JP7848040B2 - Method for improving the quality of Silkie chicken eggs, Silkie chicken eggs with enhanced antioxidant activity, Silkie chicken feed, and method for producing the same. - Google Patents

Description

Article 30, Paragraph 2 of the Patent Act applies July 25, 2021 J-STAGE homepage https://www. jstage. jst. go. jp/article/jpsa/advpub/0/advpub_0210032/_article/-char/ja/

This invention relates to a method for improving the quality of Silkie chicken eggs and to Silkie chicken eggs with enhanced antioxidant activity.

Laying hens often have supplements containing carotenoids, such as paprika and marigold, added to their feed to differentiate the color of their egg yolks. Lutein, zeaxanthin, and astaxanthin, found in these supplements, are carotenoids that have recently attracted attention not only for their coloring effect on egg yolks but also for their antioxidant properties.

On the other hand, the Silkie chicken is believed to have been introduced to Japan from China in the early Edo period. According to the Chinese herbal medicine text "Bencao Gangmu," the eggs and meat of the Silkie chicken are described as having medicinal properties. In fact, in China and Korea, the Silkie chicken has long been highly valued as an ingredient in medicinal cuisine.

The applicant in this case, the Tokyo Metropolitan Agriculture, Forestry and Fisheries Promotion Foundation, has been working on improving Silkie chickens for some time, and has created a high-egg-laying Silkie chicken called "Tokyo Ukokkei," which increases the egg-laying rate from the usual 50 to 80 eggs per year to approximately 200 eggs per year.

Furthermore, the applicant, the Tokyo Metropolitan Agriculture, Forestry and Fisheries Promotion Foundation, has revealed that the metabolism of Silkie chickens differs from that of ordinary chickens (see Non-Patent Document 1). In addition, they hold a patent for a method of suppressing the production of lipid peroxides in the meat of Silkie chickens by feeding them Angelica keiskei (see Patent Document 1).

Kojima S, et al., Food Science and Technology Research, 20: 621-628. 2014.

Patent No. 6124400

As mentioned above, it is well known that dietary carotenoids improve egg yolk color, but their effects on other egg qualities (e.g., antioxidant function) have not been sufficiently investigated.

Furthermore, the effect of dietary carotenoids on egg quality is unknown in Silkie chickens, which have a different metabolism compared to ordinary chickens. To further expand the use of Silkie chicken eggs, there was a need to develop Silkie chicken eggs with even higher added value.

This invention aims to provide a method for improving the egg quality of Silkie chickens. Furthermore, this invention aims to provide Silkie chicken eggs with enhanced antioxidant activity.

The inventors of this invention conducted extensive research to solve the above problems and, as a result, surprisingly discovered that oral administration of carotenoids to Silkie chickens resulted in the transfer of more carotenoids (particularly lutein and zeaxanthin) into Silkie chicken eggs compared to ordinary chicken eggs. Furthermore, it was found that oral administration of carotenoids enhanced the antioxidant activity of Silkie chicken eggs compared to ordinary chicken eggs. Based on these findings, the inventors completed the present invention.

In other words, the present invention provides a method for improving the quality of Silkie chicken eggs, characterized by orally feeding Silkie chickens a feed containing 8.5 mg/kg or more of paprika-derived carotenoids and/or 24.4 mg/kg or more of marigold petal-derived carotenoids for at least 7 days.

Here, "quality improvement" includes increasing the carotenoid content of egg yolks and/or enhancing the antioxidant activity (particularly singlet oxygen scavenging activity) of egg yolks.

Therefore, the method for improving the quality of Silkie chicken eggs according to the present invention may also be a method for increasing the carotenoid content of Silkie chicken eggs. Furthermore, the method for improving the quality of Silkie chicken eggs according to the present invention may also be a method for increasing the antioxidant activity (particularly singlet oxygen scavenging activity) of Silkie chicken eggs.

According to the method for enhancing the carotenoid content of Silkie chicken eggs, it is possible to obtain Silkie chicken eggs in which the zeaxanthin content in the yolk is 1 mg/100g or more, and/or the lutein content is 4 mg/100g or more.

According to the method for enhancing the antioxidant activity of Silkie chicken eggs, it is possible to obtain Silkie chicken eggs with yolk antioxidant activity more than twice as high as that of regular chicken eggs.

Also, the Silkie chicken can be called "Tokyo Silkie."

This invention provides Silkie chicken eggs with enhanced antioxidant activity, characterized in that the egg yolk has more than twice the antioxidant activity of ordinary chicken eggs.

Here, "antioxidant activity" may also refer to singlet oxygen scavenging activity.

The Silkie chicken eggs may further contain zeaxanthin at a concentration of 1 mg/100g of egg yolk and/or lutein at a concentration of 4 mg/100g of egg yolk.

This invention provides a feed for Silkie chickens, characterized by containing 8.5 mg/kg or more of carotenoids derived from paprika and/or 24.4 mg/kg or more of carotenoids derived from marigold petals.

The present invention also provides a method for producing feed for Silkie chickens, characterized by incorporating 8.5 mg/kg or more of paprika-derived carotenoids and/or 24.4 mg/kg or more of marigold petal-derived carotenoids into the feed.

Here, the feed for Silkie chickens may also be intended to enhance the antioxidant activity of the egg yolk.

According to the present invention, a method for improving the quality of Silkie chicken eggs can be provided. Specifically, by feeding Silkie chickens a diet containing a predetermined proportion of carotenoids derived from paprika and/or marigold petals, carotenoids (especially lutein and zeaxanthin) can be efficiently transferred to the eggs compared to conventional chickens. The antioxidant function of the carotenoids accumulated in the eggs in this way makes it possible to obtain Silkie chicken eggs with more than twice the antioxidant activity (especially singlet oxygen scavenging activity) compared to conventional chicken eggs.

According to this invention, it is possible to provide Silkie chicken eggs with enhanced antioxidant activity. Ingesting Silkie chicken eggs with high antioxidant function is expected to suppress lipid oxidation in the body and reduce the production of lipid peroxides, which have adverse health effects. Furthermore, due to their high antioxidant function, they are expected to be effective in maintaining or improving health by eliminating reactive oxygen species, which have been linked to cancer and accelerated aging.

This graph shows the total carotenoid content in egg yolks (Test Example 1). Data are expressed as mean ± SE (n=3). There is a significant difference between different signs within the same graph (P<0.05) ^{in a-c} . Cont = basal feed; PA = basal feed with 30 mg/kg of paprika-derived carotenoids added; MA = basal feed with 60 mg/kg of marigold petal-derived carotenoids added; AX = basal feed with 28 mg/kg of Paracoccus cell powder-derived carotenoids added. RR = Rhode Island Red; SF = Silkie chicken. This graph shows the content of (A) zeaxanthin, (B) lutein, and (C) astaxanthin in egg yolk (Test Example 1). Data are expressed as mean ± SE (n=5). There is a significant difference between different signs within the same graph (P<0.05) ^{for a-c} . Cont = basal feed; PA = basal feed with 30 mg/kg of paprika-derived carotenoids added; MA = basal feed with 60 mg/kg of marigold petal-derived carotenoids added; AX = basal feed with 28 mg/kg of Paracoccus cell powder-derived carotenoids added. RR = Rhode Island Red; SF = Silkie chicken. This graph shows the singlet oxygen scavenging activity (50% inhibitory concentration; _IC50 ) by egg yolk (Test Example 1). Data are expressed as mean ± SE (n=3). There is a significant difference between different signs within the same graph (P<0.05) ^{in a-c} . Cont = basal feed; PA = basal feed with 30 mg/kg of paprika-derived carotenoids added; MA = basal feed with 60 mg/kg of marigold petal-derived carotenoids added; AX = basal feed with 28 mg/kg of Paracoccus cell powder-derived carotenoids added. RR = Rhode Island Red; SF = Silkie chicken. This graph shows the change in egg yolk color over time for (A) Rhode Island Red and (B) Silkie chickens (Test Example 1). The data is expressed as the mean value (n=12). Cont = base feed; PA = base feed with 30 mg/kg of carotenoids derived from paprika extract added; MA = base feed with 60 mg/kg of carotenoids derived from marigold petal extract added; AX = base feed with 28 mg/kg of carotenoids derived from Paracoccus cell powder added. This graph shows the content of (A) zeaxanthin, (B) lutein, and (C) astaxanthin in egg yolk (Test Example 2). Data are expressed as mean ± SE (n=4). There is a significant difference between different signs within the same graph ( ^a-c ) (P<0.05). RR = Rhode Island Red; SF = Silkie chicken. Cont = Basic feed; PA = 60 mg/kg of paprika-derived carotenoids added; MA = 120 mg/kg of marigold petal-derived carotenoids added; AX = 56 mg/kg of Paracoccus cell powder-derived carotenoids added. This graph shows the singlet oxygen scavenging activity (50% inhibitory concentration; _IC50 ) by egg yolk (Test Example 2). Data are expressed as mean ± SE (n=3). There is a significant difference between the different signs within the same graph (P<0.05). ^RR = Rhode Island Red; SF = Silkie chicken. Cont = base feed; PA = 60 mg/kg of paprika-derived carotenoids added; MA = 120 mg/kg of marigold petal-derived carotenoids added; AX = 56 mg/kg of Paracoccus cell powder-derived carotenoids added. This graph shows the content of (A) lutein, (B) zeaxanthin, and (C) astaxanthin in egg yolk (Test Example 3). Data are expressed as mean ± SE (n=5). There is a significant difference between the same graphs with different signs ( ^a-c ) (P<0.05). RR = Rhode Island Red; SF = Silkie chicken. Cont = basal feed; MA+PA = basal feed with 30 mg/kg of carotenoids derived from marigold petals and 15 mg/kg of carotenoids derived from paprika added; MA+AX = basal feed with 30 mg/kg of carotenoids derived from marigold petals and 14 mg/kg of carotenoids derived from Paracoccus bacteria powder added. This graph shows the singlet oxygen scavenging activity (50% inhibitory concentration; _IC50 ) by egg yolk (Test Example 3). Data are expressed as mean ± SE (n=3). There is a significant difference between the same graph with different signs in ^a-c (P<0.05). RR = Rhode Island Red; SF = Silkie chicken. Cont = basal feed; MA+PA = basal feed with 30 mg/kg of carotenoids derived from marigold petals and 15 mg/kg of carotenoids derived from paprika added; MA+AX = basal feed with 30 mg/kg of carotenoids derived from marigold petals and 14 mg/kg of carotenoids derived from Paracoccus bacteria powder added.

The following describes this embodiment in detail.

The method for improving the quality of Silkie chicken eggs according to this embodiment is characterized by orally administering carotenoids derived from paprika and/or carotenoids derived from marigold petals to Silkie chickens for at least 7 days.

The Silkie chicken is a breed of chicken. However, unlike common chickens, Silkies have distinctive physical characteristics such as an extra toe and downy feathers. Furthermore, their skin, meat, and even their bones are black.

Specifically, an example of a Silkie chicken is the "Tokyo Ukokkei," a high-egg-laying breed developed by the Tokyo Metropolitan Agriculture, Forestry and Fisheries Promotion Foundation, the applicant in this case. This "Tokyo Ukokkei" is a breed with superior egg-laying ability.

Silkie chickens used are egg-laying hens. Generally, egg-laying hens reach sexual maturity and begin laying eggs around 5 months after hatching (5 months of age), and are used for egg production for about 1 to 1.5 years. Therefore, egg-laying hens that are 5 months to 2 years old are preferably used.

"Paprika-derived carotenoids" refers to carotenoids contained in the fruit of paprika, a plant belonging to the nightshade family. The form in which carotenoids are administered is not particularly limited, as long as it does not pose a problem for feeding to Silkie chickens. It may also be in the form of dried and ground paprika powder or paprika extract.

"Paprika extract" refers to a product obtained by extracting carotenoids, including zeaxanthin, from paprika fruit, and the extraction method is not limited. Furthermore, paprika extract may also be a paprika extract product obtained by saponification after extracting carotenoids with an organic solvent.

Furthermore, powdered compound feed prepared by mixing paprika powder or paprika extract with multiple feed ingredients can also be used as "paprika-derived carotenoids." Specific examples of such compound feeds include RunRed (registered trademark) (Elanco Japan Co., Ltd.), ColorUp (Kokin Chemical Co., Ltd.), ChiliRed 10, Pixafil Red HY-C15 (Yamato Chemical Co., Ltd.), and CapsaRed (Asuka Animal Health Co., Ltd.).

"Carotenoids derived from marigold petals" refers to carotenoids contained in the petals of marigolds, a plant belonging to the Asteraceae family. The form in which carotenoids are administered is not particularly limited, as long as it does not pose any problems for feeding to Silkie chickens. It may be in the form of dried and crushed marigold petal powder or marigold petal extract.

"Marigold petal extract" refers to a product obtained by extracting carotenoids, including lutein, from marigold petals, and the extraction method is not limited. Furthermore, the marigold petal extract may also be a marigold petal extract obtained by saponification after extracting carotenoids with an organic solvent.

Furthermore, powdered compound feed prepared by mixing marigold petal powder or marigold petal extract with multiple feed ingredients can also be used as a "carotenoid derived from marigold petals." Specific examples of such compound feeds include Xan Yellow (registered trademark) (Elanco Japan Co., Ltd.), Color Up Yellow S (Kokin Chemical Co., Ltd.), and Dynefil S20 (Yamato Chemical Co., Ltd.). Zeaxanthin may also be present in marigold petal extract.

Typically, carotenoids derived from paprika and/or marigold petals are added to and mixed into the basal feed and administered orally. There are no special restrictions on the basal feed; any feed commonly used for laying hens is acceptable, and commercially available compound feeds can be used as desired.

The amount of paprika-derived carotenoids in the basal feed can be 8.5 mg/kg or more, preferably 8.5 to 90 mg/kg, more preferably 15 to 60 mg/kg, and particularly preferably 30 to 60 mg/kg, as total carotenoids. Alternatively, the amount of paprika-derived carotenoids in the basal feed can be 0.714 mg/kg or more, preferably 0.714 to 7.56 mg/kg, more preferably 1.26 to 5.04 mg/kg, and particularly preferably 2.52 to 5.04 mg/kg, as zeaxanthin.

The amount of carotenoids derived from marigold petals in the basal feed can be 24.4 mg/kg or more, preferably 24.4 to 160 mg/kg, more preferably 30 to 120 mg/kg, and particularly preferably 60 to 120 mg/kg, as total carotenoids. Alternatively, the amount of marigold petal extract in the basal feed can be 14.52 mg/kg or more, preferably 14.52 to 95.2 mg/kg, more preferably 17.85 to 71.4 mg/kg, and particularly preferably 35.7 to 71.4 mg/kg, as lutein.

If the amount of paprika and/or marigold petal-derived carotenoids in the basal feed is below the above range, a sufficient improvement in egg quality cannot be obtained. On the other hand, if the amount of paprika and/or marigold petal-derived carotenoids in the basal feed exceeds the above range, not only is the advantage of Silkie chickens over general chickens lost, but the improvement in egg quality commensurate with the amount added is not obtained, making it uneconomical and therefore undesirable. Furthermore, paprika-derived carotenoids and marigold petal-derived carotenoids can be used in combination within the above-mentioned range.

The period for administering carotenoids derived from paprika and/or marigold petals can be at least 7 days, preferably 10 days or more, and more preferably 14 days or more. If the administration period is too short, sufficient improvement in egg quality may not be achieved.

The above-described method for improving the quality of Silkie chicken eggs can enhance the carotenoid content (particularly lutein and zeaxanthin) in the egg yolk, thereby providing Silkie chicken eggs with high antioxidant activity.

Therefore, according to this embodiment, a method for increasing the carotenoid content of Silkie chicken eggs is provided, characterized by orally administering paprika-derived carotenoids and/or marigold petal-derived carotenoids to Silkie chickens for at least 7 days.

According to the method for enhancing the carotenoid content of Silkie chicken eggs, it is possible to obtain Silkie chicken eggs in which the zeaxanthin content in the yolk is 1 mg/100g or more, preferably 1.2 mg/100g or more of egg yolk, and/or the lutein content is 4 mg/100g or more, preferably 5 mg/100g or more of egg yolk. The carotenoid content can be measured by the method described in Example (Test Example 1).

Furthermore, this embodiment provides a method for enhancing the antioxidant activity of Silkie chicken eggs, characterized by orally administering paprika-derived carotenoids and/or marigold petal-derived carotenoids to Silkie chickens for at least seven days.

In this specification, "antioxidant activity" specifically refers to the scavenging activity against reactive oxygen species, preferably singlet oxygen scavenging activity.

According to the method for enhancing the antioxidant activity of Silkie chicken eggs, it is possible to obtain Silkie chicken eggs with yolk antioxidant activity more than twice as high as that of ordinary chicken eggs. The antioxidant activity (singlet oxygen scavenging activity) of the yolk can be measured by the method described in Example (Test Example 1).

The Silkie chicken eggs of this embodiment have an enhanced carotenoid content (particularly lutein and zeaxanthin), resulting in improved egg yolk color. Furthermore, because the antioxidant activity of these Silkie chicken eggs is enhanced, consuming them is expected to have functional benefits such as suppressing the production of lipid peroxides in the body and eliminating reactive oxygen species.

Furthermore, according to this embodiment, a feed for Silkie chickens is provided that contains the carotenoids derived from paprika and/or the carotenoids derived from marigold petals in the aforementioned amounts. This feed for Silkie chickens can be produced by blending the carotenoids derived from paprika and/or the carotenoids derived from marigold petals into the feed in the aforementioned amounts. Here, "feed" refers to the same as the basic feed described above.

As mentioned above, orally administering this feed for Silkie chickens can enhance the carotenoid content of the egg yolk, thereby increasing the antioxidant activity of the egg yolk, particularly its singlet oxygen scavenging activity. Therefore, this feed for Silkie chickens can be used to improve the quality of Silkie chicken eggs, to increase the carotenoid content in the egg yolk, or to enhance the antioxidant activity of the egg yolk.

This embodiment will be specifically described below with reference to examples and comparative examples.

(Example Test 1)
The effects of dietary carotenoids on egg yolk were investigated using laying hens. Forty 60-week-old Rhode Island Red chickens and 40 Silkie chickens ("Tokyo Ukokkei") were randomly divided into four groups, with five chickens in each group for two replicates. Each chicken was kept individually in an open-air cage for 28 days, and water and basal feed were freely available.

- Control group (Cont): Only commercially available chicken feed (crude protein 17%, metabolizable energy 2800 kcal/kg, JA Higashi Nippon Kumiai Feed Co., Ltd.) was given as the basal feed.
- Paprika extract group (PA): The basal feed was supplemented with a mixed feed containing powdered paprika extract (Runred®, Elanco Japan Co., Ltd.; carotenoid content: 5g/kg). The amount added was set to 30mg/kg of total carotenoids in the feed (2.5mg/kg of zeaxanthin).
- Marigold petal extract group (MA): The basal feed was supplemented with a mixed feed containing powdered marigold petal extract (Xan Yellow®, Elanco Japan Co., Ltd.; carotenoid content: 20 g/kg). The amount added was set to 60 mg/kg of total carotenoids in the feed (36 mg/kg of lutein).
- Paracoccus cell powder (AX): The basal feed was supplemented with a powdered astaxanthin-containing mixed feed (Panaferd®-P, ENEOS Corporation; carotenoid content: 35 g/kg) produced using Paracoccus bacteria. The amount added was set to 28 mg/kg of total carotenoids in the feed (16 mg/kg of astaxanthin).

Egg yolk, total carotenoid content in the yolk, and singlet oxygen scavenging activity were measured using eggs collected 22 to 28 days (4 weeks) after the start of the study.

(Method for measuring egg yolk)
Egg yolk color was measured using a spectrophotometer (CM-508b; Konica Minolta, Inc.), and the lightness (L ^* value), redness (a ^* value), yellowness (b ^* value), and the ratio of redness to yellowness (a/b) in the CIELAB system were calculated. Egg yolk color was also measured using the Roche York Color Fan (RYCF) score.

(Method for measuring total carotenoid content and singlet oxygen scavenging activity)
For the quantification of total carotenoids in egg yolk, eggs laid after the completion of a 4-week study were used. After weighing the egg yolk, 15 mL of acetone was added to the yolk and stirred at room temperature (26 ± 2°C) for 15 minutes. The supernatant was collected, and 30 mL of dichloromethane-methanol (2:1, v/v) was added and the mixture was stirred at room temperature for 30 minutes to extract the carotenoids (this process was repeated three times). 100 mL of each extract was mixed with 100 mL of hexane and 100 mL of water, and the mixture was separated using a separatory funnel. The upper layer (yellow) was collected and dehydrated with anhydrous sodium sulfate, and the total carotenoid content was calculated using the optical density at λmax = 450–470 nm. For quantification, the following extinction coefficients and absorbances at 1% concentration were used: 2400 for the control group, 2072 for the PA group, 2500 for the MA group, and 2100 for the AX group (see Britton et al., Carotenoids Handbook, Birkhauser, 2004).

Next, to investigate the singlet oxygen scavenging activity, each extract obtained above was concentrated to dryness and subjected to silica gel column chromatography (Silica gel 60, Kanto Chemical Co., Ltd.; 10 mm i.d. × 150 mm; solvent: hexane). Development was performed stepwise using hexane (50 mL), hexane-acetone 1:1 (40 mL), and acetone (50 mL). The eluents containing carotenoids in hexane-acetone (Fraction I) and acetone (Fraction II) were collected and concentrated to obtain a yellow oil.

Singlet oxygen scavenging activity was measured by the photo-oxidation reaction of linoleic acid using methylene blue as a sensitizer (see Kobayashi M and Sakamoto Y, Biotechnology Letters, 21: 265-269, 1999). To evaluate the carotenoid extract from egg yolk, 80 μL of 0.025 mM methylene blue-ethanol solution, 100 μL of 240 mM linoleic acid-ethanol solution, and 140 μL of ethanol with or without 40 μL of extract (total carotenoid solutions of 50 μM, 200 μM, and 400 μM in dichloromethane, resulting in final total carotenoid concentrations of 5 μM, 20 μM, and 40 μM, respectively) were placed in microglass vials (5.0 mL). The vials were tightly sealed with screw caps and septums, and the mixtures were irradiated in a cardboard box at 7000 lux at 22°C for 3 hours.

Next, the amount of conjugated linoleic acid (CLA) produced was estimated by diluting 100 μL of the reaction mixture to 3.0 mL with ethanol and measuring the absorbance at 235 nm (see Hirayama et al., Lipids, 29: 149-150, 1994). The value for the case without carotenoid addition was determined, and the singlet oxygen scavenging activity was calculated based on this reference value. The activity was evaluated using the egg yolk concentration (μg/mL) that showed a 50% inhibitory effect on the production of conjugated linoleic acid from linoleic acid, and was expressed as the average of three experimental data points. A lower value indicates higher singlet oxygen scavenging activity.

(High-performance liquid chromatography (HPLC) analysis of carotenoids in egg yolk)
The zeaxanthin, lutein, and astaxanthin content was analyzed using egg yolks from eggs laid after a 4-week trial. The analysis of zeaxanthin and lutein was performed as follows: First, each egg yolk sample (0.5 g) was mixed with 3% pyrogallol-ethanol solution (10 mL), 1% sodium chloride solution (0.5 mL), and 60% potassium hydroxide solution (1.0 mL), and the oils in the sample were saponified by stirring at 70°C for 30 minutes. Next, this mixture was cooled, and 1% sodium chloride solution (22.5 mL) and ethyl acetate-hexane (1:9, v/v; 15 mL) were added, followed by shaking for 5 minutes.

The upper layer of the solution was collected, and 15 mL of ethyl acetate-hexane (1:9, v/v) was added to the lower layer. The mixture was shaken for 5 minutes, and the upper layer was collected. This procedure was repeated twice. The extracted upper layers were mixed and concentrated to dryness to obtain a yellow oil. This yellow oil was dissolved in 10 mL of ethanol and filtered through a Millipore filter disc (Merck) with a pore size of 0.45 μm. 10 μL of the obtained solution was subjected to HPLC (1260 Infinity II LC system, Agilent) equipped with a photodiode array detector (1260 diode array detector HS, Agilent) for analyzing the carotenoid content. An Inertsil® ODS-3V column (GL Sciences; 4.6 mm i.d. × 150 mm) was used, and the mixture was developed with 98% methanol at a flow rate of 0.5 mL/min at 40°C. Zeaxanthin and lutein were eluted with retention times (Rt) of 7.7 minutes and 6.8 minutes, respectively.

The astaxanthin analysis was performed as follows: 1.0 g of each egg yolk sample was dissolved in 1 mL of deionized water and 5 mL of acetone containing 1.2 mM butylated hydroxytoluene (BHT). 10 mL of acetone containing 1.2 mM BHT was added to the mixture, and after thorough stirring, the mixture was centrifuged at 3000 rpm for 10 minutes to collect the upper layer of the solution. Next, 5 mL of acetone containing 1.2 mM BHT was added to the lower layer and thoroughly stirred, and then 10 mL of acetone containing 1.2 mM BHT was added. The mixture was centrifuged at 3000 rpm for 10 minutes to collect the upper layer of the solution. The extracted upper layer was mixed, and 1.2 mM BHT-containing acetone was added to obtain 50 mL of solvent. This solvent was subjected to silica gel HPLC (COSMOSIL® Cholester, Nacalai Tesque; 4.6 mm i.d. × 250 mm) at 35°C. A mixed solvent of acetone, deionized water, and tetrahydrofuran (770:215:15, v/v) was used as the mobile phase, and the sample was developed at a flow rate of 1.0 mL/min.

Astaxanthin was eluted at Rt values of 13.9 min and 14.8 min. Carotenoid content was calculated using absorbance and calibration curves at 455 nm for zeaxanthin and lutein, and at 480 nm for astaxanthin.

(statistical analysis)
Results were expressed as mean ± SE. Two-way ANOVA was performed on total carotenoid content in egg yolk, individual carotenoid content, egg yolk, and singlet oxygen scavenging activity to analyze the interaction between breed and feed. Tukey's method was used for multiple comparisons. All statistical analyses were performed using R software (http://www.R-project.org/; Ihaka and Gentleman, 1996), with a significance level of P<0.05.

(result)
Table 1 shows the values for egg yolk. Feed had a significant effect on all items (P<0.001). Breed had a significant effect on L ^* , b ^* , and a/b values (P<0.001). A significant interaction was observed between breed and feed for ^a* and a/b values. Furthermore, the addition of carotenoids increased a ^* values and RYCF scores. In other words, the a ^* values and RYCF scores in the treatment group were significantly higher than in the control group (P<0.05). In addition, the L ^* and b ^* values of Silkie chickens were higher than those of Rhode Island Red chickens.

The data is expressed as mean ± SE (n=12).
^a-e: Significant difference between different signs in the same column (P<0.05), *: P<0.05, **: P<0.01, ***: P<0.001, NS: No significant difference.
L ^* = lightness; a ^* = redness; b ^* = yellowness; RYCF score = Roche York Color Fun score (1-15).
RR = Rhode Island Red; SF = Silkie chicken.
Cont = Basic feed; PA = Paprika extract; MA = Marigold petal extract; AX = Paracoccus cell powder.

Figure 1 is a graph showing the total carotenoid content in egg yolks. Data are expressed as mean ± SE (n = 3). There is a significant difference between ^a-c with different signs (P < 0.05). Cont = basal feed; PA = basal feed with 30 mg/kg of paprika-derived carotenoids added; MA = basal feed with 60 mg/kg of marigold petal-derived carotenoids added; AX = basal feed with 28 mg/kg of Paracoccus cell powder-derived carotenoids added. RR = Rhode Island Red; SF = Silkie chicken.

The total carotenoid content in egg yolks was also higher in Silkie chickens than in Rhode Island Red chickens. Specifically, in Silkie chickens, the PA, MA, and AX groups accumulated significantly more carotenoids than the control group (P<0.05), but no significant difference was observed between the different treatment groups in Rhode Island Red eggs (Figure 1). Furthermore, eggs from the MA group of Silkie chickens contained more carotenoids than eggs from the PA and AX groups of Rhode Island Red chickens, and eggs from both control groups (P<0.05; Figure 1).

Figure 2 is a graph showing the content of (A) zeaxanthin, (B) lutein, and (C) astaxanthin in egg yolk. Data are expressed as mean ± SE (n=5). There is a significant difference between ^a-c with different signs (P<0.05). Cont = basal feed; PA = basal feed with 30 mg/kg of paprika-derived carotenoids added; MA = basal feed with 60 mg/kg of marigold petal-derived carotenoids added; AX = basal feed with 28 mg/kg of Paracoccus cell powder-derived carotenoids added. RR = Rhode Island Red; SF = Silkie chicken.

The zeaxanthin, lutein, and astaxanthin content in egg yolks increased in the PA, MA, and AX groups of both breeds, respectively (Figure 2). Zeaxanthin and lutein content in the PA and MA groups were significantly higher in Silkie chickens than in Rhode Island Red chickens (Figure 2(A) and (B); P<0.05). Conversely, no significant differences in astaxanthin content were observed between breeds.

Table 2 shows the effects of breed and feed on carotenoid content in egg yolk and singlet oxygen scavenging activity by egg yolk. Significant differences were observed in total carotenoid content, zeaxanthin content, lutein content, astaxanthin content, and singlet oxygen scavenging activity (P<0.001). Significant differences were observed in total carotenoid content, zeaxanthin content, lutein content, and singlet oxygen scavenging activity (P<0.001, P<0.01, P<0.001, and P<0.05, respectively). Furthermore, a significant interaction was observed between the effects of breed and feed on lutein content (P<0.01).

Breed: Rhode Island Red, Silkie chicken.
Feed = Basic feed, paprika extract, marigold petal extract, Paracoccus cell powder.
*: P<0.05, **: P<0.01, ***: P<0.001, NS: No significant difference.

Figure 3 is a graph showing the singlet oxygen scavenging activity (50% inhibitory concentration; _IC50 ) by egg yolk. Data are expressed as mean ± SE (n=3). There is a significant difference between ^a-c with different signs (P<0.05). Cont = basal feed; PA = basal feed with 30 mg/kg of paprika-derived carotenoids added; MA = basal feed with 60 mg/kg of marigold petal-derived carotenoids added; AX = basal feed with 28 mg/kg of Paracoccus cell powder-derived carotenoids added. RR = Rhode Island Red; SF = Silkie chicken.

All treatment groups showed significantly different singlet oxygen scavenging activity compared to the Rhode Island Red control group (Figure 3; P<0.05). No significant differences were observed between treatment groups (Figure 3). The Silkie chicken AX group showed a significant difference compared to the control group (Figure 3; P<0.05).

Figure 4 is a graph showing the results of measuring egg yolk color (RYCF score) every week. (A) shows the change in egg yolk color over time for Rhode Island Red chickens, and (B) shows the change for Silkie chickens. The data is expressed as average values (n=12). Cont = basal feed; PA = basal feed supplemented with 30 mg/kg of paprika-derived carotenoids; MA = basal feed supplemented with 60 mg/kg of marigold petal-derived carotenoids; AX = basal feed supplemented with 28 mg/kg of Paracoccus cell powder-derived carotenoids.

In all treatment groups, egg yolk levels increased within one week of starting administration and then stabilized (Figure 4). A correlation is thought to exist between egg yolk levels and the amount of carotenoids (lutein, zeaxanthin, astaxanthin) in the egg yolk (Akiba Y, et al., Japanese poultry science, 37: 77-85. 2000; Islam KMS, et al., Journal of Poultry Science, 54: 271-277, 2017). Therefore, oral administration for at least seven days is considered to exert a sufficient effect (enhancement of transfer to egg yolk and antioxidant activity in egg yolk).

(Consideration)
This study investigated the effect of dietary carotenoids on egg quality in laying hens. Total carotenoid accumulation in the yolks of Silkie chickens was higher than that of Rhode Island Red chickens. This result suggests that dietary carotenoids are more likely to transfer to the yolks of Silkie chickens compared to those of Rhode Island Red chickens, a common laying hen. In particular, a significant interaction was observed between the effects of breed and feed on lutein content. This indicates that the combination of marigold petal extract-containing feed and Silkie chickens further promotes lutein accumulation in the yolks.

The supplementation of dietary carotenoids enhanced singlet oxygen scavenging activity in egg yolks, and Silkie chickens accumulated more carotenoids in their yolks than the common Rhode Island Red hen. This was also observed in the control group. Furthermore, Paracoccus cell powder contains adonirubin and adonixanthin, which possess high antioxidant activity comparable to astaxanthin. Therefore, adonirubin and adonixanthin may also have influenced the singlet oxygen scavenging activity in the AX group.

The RYCF score and a ^* value were significantly higher in the marigold petal extract group compared to the control group in both varieties (P<0.05). In the paprika extract group, the RYCF score, a ^* value, and a/b value were significantly higher (P<0.05), but the L ^* value was lower than the control group in both varieties. In both varieties, the RYCF score of the Paracoccus cell powder group was 14 or higher. In the Paracoccus cell powder group, the a ^* value and a/b value were also significantly higher, but the L ^* value was lower than the control group. The RYCF score and a/b value are used to evaluate egg yolk color.

Therefore, these results indicate that dietary carotenoids have the effect of improving egg yolk quality and enhancing the singlet oxygen scavenging activity of the egg yolk. Furthermore, it was suggested that Silkie chickens are a particularly effective breed for improving egg quality using dietary carotenoids.

(Test example 2)
In this study, we investigated the effect of increasing the amount of dietary carotenoids given to laying hens on egg quality.

(method)
Forty 55-week-old Silkie chickens (Tokyo Silkie) and 40 Rhode Island Red chickens (a common laying hen) were used in the experiment. The 40 chickens were randomly divided into four groups, with five chickens in each group for two replicates. The amount of carotenoids administered to each treatment group was twice that of Experiment 1.

- Control group (Cont): Only commercially available chicken feed (crude protein 17%, metabolizable energy 2800 kcal/kg, JA Higashi Nippon Kumiai Feed Co., Ltd.) was given as the basal feed.
- Paprika extract group (PA): The basal feed was supplemented with a mixed feed containing powdered paprika extract (Runred®, Elanco Japan Co., Ltd.). The amount added was set to 60 mg/kg of total carotenoids in the feed (5.04 mg/kg of zeaxanthin).
- Marigold petal extract group (MA): The basal feed was supplemented with a mixed feed containing powdered marigold petal extract (Xan Yellow®, Elanco Japan Co., Ltd.). The amount added was set to 120 mg/kg of total carotenoids in the feed (71.40 mg/kg of lutein).
- Paracoccus cell powder (AX): The basal feed was supplemented with a powdered astaxanthin-containing mixed feed (Panaferd®-P, ENEOS Corporation) produced using Paracoccus bacteria. The amount added was set to 56 mg/kg of total carotenoids in the feed (31.04 mg/kg of astaxanthin).

The test period was four weeks, and the rearing method was the same as in Test Example 1, except for the amount of carotenoid supplementation. The body weight of the test chickens was measured on the start and end of the test, and the amount of remaining feed was measured every seven days. The weight of all eggs laid during the test period was measured, and egg quality was examined every seven days. The amounts of zeaxanthin, lutein, and astaxanthin in the egg yolk were measured by HPLC. Singlet oxygen scavenging activity was evaluated by the egg yolk concentration that showed a 50% inhibitory effect on the development of conjugated linoleic acid (CLIN) from linoleic acid. A lower value indicates higher singlet oxygen scavenging activity. The above measurement methods and statistical analysis were performed in the same manner as in Test Example 1.

(result)
Table 3 shows the comparison results for egg yolk. In a two-way ANOVA, the effect of feed was observed in all items for egg yolk, and the effect of breed was observed in the L ^* value (brightness), b ^* value (yellowness), and a/b (an objective indicator said to reflect the color of egg yolk well). Furthermore, since an interaction was observed in a/b, it was suggested that there is an effect not only on the difference in carotenoid accumulation among chicken breeds, but also on the combination of breed and feed (interaction) (Table 3).

The data is expressed as mean ± SE (n=10).
^a-e: Significant difference between different signs in the same column (P<0.05), ***: P<0.001, NS: No significant difference.
L ^* = lightness; a ^* = redness; b ^* = yellowness; RYCF score = Roche York Color Fun score (1-15).
RR = Rhode Island Red; SF = Silkie chicken.
Cont = Basic feed; PA = Paprika extract; MA = Marigold petal extract; AX = Paracoccus cell powder.

Figure 5 is a graph showing the content of (A) zeaxanthin, (B) lutein, and (C) astaxanthin in egg yolk. Data are expressed as mean ± SE (n=4). There is a significant difference between different signs within the same graph ( ^a-c ) (P<0.05). RR = Rhode Island Red; SF = Silkie chicken. Cont = basal feed; PA = basal feed with 60 mg/kg of paprika-derived carotenoids added; MA = basal feed with 120 mg/kg of marigold petal-derived carotenoids added; AX = basal feed with 56 mg/kg of Paracoccus cell powder-derived carotenoids added.

Regarding the transfer of carotenoids into egg yolk, all three experimental groups showed greater transfer than the control group, and in two experimental groups (PA and MA), Silkie chickens showed greater transfer than Rhode Island Red chickens (generic chickens). The Silkie chicken PA group contained zeaxanthin at a concentration of 1 mg/100g or more in the egg yolk. Furthermore, the Silkie chicken MA group contained lutein at a concentration of 4 mg/100g or more in the egg yolk (Figure 5).

Table 4 shows the effects of breed and feed on carotenoid content in egg yolk and singlet oxygen scavenging activity by egg yolk. Two-way ANOVA revealed no significant differences in total carotenoid content in egg yolk, similar to those observed in Test Example 1, suggesting an upper limit on carotenoid supplementation in feed. Differences in zeaxanthin and lutein were observed not only due to feed but also to breed. In particular, lutein showed an effect due to the combination of breed and feed (interaction), demonstrating a synergistic effect of carotenoid (lutein) supplementation in Silkie chickens. Furthermore, the observation of effects on singlet oxygen scavenging activity due to the combination of breed and feed (interaction), in addition to the effect of feed, also indicates the superiority of Silkie chickens (Table 4).

Breed: Rhode Island Red, Silkie chicken.
Feed = Basic feed, paprika extract, marigold petal extract, Paracoccus cell powder.
†: P<0.1, *: P<0.05, **: P<0.01, ***: P<0.001, NS: No significant difference.

Figure 6 is a graph showing the singlet oxygen scavenging activity (50% inhibitory concentration; _IC50 ) by egg yolk. Data are expressed as mean ± SE (n=3). There is a significant difference between the same graph with different signs in ^a-d (P<0.05). RR = Rhode Island Red; SF = Silkie chicken. Cont = basal feed; PA = basal feed with 60 mg/kg of paprika-derived carotenoids added; MA = basal feed with 120 mg/kg of marigold petal-derived carotenoids added; AX = basal feed with 56 mg/kg of Paracoccus cell powder-derived carotenoids added.

The singlet oxygen scavenging activity of egg yolk was higher in the treated group than in the control group (Figure 6). This indicates that carotenoid supplementation can increase the added value of Silkie chicken eggs.

Table 5 shows the egg-laying rate, egg weight, feed intake, and feed conversion ratio. For Silkie chickens, egg weight significantly increased in all treatment groups, and egg-laying rate and feed intake significantly increased in the PA group (P<0.05, Table 5).

The data is expressed as mean ± SE (n=10).
^a-c show a significant difference between different signs in the same column (P<0.05).
RR = Rhode Island Red; SF = Silkie chicken.
Cont = Basic feed; PA = Paprika extract; MA = Marigold petal extract; AX = Paracoccus cell powder.

(Consideration)
Based on the results of this study (such as the transfer of zeaxanthin and lutein to egg yolk and the enhancement of singlet oxygen scavenging activity), it was found that the upper limit for carotenoid intake (the amount beyond which further increases in the carotenoid concentration in the feed do not lead to parallel improvements in performance and the unique advantages of Silkie chickens are considered to decrease) is 60 mg/kg of total carotenoids from paprika (5.04 mg/kg as zeaxanthin), 120 mg/kg of total carotenoids from marigold petals (71.40 mg/kg as lutein), and 56 mg/kg of total carotenoids from Paracoccus cell powder (31.04 mg/kg as astaxanthin).

Furthermore, the daily intake of each carotenoid per chicken (the actual amount consumed by the chicken) was as follows: Silkie chickens consumed 0.32 mg of zeaxanthin (PA), 4.21 mg of lutein (MA), and 1.83 mg of astaxanthin (AX). Rhode Island Red chickens (general breed) consumed 0.57 mg of zeaxanthin (PA), 7.98 mg of lutein (MA), and 3.55 mg of astaxanthin (AX).

(Test example 3)
This study investigated the effect of supplemental feeding of dietary carotenoids on egg quality in laying hens.

(method)
Thirty 60-week-old Silkie chickens (Tokyo Silkie) and 30 Rhode Island Red laying hens were used in the experiment. The 30 chickens were randomly divided into the following three groups. The control group was fed only basal feed. In the test groups, two types each of marigold petal extract ((registered trademark), Elanco Japan Co., Ltd., MA), paprika extract (Lanred (registered trademark), Elanco Japan Co., Ltd., PA), and Paracoccus cell powder (Panaferd (registered trademark)-P, ENEOS Corporation, AX) were added to the basal feed to investigate the effects of concomitant carotenoid supplementation. The amount of MA, PA, and AX given was set to 0.5, with the amount of carotenoid supplementation in Test Example 1 set to 1.

- Control group (Cont): Only basal feed (commercially available chicken feed; crude protein 17%, metabolizable energy 2800 kcal/kg; JA Higashi Nippon Kumiai Feed Co., Ltd.) was given.
- MA+PA group: Marigold petal extract was added to the basal feed at a concentration of 30 mg/kg as total carotenoids (17.85 mg/kg as lutein), and paprika extract was added at a concentration of 15 mg/kg as total carotenoids (1.26 mg/kg as zeaxanthin), and both were administered in combination.
- MA+AX group: Marigold petal extract was added to the basal feed at a total carotenoid level of 30 mg/kg (17.85 mg/kg as lutein), and astaxanthin-containing mixed feed produced using Paracoccus bacteria was added at a total carotenoid level of 14 mg/kg (7.76 mg/kg astaxanthin), and both were fed in combination.

The test period was four weeks, and the rearing method was the same as in Test Example 1, except for the amount of carotenoid supplementation. The body weight of the test chickens was measured on the start and end of the test, and the amount of remaining feed was measured every seven days. The weight of all eggs laid during the test period was measured, and egg quality was examined every seven days. The amounts of zeaxanthin, lutein, and astaxanthin in the egg yolk were measured by HPLC. Singlet oxygen scavenging activity was evaluated by the egg yolk concentration that showed a 50% inhibitory effect on the development of conjugated linoleic acid (CLIN) from linoleic acid. A lower value indicates higher singlet oxygen scavenging activity. The above measurement methods and statistical analysis were performed in the same manner as in Test Example 1.

(result)
Table 6 shows the comparative results for egg yolk. Two-way ANOVA revealed that the feed had an effect on all parameters, while the breed had an effect on the L ^* value (brightness), a ^* value (redness), and b ^* value. The effect of breed on egg yolk suggests differences in the ability of breeds to transfer carotenoids to the yolk. No interaction effect on egg yolk was observed between breed and feed combinations in combined feeding (Table 6).

The data is expressed as mean ± SE (n = 6).
^a-c: Significant difference between different signs in the same column (P<0.05), †: P<0.1, *: P<0.05, ***: P<0.001, NS: No significant difference.
L ^* = lightness; a ^* = redness; b ^* = yellowness; RYCF score = Roche York Color Fun score (1-15).
RR = Rhode Island Red; SF = Silkie chicken.
Cont = Basic feed; PA = Paprika extract; MA = Marigold petal extract; AX = Paracoccus cell powder.

Figure 7 is a graph showing the content of (A) lutein, (B) zeaxanthin, and (C) astaxanthin in egg yolk. Data are expressed as mean ± SE (n=5). There is a significant difference between different signs within the same graph ( ^a-c ) (P<0.05). RR = Rhode Island Red; SF = Silkie chicken. Cont = basal feed; MA+PA = basal feed supplemented with 30 mg/kg of carotenoids derived from marigold petals + 15 mg/kg of carotenoids derived from paprika; MA+AX = basal feed supplemented with 30 mg/kg of carotenoids derived from marigold petals + 14 mg/kg of carotenoids derived from Paracoccus bacteria powder.

Regarding the content of each carotenoid in egg yolk, Silkie chickens showed higher values than general chickens, except for astaxanthin. In all Silkie chicken treatment groups, the egg yolks contained at least 1 mg/100g of zeaxanthin and at least 4 mg/100g of lutein (Figure 7).

Table 7 shows the effects of breed and feed on carotenoid content in egg yolk and singlet oxygen scavenging activity by egg yolk. Two-way ANOVA revealed significant differences between breed and feed for all parameters. In particular, significant differences were observed in total carotenoid content by breed, similar to the results in Test Example 1 (P<0.01). Significant differences were also observed in zeaxanthin and lutein by breed (P<0.001). Furthermore, the significant difference observed in singlet oxygen scavenging activity by breed (P<0.001) also demonstrates the superiority of the Silkie chicken (Table 7).

Breed: Rhode Island Red, Silkie chicken.
Feed = Basic feed, marigold petal extract + paprika extract, marigold petal extract + Paracoccus cell powder.
*: P<0.05, **: P<0.01, ***: P<0.001, NS: No significant difference.

Figure 8 is a graph showing the singlet oxygen scavenging activity (50% inhibitory concentration; _IC50 ) by egg yolk. Data are expressed as mean ± SE (n=3). There is a significant difference between the same graph with different signs in ^a-c (P<0.05). RR = Rhode Island Red; SF = Silkie chicken. Cont = basal feed; MA+PA = basal feed supplemented with 30 mg/kg of carotenoids derived from marigold petals + 15 mg/kg of carotenoids derived from paprika; MA+AX = basal feed supplemented with 30 mg/kg of carotenoids derived from marigold petals + 14 mg/kg of carotenoids derived from Paracoccus bacteria powder.

The singlet oxygen scavenging activity of egg yolk was increased in the treated group compared to the control group. Furthermore, Silkie chicken egg yolk showed higher reactive oxygen species scavenging activity than that of normal chickens. In all Silkie chicken treated groups, the _IC50 value was less than half that of the normal chicken control group, and therefore the singlet oxygen scavenging activity of Silkie chicken eggs was more than twice that of normal chicken eggs (Figure 8).

(Consideration)
In this study, the results of combined carotenoid feeding showed the same superiority of Silkie chickens as in Study 1, in terms of the transfer of carotenoids (lutein and zeaxanthin) to egg yolk and the singlet oxygen scavenging activity of egg yolk. Combined carotenoid feeding was performed by combining two of the following amounts in the feed: zeaxanthin at 1.26 mg/kg (PA), lutein at 17.85 mg/kg (MA), and astaxanthin at 7.76 mg/kg (AX). The daily intake of each carotenoid per chicken (the amount actually consumed by the chicken) was 1.08 mg of lutein and 0.08 mg of zeaxanthin for the MA+PA regimen, and 1.01 mg of lutein and 0.44 mg of astaxanthin for the MA+AX regimen. In Rhode Island Red (common chicken), MA+PA contained 1.82mg of lutein and 0.13mg of zeaxanthin, while MA+AX contained 1.90mg of lutein and 0.82mg of astaxanthin.

In this study, even though the amount of paprika-derived carotenoids added to the basal feed was 15 mg/kg, which was 1/2 and 1/4 of that used in study examples 1 and 2, respectively, the amount of zeaxanthin transferred to the egg yolk of Silkie chickens was 1.25 mg/100g yolk, which was significantly higher than in both the Cont and general chicken study groups (Figure 7(B)). It was estimated that to achieve Silkie chicken eggs with a zeaxanthin content of 1.0 mg/100g yolk or higher, the amount of paprika-derived carotenoids added to the basal feed should be less than that used in this study, approximately 8.5 mg/kg or more.

Furthermore, regarding lutein, even though the amount of marigold petal-derived carotenoids added to the basal feed in this study was 30 mg/kg, which was 1/2 and 1/4 of that in study examples 1 and 2, respectively, the amount of lutein transferred to the egg yolk of Silkie chickens was 4.64 mg/100g egg yolk, which was significantly higher than in the Cont study (Figure 7(A)). It was estimated that to achieve Silkie chicken eggs with a lutein content of 4.0 mg/100g egg yolk or higher, the amount of marigold petal-derived carotenoids added to the basal feed should be less than that in this study, approximately 24.4 mg/kg or more.

This invention reveals that feeding laying hens a diet rich in carotenoids improves egg yolk color and enhances the antioxidant activity of the yolk. In particular, it was found that Silkie chickens accumulate more carotenoids (lutein and zeaxanthin) in their egg yolks than conventional chickens. Therefore, Silkie chickens are considered a suitable breed for improving egg quality by adding carotenoids to their feed. Consequently, this invention is expected to expand the use of Silkie chicken eggs and contribute to the food and health food industries.

Claims (7)

A method for improving the quality of Silkie chicken eggs, characterized by orally feeding Silkie chickens a feed containing 8.5 mg/kg or more of carotenoids derived from paprika and/or 24.4 mg/kg or more of carotenoids derived from marigold petals for at least 7 days. The method according to claim 1 , which allows for obtaining a Silkie chicken egg having a zeaxanthin content of 1 mg/100 g egg yolk or more, and/or a luten content of 4 mg/100 g egg yolk or more. The method according to claim 1 or 2, wherein the Silkie chicken is a Tokyo Silkie chicken. A feed for Silkie chickens, characterized by containing 8.5 mg/kg or more of carotenoids derived from paprika and/or 24.4 mg/kg or more of carotenoids derived from marigold petals. The feed according to claim 4 , which is for enhancing the antioxidant activity of egg yolk. A method for producing feed for Silkie chickens, characterized by incorporating 8.5 mg/kg or more of paprika-derived carotenoids and/or 24.4 mg/kg or more of marigold petal-derived carotenoids into the feed. The manufacturing method according to claim 6 , wherein the feed for Silkie chickens is for enhancing the antioxidant activity of egg yolks.