 ± 2.6##	82.3 ± 6.7
CL-3-801 + control yeast powder (M3)	–	4.9 ± 0.5	410 ± 20**	7.3 ± 2.0**	79.6 ± 6.7
CL-3-801 + carotenoid producing yeast powder (M4, M5)	10	4.4 ± 1.0	390 ± 30#	8.4 ± 1.5***	89.2 ± 4.4
	20	4.5 ± 0.7	410 ± 20**	9.7 ± 1.0***	88.1 ± 4.5
	45	4.8 ± 0.9	410 ± 20**	7.5 ± 1.0**	79.4 ± 5.8
	90	5.2 ± 0.8**	430 ± 50***	6.9 ± 2.0*	83.2 ± 10.5
	190	4.7 ± 0.7	410 ± 10**	8.0 ± 1.9**	86.7 ± 3.2

* P < 0.05, ** P < 0.01, *** P < 0.001 compared to Basal Feed with no color agent (M2).

^# P < 0.05, ^## P < 0.01, ^### P < 0.001 compared to Commercial Diet (M1).

As expected, eggs from hens fed the commercial feed, containing color agents Carophyll yellow (1.5 mg/kg feed) and Carophyll red (4.5 mg/kg feed), (M1 group) had a significant increase in yolk color. The average color fan score was 9 for the M1 group, compared to a score of 6 for the no color agent (M2) or control yeast (M3) groups. Carotenoid addition from yeast at 10 and 20 mg carotenoids/kg feed (M4 group) did not significantly increase yolk color compared to no added color agent groups (M2 and M3) (Fig. 2). However, increasing the dried yeast to provide 45, 90, and 190 mg carotenoids/kg feed resulted in a significant increase in yolk color compared to eggs from hens on a diet without color agents. The yolk color score was increased from 6 to 7.2, 7.5, and 8.2 for feed containing yeast equivalent to 45, 90, and 190 mg carotenoids/kg feed, respectively (Fig. 2C). These results indicate that feed supplementation of yeast-produced carotenoids is capable of improving yolk color.

Feeding Trial 2: These experiments utilized pre-formed pellet feed formulated without added oil. The strategy to combine the yeast derived carotenoids with the pellet feed was to use an electric sprayer to cover the pellet feed with a soybean oil or carotenoid extract-oil. An additional potential advantage was that the inclusion of oil may promote bioavailability of carotenoids in the feed preparation⁴⁹. CL-3-802 feed, containing the commercial color agents Carophyll yellow (1.5 mg/kg feed) and Carophyll red (4.5 mg/kg feed), was used as a positive control (T1). The negative control (T2) group was fed experimental feed CL-3-803 without added color agents.

The carotenoid fraction was resuspended with soybean oil and used for treatment group T3. Feeding Trial 2 utilized the control feeds (T1 and T2), and pellet feed prepared containing 35 mg carotenoids/kg feed using the carotenoid oil-mixture. Soybean oil only or soybean oil containing carotenoid supplements was added to each feed at 20 mL/kg feed. The results from Trial 2 were promising, with an average yolk color score of 8.3 from the T3 group. However, these results were inconclusive as to the cause of the enhanced bioavailability. The lycopene provide from the carotenoid extract-oil may have enhance bioavailability compared to whole yeast. Alternatively, the presence of the soybean oil may enhance the bioavailability of any carotenoid provided in the feed preparation. A third feeding trial was performed to examine the effects of the carotenoid extraction and presence of soybean oil on bioavailability in feed.

Feeding Trial 3: To examine effects on bioavailability different carotenoid preparations were examined. In the third feeding trial and the control feeds (T1 and T2) and treatment groups (T3, T4, and T5) prepared with experimental feed CL-3-803, without added color agents were used. The T3 group was supplemented with several concentration of the carotenoid yeast-oil mixture. Yeast with ruptured cell wall (group T4) and intact yeast (group T5) were also resuspended in soybean oil for use in feed. Feed was supplemented with each type of carotenoid source at concentrations of 17.5, 35, and 70 mg carotenoids/kg feed. Soybean oil only or soybean oil containing carotenoid supplements was added to each feed at 20 mL/kg feed (Fig. 3).

Fig. 3.

Preparation of CL-3-803 pellet feed supplemented with carotenoid extract-oil. (A) Carotenoid extract-oil preparation from carotenoid producing yeast. Extracts were prepared from 30 L of yeast grown in SC medium lacking uracil in shake flasks. (B) Photographs of commercial feed (CL-3-802) and basal pellet feed (CL-3-803) with soybean oil added (upper panels). Feed CL-3-803 supplemented with the indicated concentration of the carotenoid-oil extract. Soybean oil and carotenoid-oil extract was applied using an electric oil sprayer.

Feed intake of hens was improved in carotenoid supplemented groups (T3, T4, and T5) compared to the commercial diet (T1) and basal feed (T2). The increased feed intake is likely linked to the increased egg production from hen fed the carotenoid supplemented pellet feed. Egg mass was not substantially impacted by feed utilized in this feeding trial. However, eggs from hens given feed supplemented with carotenoids from broken yeast-oil (T4) exhibited a small reduction in egg mass compared to other groups (Table 3). Other egg parameters were not substantially altered by carotenoid supplementation. Although, eggshell thickness, and Haugh unit showed a significant change in some groups, the absolute change in these parameters was small. No significant change was detected in albumen height for any test group. It was observed that inclusion of 70 mg carotenoids/kg feed as either broken yeast-oil or yeast-oil caused a statistically significant reduction in eggshell strength, although the cause of this effect is not clear (Table 4). The lack of serious adverse effects on laying hens or egg parameters from the oil-based carotenoid formulation suggests these preparations can be safely used in feed.

Table 3.

Performance of hens on pellet feed formulations.

	Carotenoid supplement (mg/kg feed)	Feed intake (g/bird/day)	Egg production (%)	Egg mass (g)
CL-3-802 Commercial diet (T1)	-	108 ± 3.2***	87 ± 7.2**	62.5 ± 3.3
CL-3-803 Basal feed (T2)	-	118 ± 2.5###	91 ± 3.1##	61.5 ± 1.5
CL-3-803 + carotenoid extract-oil (T3)	17.5	128 ± 1.1***, ###	92 ± 4.3##	62.1 ± 1.3
	35	122 ± 2.7###	94 ± 2.8##	61.9 ± 1.7
	70	121 ± 4.2###	98 ± 2.0**, ##	64.6 ± 1.2*, #
CL-3-803 + broken yeast-oil (T4)	17.5	118 ± 3.4###	92 ± 2.8##	60.1 ± 1.0#
	35	113 ± 2.8**, ###	96 ± 2.6**, ##	59.1 ± 1.1*, #
	70	117 ± 3.7###	86 ± 4.5**	59.7 ± 1.4#
CL-3-803 + yeast-oil (T5)	17.5	127 ± 4.3**, ###	98 ± 2.0**, ##	61.7 ± 0.7
	35	120 ± 3.1###	94 ± 2.8*,##	60.9 ± 0.6#
	70	118 ± 2.9###	98 ± 2.0**, ##	62.6 ± 0.9

* P < 0.05, ** P < 0.01, *** P < 0.001 compared to Basal Feed with no color agent (T2).

^# P < 0.05, ^## P < 0.01, ^### P < 0.001 compared to Commercial Diet (T1).

Table 4.

Quality of chicken eggs from hens provided pellet feed formulations.

	Carotenoid supplement (mg/kg feed)	Eggshell strength (kgf)	Eggshell thickness (µm)	Albumen height (mm)	Haugh unit
CL-3-802 Commercial diet (T1)	–	4.7 ± 0.5	410 ± 14	8.8 ± 0.5	92.5 ± 2.9
CL-3-803 Basal feed (T2)	–	4.7 ± 0.3	390 ± 9	8.5 ± 0.4	91.2 ± 2.3
CL-3-803 + carotenoid extract-oil (T3)	17.5	4.6 ± 0.3	410 ± 5	8.5 ± 0.3	91.3 ± 1.2
	35	4.6 ± 0.3	400 ± 5	8.3 ± 0.3	90.5 ± 1.8#
	70	4.7 ± 0.1	410 ± 2	8.7 ± 0.1	91.9 ± 0.7
CL-3-803 + broken yeast-oil (T4)	17.5	4.7 ± 0.2	400 ± 5	8.8 ± 0.3	93.7 ± 1.6*
	35	4.6 ± 0.2	420 ± 5**	8.4 ± 0.4	91.2 ± 2.5
	70	4.4 ± 0.1**, ##	390 ± 11	8.3 ± 0.4	90.5 ± 2.4#
CL-3-803 + yeast-oil (T5)	17.5	4.7 ± 0.2	410 ± 3	8.5 ± 0.4	91.4 ± 2.2
	35	4.9 ± 0.2	390 ± 4	8.4 ± 0.5	90.7 ± 2.6#
	70	4.3 ± 0.2**, ##	400 ± 4	8.5 ± 0.4	91.1 ± 2.2

* P < 0.05, ** P < 0.01, *** P < 0.001 compared to Basal Feed with no color agent (T2).

^# P < 0.05, ^## P < 0.01, ^### P < 0.001 compared to Commercial Diet (T1).

Daily monitoring of eggs in each experimental group revealed that the yolk color of eggs from hens fed the commercial diet (T1) rapidly increased from 5.7 seen with basal feed (T2), reaching a score of 9.8 after the first week (Fig. 4). Yolk color was only increased in pellet feed supplemented with 17.5 mg carotenoid/kg feed presented as the carotenoid extract-oil. In contrast to commercial feed, the yolk color score was observed to gradually increase in the T3 group, rising to a yolk color score of 7.2 at the end of the third week. Increasing the carotenoid content to 35 and 70 mg/kg feed resulted in increased yolk color scores in eggs from hens given feed with each type of supplement. The carotenoid extract-oil was substantially more effective in promoting yolk coloration, compared to broken or intact yeast. The carotenoid extract-oil supplement improved the yolk color score to 9.0 and 10.2 with 35 and 70 mg carotenoid/kg feed, respectively. The color score in the T3 group at 70 mg carotenoid/kg feed was similar to that in the T1 commercial feed group. Results from Trial 2 for groups T1, T2, and T3 (35 mg carotenoid extract-oil/kg feed) are combined with the data from Trial 3.

Fig. 4.

Yolk color scores for eggs from hen provided feed containing yeast-based carotenoid preparations. (A) Yolk color scores for each group provided pellet feed were recorded daily, results are from 5 to 6 eggs for each treatment group. Marks for statistical analysis are color coded for each group. (B) Analysis of yolk color scores for eggs collected during the third week of each concentration tested, results are from at least 30 eggs for each treatment group. Results are the mean ± standard deviation. Statistical analysis employed one-way ANOVA with post-hoc Tukey test with differences relative to the basal feed group (CL-3-803 with no color agent added), * P < 0.05, ** P < 0.01, and *** P < 0.001.

Providing feed supplemented with broken yeast-oil or yeast-oil resulted in a statistically significant increase in the yolk color score. Eggs from hens fed the broken yeast-oil supplemented feed produced eggs with a yolk color score of 6.6 for both 35 and 70 mg lycopene/kg feed. Yolk color scores for eggs from hens fed the yeast-oil supplement were similar to broken yeast-oil preparation at 6.7 and 6.9 for 35 and 70 mg carotenoids/kg feed, respectively (Fig. 4).

Visual examination of eggs from the T3 group at 70 mg carotenoids/kg feed revealed red-orange coloration of egg yolk that is comparable to the T1 commercial feed group, consistent with the yolk color score (Fig. 5A). The effect of cooking lycopene-fortified eggs was examined using the preparation of soft-boiled eggs. Eggs were boiled for 8 min and then placed in room temperature water to cool. The coloration of boiled eggs was consistent with that observed for fresh eggs. Boiled eggs from hens provided feed supplemented with Carophyll Yellow and Carophyll Red (T1) had a similar appearance as eggs from the T3 group produced using feed containing 70 mg carotenoids/kg feed (Fig. 5B). An informal taste testing of eggs with ten volunteers indicated that lycopene fortification did not introduce additional flavors to the eggs.

Fig. 5.

Visible color enhancement of egg yolk from hens feed yeast-based carotenoid preparations. (A) Photographs of egg yolks on the final day of the feeding trial for each concentration of carotenoid supplement. (B) Soft boiled eggs from each group on the final day from the 70 mg carotenoid/kg feed groups compared to eggs from hens fed commercial feed (CL-3-802) or basal feed without color agent added (CL-3-803).

Overall, these finding indicate that the carotenoid extract-oil exhibits enhanced bioavailability compared to carotenoids provided as intact or broken yeast. The enhanced bioavailability was not simply due to the presence of oil in the feed mixture.

Lycopene fortification of egg yolk

HPLC analysis of freeze-dried egg yolk revealed the presence of lycopene in egg yolk from hens provided pellet feed supplemented with carotenoid extract-oil (T3). As expected, lycopene was not detected in egg yolk from the T1 or T2 groups. The yolk color was increased in eggs from hens given feed supplemented with broken yeast-oil (T4) or yeast-oil (T5); however, lycopene was not detected in yolk from these groups. Lycopene content of yolk from the T4 and T5 groups may be below the limit of detection of the analysis. In addition, beta-carotene was not detected in yolk from any of the test groups (Table 5).

Table 5.

Carotenoid content of egg yolk (µg carotenoid/g dry yolk).

	Carotenoid supplement (mg/kg feed)	Lycopene (µg/g yolk)	β-Carotene (µg/g yolk)
CL-3-802 Commercial diet (T1)	-	ND	ND
CL-3-803 Basal feed (T2)	-	ND	ND
CL-3-803 + carotenoid extract-oil (T3)	17.5	17.4 ± 0.2	ND
	35	23.6 ± 0.4	ND
	70	32.5 ± 0.5	ND
CL-3-803 + broken yeast-oil (T4)	17.5	ND	ND
	35	ND	ND
	70	ND	ND
CL-3-803 + yeast-oil (T5)	17.5	ND	ND
	35	ND	ND
	70	ND	ND

Not Detected (ND), below the limit of detection (LOD). The carotenoid content of the supplement is listed for groups T3, T4, and T5. (-) Indicates no carotenoid supplement added. LOD and LOQ values are as follows: Lycopene LOD, 5.0 µg/g yolk; lycopene LOQ, 15.1 µg/g yolk; beta-carotene LOD 3.4 µg/g yolk; and beta-carotene LOQ 10.3 µg/g yolk.

Discussion

Carotenoids and yolk color

Yolk color is an important component of consumer acceptance and is often associated with the nutritive value of eggs. Typically, deep yellow to orange-yellow yolks are preferred by consumers, and the presence of carotenoids is a major determinant for egg yolk coloration^20,50. Canthaxanthin and β-apo-8’-carotenoic acid ethyl ester (apo-ester), produced through chemical synthesis, are the most commonly used color agents in poultry feed for pigmentation of egg yolk^51,52. Although capable of enhancing yolk coloration, canthaxanthin and apo-ester do not appear to provide nutritional benefits to egg consumers^53,54.

There is increasing interest in the use of natural dyes as color agents that also provide added health benefits in both human foods and animal feed^55,56. The carotenoid lycopene has commercial appeal due to its vibrant red color⁵⁷. In addition, lycopene has the highest antioxidant potential among carotenoids^11,58 and health benefits in humans have been associated with the consumption of lycopene^59–61.

The ability to fortify egg yolk with lycopene provides the opportunity to produce functional foods with enhanced nutrient composition⁶². Lycopene present in egg yolk exhibits greater bioavailability compared to other sources, such as fresh tomatoes or tomato paste, making it a good choice as a carrier of this carotenoid for human consumption⁶³. These properties suggest that the production of lycopene-fortified eggs may have utility in promoting improved health of consumers.

In the context of animal well-being, lycopene has been extensively studied in poultry, with positive effects observed in reducing abdominal fat accumulation and blood lipid levels in chickens⁶⁴. The addition of carotenoids to poultry diets has also shown benefits in improving health parameters in heat-stressed chickens⁶⁵. In contrast to beta-carotene, lutein, and zeaxanthin which are naturally present in major poultry feed ingredients, lycopene must be supplemented⁶⁶.

Comparative efficacy of lycopene preparations

Although lycopene supplements are not typically utilized as a color agent in poultry feed, this carotenoid can accumulate in egg yolk to enhance pigmentation. However, the low bioavailability of lycopene from dietary sources, such as fresh tomato or tomato paste, can limit the ability to fortify egg yolk with high concentrations of lycopene^67,68. Reports on the effectiveness of lycopene supplementation to enhance yolk color vary, although an increase in the yolk color score of two to three points has been documented^69–71. In these reports, the lycopene content of egg yolk ranged from 0.3 to 4 µg/g yolk with 20 to 257 mg lycopene/kg feed, with the source of lycopene appearing to have a significant impact on its accumulation in yolk^69–72.

In this study, improved yolk color was observed in eggs from hens given feed supplemented with intact yeast containing carotenoids, provided in mash feed, at concentrations of 45 mg carotenoids/kg feed or higher. A yolk color score of almost eight was observed in eggs from hens fed mash feed supplemented with 190 mg carotenoids/kg feed, provided as whole yeast. This was an increase of two points compared to eggs from hens fed a basal feed without color agents added.

Preparations of pellet feed supplemented with either intact or broken yeast suspension in soybean oil enhanced yolk color to a similar level as intact carotenoid yeast in the mash feed. These feed preparations produced only a 1.0 to 1.2 point increase in the yolk color on the DSM color fan. In contrast, the use of a carotenoid extract-oil supplement in pellet feed was more efficient at enhancing yolk coloration. Yolk color was increased 4.5 points, to a final score of 10.2 on the color fan in eggs from hens given feed containing 70 mg carotenoids/kg feed (56 mg lycopene/kg feed and 14 mg beta-carotene/kg feed) provided as the carotenoid extract-oil mixture, the highest concentration tested. The coloration of eggs produced with feed containing the carotenoid extract-oil mixture at 70 mg carotenoids/kg feed was equivalent that seen with the commercial diet containing Carophyll Red (4.5 mg/kg feed) and Carophyll Yellow (1.5 mg/kg feed). These results demonstrate the high bioavailability of the carotenoid extract-oil preparation.

Feed formulation considerations

The use of whole yeast in mash feed as a lycopene source could enhance yolk pigmentation. However, several problems were identified when using mash feed that may limit successful fortification of the feed. The yeast powder did not bind well with the mash feed making uniform feeding difficult. Hens also appeared to select larger pieces, such as corn fragments, further limiting consumption of the powder or granular components. Based on these results, it appeared that the use of mash feed was not optimal. In addition, carotenoids provided as dried yeast in the mash feed appear to have limited bioavailability, reducing their ability to promote yolk pigmentation.

To overcome the concerns with the use of mash feed, a pellet feed was utilized. Pellet feed has the advantage of being easier to handle than mash feed and less food is wasted with pellets. In addition, feeding can be more consistent since hens are unable to pick specific components for consumption. Using an electric oil sprayer, it was possible to prepare pellet feed supplemented with carotenoid extracts prepared from yeast as well as intact and broken yeast cells suspended in soybean oil as a feed-compatible solvent.

Bioavailability mechanisms

Our preparations of carotenoids, either as intact yeast or carotenoid-enriched yeast extracts, exhibited distinct bioavailability as a component of poultry feed. Release of lycopene, capable of being incorporated into egg yolk, was limited in feed supplemented with yeast or yeast-oil suspensions, even with the cell wall ruptured. An important factor limiting the bioavailability of carotenoids from plant sources is their localization within chloroplasts and the presence of the plant cell wall, which are resistant to digestion^70,73.

Lycopene in yeast cells appears to be similarly contained in digestion-resistant structures, limiting release from the yeast present in the feed matrix. The cell wall of baker’s yeast provides physical protection against the environment and is composed of a highly cross-linked mannoprotein outer layer that stabilizes the underlying beta-glucan network⁷⁴. The beta-glucan and mannoproteins can act as adsorbents⁷⁵, and lycopene may bind to the cell wall components through hydrophobic interactions, preventing release into the digestive system. This adsorbent effect of the yeast cell wall would be expected to persist even in lysed yeast. The properties of the cell wall proteins may have a major impact on limiting the bioavailability of lycopene in intact and broken yeast cells.

In contrast to the effect of yeast cell wall components, the presence of dietary oil is expected to enhance the bioavailability of lycopene and other carotenoids. Inclusion of oil with lycopene from food sources, such as tomato products, is known to enhance carotenoid bioavailability⁷⁶. Absorption of lycopene and beta-carotene into intestinal cells is dependent on the incorporation into micelles, formed from bile salts and dietary fats. The dietary fat appears to function both to enhance the production of bile salts as well as to sequester lycopene⁷⁷. The inclusion of oil with the yeast carotenoid extracts provides a ready source of fat for micelle assembly, enhancing the incorporation of lyophilic carotenoids.

The carotenoid extract-oil containing feed formulations exhibited high bioavailability compared to previous reports and relative to oil suspension of whole or broken carotenoid-producing yeast. Inclusion of oil with the whole or broken yeast was not sufficient to overcome the limited bioavailability, likely due to the presence of yeast cell wall components. The extraction process utilized to isolate carotenoids from engineered yeast would be expected to disrupt cellular components that limit lycopene bioavailability. Removal of the cell wall proteins may be a key factor in producing lycopene with high bioavailability.

Fortification of egg yolk with lycopene

The content of beta-carotene in egg yolk was below the limit of detection with all concentrations of carotenoid extract-oil supplement, indicating that in our feeding trials, enhanced yolk coloration was primarily due to lycopene accumulation. It is possible that the beta-carotene supplement was metabolized by hens, preventing its incorporation into egg yolk. Lycopene fortification at 32.5 µg/g yolk was found with feed containing 70 mg carotenoids/kg feed using the carotenoid extract-oil mixture. This level of lycopene fortification was significantly improved compared to previous reports, approximately eight-fold higher than 4 µg/g reported by Olson et al. in which lycopene powder in a gelatin carrier was used⁷¹. The average dry yolk weight was 14 g; thus the lycopene content of each egg from hens fed the yeast extract-oil supplement could reach 450 µg.

Differences in lycopene isomeric composition and egg yolk matrix effects may have contributed to an underestimation of total yolk lycopene content. The lycopene cis-isomer predominantly accumulates in egg yolk, and while extraction of the cis-isomer is typically more efficient compared to all-trans lycopene⁷⁸, matrix effects from the egg yolk can limit lycopene extraction. The high fat content of the egg yolk can saturate organic solvents, limiting the extraction of carotenoids. A saponification step³³ was used to modify the fats, reducing their hydrophobic character, to facilitate the extraction of lycopene or other carotenoids from egg yolk. An additional factor that could limit lycopene quantitation is the distinct absorption intensity between lycopene isomers. Cis-lycopene exhibits a weaker absorption intensity compared to all-trans lycopene. Our analysis utilized all-trans lycopene standards, and this may lead to an underestimation of lycopene content in extracts from egg yolk. Therefore, the lycopene concentrations observed in this study (17.4–32.5 µg/g yolk) likely represent minimum estimates of total lycopene deposition rather than absolute values.

Although the fat in the egg yolk may lead to difficulties in lycopene determination, the presence of lycopene in the fat-rich matrix of the egg yolk has high bioavailability in humans⁷⁹. The potential of high bioavailable lycopene makes fortified eggs a desirable functional food with health-promoting properties.

Limitations and future considerations

A limitation of this study is the relatively small number of hens included in each feeding trial. Several studies examining changes in yolk pigmentation have successfully utilized similar population sizes of laying hens^69,80,81. Six to eight hens were used in each of our test groups, providing at least 30 samples per group for measurement of egg parameters. Power analysis of each of the egg parameters using the biologically relevant effect size, the minimum difference between two groups that would be of biological interest, indicates that the egg sample size was sufficient to provide statistical confidence in the results presented. Although a larger study may be beneficial to confirm the effects of the yeast-derived lycopene oil as a feed supplement in an industrial setting.

Analysis of the scalability of the extraction of lycopene from yeast from lab scale to industrially relevant quantities for feed supplements would also require additional studies. While the extraction method employed in this study was sufficient for lab-scale production of carotenoid-oil extracts, modifications are likely necessary for industrial utilization. The potential for variability in carotenoid content from each batch would be another factor that would need to be resolved to allow the preparation of carotenoid extract-oil at industrial levels.

Regulatory frameworks such as the EFSA in Europe or the FDA in the USA classify a concentration of 60 mg lycopene/kg feed as high for poultry. To market the fortified feed or resulting lycopene-enriched eggs, regulatory approval would be required. Previous studies indicate that lycopene at 60 mg/kg feed is generally safe; however, this concentration would be considered a pharmacological dose designed to change the composition of eggs. Typically, the health of hen would be followed for 90 days to assess changes in kidney, liver, and metabolic function. In addition, documentation of yolk deposition analysis and stability of lycopene in feed formulations would be required to ensure hens were not adversely affected by the supplement⁸².

Validation of lycopene fortified eggs as a functional food in humans would require more rigorous testing. Analysis of the bioavailability of lycopene following consumption of fortified eggs would require monitoring of lycopene in the blood of participants. In addition, efficacy studies to evaluate the ability of lycopene in fortified eggs to lower markers of oxidative stress would also be important. A key aspect of the validation process would be long-term safety and toxicology assessments. The impact of ingestion of lycopene fortified eggs on lipid profile, liver and kidney function, as well as allergenicity, would need to be considered. Finally, sensory analysis of lycopene fortified eggs for off-flavors, changes in texture, aroma, flavor intensity, and aftertaste would be a critical evaluation to judge consumer acceptance prior to attempts to market the product^83,84. In this study, the enhanced yolk coloration in eggs from hens fed carotenoid-oil extract-supplemented feed was found to be stable in soft-boiled eggs. However, other cooking techniques were not evaluated during this study, and future analysis would be required to examine lycopene stability in fried, scrambled, poached, and baked eggs. Preliminary sensory testing of soft-boiled eggs with ten volunteers indicated that eggs fortified with lycopene from hens fed the carotenoid extract-oil mixture had a similar flavor, appearance, and aroma compared to eggs produced using the commercial diet. More detailed sensory testing with a larger group would be required prior to potential marketing of a lycopene-fortified egg product.

Conclusions

In combination with the oil-based delivery, the feed formulation used in the current study provided a source of lycopene with high bioavailability. To our knowledge, lycopene fortification at 32.5 µg/g yolk is the highest value reported for feed supplemented with lycopene-enriched extracts. Importantly, the carotenoid preparations did not adversely affect egg quality or the health and productivity of laying hens. Overall, our findings demonstrate that the new formulation of yeast-derived lycopene oil has the potential for use as an alternative color agent for yolk pigmentation with the added health-promoting benefits associated with lycopene fortification.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization, C.C., M.W., P.S., L.T.J. and A.N.J.; Methodology, C.C., M.W., P.J., P.S., L.T.J., A.N.J.; Formal Analysis, C.C., M.W., P.J., M.L., L.T.J.; Investigation, C.C., M.L., P.S., and L.T.J.; Writing – Original Draft Preparation, C.C. and L.T.J.; Writing – Review & Editing, C.C. and L.T.J.; Visualization, C.C., M.W., P.S., and L.T.J.; Supervision, L.T.J., A.N.J., M.W., and P.S.; Project Administration, L.T.J., M.W., and P.S.; Funding Acquisition, L.T.J., and A.N.J.

Funding

This research project received funding support from Mahidol University under the New Discovery and Frontier Research Grant: NDFR 15/2564 (LTJ); NDFR 05/2563 (ANJ); National Research Council of Thailand (NRCT): N42A670552 (LTJ); Thailand Science Research and Innovation (TSRI): RSA6280058 (ANJ); The National Science, Research and Innovation Fund (NSRF) via the Program Management Unit for Human Resources and Institutional Development, Research and Innovation: grant number B38G680002; The CIF and CNI Grant, Faculty of Science, Mahidol University (LTJ); and the Faculty of Science, Mahidol University. Charoen Pokphand Foods Public Company Limited (CPF) provided support and facilities for experiments using laying hens.

Data availability

Data is available from the corresponding author Laran Jensen (email: laran.jen@mahidol.ac.th) upon request.

Declarations

Competing interests

The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. A patent is pending for L.T.J. as follows: The process of preparing carotenoids for use as ingredients in animal feed. Thai Patent 2501000135, filed January 01, 2025. Patent pending. All other authors have no conflicts of interest.