Possibility of Using Astaxanthin-Rich Dried Cell Powder from Paracoccus Carotinifaciens to Improve Egg Yolk Pigmentation of Laying Hens

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Article Possibility of Using Astaxanthin-Rich Dried Cell Powder from Paracoccus carotinifaciens to Improve Egg Yolk Pigmentation of Laying Hens

Masaki Honda 1,* , Yuki Kawashima 2,*, Kazuaki Hirasawa 2, Takeshi Uemura 2, Sun Jinkun 3 and Yoshiaki Hayashi 3,* 1 Faculty of Science & Technology, Meijo University, Shiogamaguchi, Tempaku-ku, Nagoya 468-8502, Japan 2 Biotechnology R&D Group, JXTG Nippon Oil & Energy Corporation, Chidori-cho, Naka-ku, Yokohama 231-0815, Japan; [email protected] (K.H.); [email protected] (T.U.) 3 Experimental Farm, Faculty of Agriculture, Meijo University, Hishigaike, Takaki-cho, Kasugai 486-0804, Japan; [email protected] * Correspondence: [email protected] (M.H.); [email protected] (Y.K.); [email protected] (Y.H.)

Received: 29 April 2020; Accepted: 29 May 2020; Published: 2 June 2020

Abstract: The study investigated egg quality aspects such as astaxanthin concentration, E/Z-isomer ratio, and yolk color in laying hens fed with astaxanthin-containing diets. Dried Paracoccus carotinifaciens cell powder (Panaferd-AX) and ﬁne cell powder (Panaferd-P) were used as sources of astaxanthin, with average particle diameters of approximately 100 µm and 10 µm, respectively. Paracoccus carotinifaciens contains valuable rare carotenoids such as adonirubin and adonixanthin, and thus the concentrations of these carotenoids in egg yolk were also evaluated. The E/Z-isomer ratios of the egg yolk carotenoids were determined by normal-phase high-performance liquid chromatography (HPLC) with an improved solvent system. Feeding diets containing P. carotinifaciens resulted in increased concentrations of astaxanthin, adonirubin, and adonixanthin in egg yolk, as well as a marked increase in the yolk color fan score; values associated with the Panaferd-P-containing diet were higher than those associated with the Panaferd-AX-containing diet. For example, the astaxanthin concentration in egg yolks of hens fed with the Panaferd-AX- and Panaferd-P-containing diets for 21 days were 1.21 µg/g and 1.85 µg/g, respectively. This indicates that the pulverization treatment of the P. carotinifaciens powder increased the eﬃciency of carotenoid accumulation in the egg yolk. Moreover, more than 95% of astaxanthin in P. carotinifaciens was present as the all-E-isomer. However, approximately 25% of astaxanthin in egg yolk was present as the Z-isomers. In recent years, astaxanthin Z-isomers have attracted substantial attention as they exhibit a greater bioavailability and bioactivity than the all-E-isomer. These data are important not only for understanding egg yolk pigmentation but also for improving the nutritional value of hens’ egg yolk through the addition of P. carotinifaciens to their diet.

Keywords: Paracoccus carotinifaciens; astaxanthin; adonirubin; adonixanthin; geometric isomer; egg yolk

1. Introduction The color of egg yolk is an important factor in determining whether the product will be acceptable to the consumer. Yolk with a high pigment content is in demand for egg-containing processed foods such as noodles, baked goods, and mayonnaise. Carotenoids are principally responsible for the yolk color, but because chickens cannot biosynthesize carotenoids, they obtain carotenoids from their diet [1–3]. Dietary sources of carotenoids have traditionally been corn,

Symmetry 2020, 12, 923; doi:10.3390/sym12060923 www.mdpi.com/journal/symmetry Symmetry 2020, 12, 923 2 of 13 alfalfa, and marigold flower, which contain lutein and zeaxanthin [1,4,5]. In recent years, the use of astaxanthinSymmetry 2020 (3,3’-dihydroxy-, 12, x FOR PEER REVIEWβ,β-carotene-4,4’-dione; Figure1a) as a carotenoid for improving2 of 12 egg yolk pigmentation has attracted substantial attention [6–8]. Astaxanthin efficiently pigments egg astaxanthin (3,3’-dihydroxy-β,β-carotene-4,4’-dione; Figure 1a) as a carotenoid for improving egg yolk and shows a strong antioxidant activity that is 10 times higher than β-carotene and 100 times yolk pigmentation has attracted substantial attention [6–8]. Astaxanthin efficiently pigments egg yolk α higherand shows than a -tocopherolstrong antioxidant [9,10]. activity Furthermore, that is 10 carotenoid times higher consumption than β-carotene off anders various100 times health higher benefits suchthan as α decreased-tocopherol risks[9,10]. of Furthermore, cardiovascular carotenoid disease, consumption certain cancers, offers va andrious eye health disease benefits [9– such11]. Theas yeast Phadecreasedffia rhodozyma risks ofhas cardiovascular been used disease, as a source certain of cancers, astaxanthin and eye to disease allow pigmentation[9–11]. The yeast of Phaffia egg yolk for commercialrhodozyma has purposes been used [12 as, 13a source]. In thisof astaxanthin study, we to evaluatedallow pigmentation the use of of egg an yolk aerobic for commercial Gram-negative microorganism,purposes [12,13].Paracoccus In this study, carotinifaciens we evaluatedASB-57 the use strain of an [ 14aerobic], as aGram-negative source of astaxanthin microorganism, (3S,3’S form). ParacoccusParacoccus carotinifaciens carotinifacienscontains ASB-57 notstrain only [14], high as concentrationsa source of astaxanthin of astaxanthin (3S,3’S but form). also twoParacoccus valuable rare carotenoids,carotinifaciens adonirubin contains not (3-hydroxy- only highβ concentrations,β-carotene-4,4’-dione, of astaxanthin 3S form; but also Figure two1b) valuable and adonixanthin rare carotenoids, adonirubin (3-hydroxy-β,β-carotene-4,4’-dione, 3S form; Figure 1b) and adonixanthin (3,3’-dihydroxy-β,β-carotene-4-one, 3S,3’R form; Figure1c) [ 15,16]. These rare carotenoids have over (3,3’-dihydroxy-β,β-carotene-4-one, 3S,3’R form; Figure 1c) [15,16]. These rare carotenoids have over 2.5 times more antioxidant activity than astaxanthin [17]. Moreover, microorganisms belonging to the 2.5 times more antioxidant activity than astaxanthin [17]. Moreover, microorganisms belonging to genesthe genesParacoccus Paracoccusare advantageous are advantageous in their in their proliferation proliferation rates, rates, and and their their carotenoid carotenoid productivityproductivity is high comparedis high compared to that of to yeasts. that of Thus, yeasts. the Thus, use the of P. use carotinifaciens of P. carotinifaciensshould should provide provide advantages advantages in terms in of the nutritionalterms of the value nutritional and production value and prod costuction of eggs cost compared of eggs compared to P. rhodozyma to P. rhodozyma[18]. Dried [18]. P.Dried carotinifaciens P. cellcarotinifaciens powder was cell recently powder commercializedwas recently commercialized by JXTG Nippon by JXTG Oil Nippon & Energy Oil & Corporation Energy Corporation (Tokyo, Japan) for(Tokyo, use in Japan) the pigmentation for use in the pigmentation of hens’ egg of hens yolk’ egg and yolk seafoods and seafoods such such as salmon, as salmon, trout, trout, and and shrimp. Theseshrimp. cell These powders cell arepowders sold underare sold the under names thePanaferd-AX names Panaferd-AX and Panaferd-P. and Panaferd-P. Panaferd-P Panaferd-P is obtained is by finelyobtained pulverizing by finely Panaferd-AX;pulverizing Panaferd-AX; the average the particle average diametersparticle diameters of Panaferd-AX of Panaferd-AX and Panaferd-Pand Panaferd-P are approximately 100 μm and 10 μm, respectively [8,15]. Several studies indicated that are approximately 100 µm and 10 µm, respectively [8,15]. Several studies indicated that the the pulverization treatment of ingredients containing carotenoids, such as red pepper (Capsicum pulverization treatment of ingredients containing carotenoids, such as red pepper (Capsicum frutescens) frutescens) and P. rhodozyma, enhanced the yolk pigmentation efficiency of laying hens [19,20]. andTherefore,P. rhodozyma there, is enhanced a possibility the that yolk Panaferd-P pigmentation has a ehigherfficiency pigmentation of laying hensefficiency [19,20 than]. Therefore, Panaferd- there is a possibilityAX. that Panaferd-P has a higher pigmentation efficiency than Panaferd-AX.

FigureFigure 1. 1.Chemical Chemical structures of of astaxanthin astaxanthin isomers: isomers: (a) (all- (a)E (all-)-astaxanthin;E)-astaxanthin; (b) (all- (bE))-adonirubin; (all-E)-adonirubin; (c)(c (all-) (all-E)-adonixanthin;E)-adonixanthin; ( (dd)) (9 (9ZZ)-astaxanthin;)-astaxanthin; (e) ((15e) (15Z)-astaxanthin.Z)-astaxanthin.

TheFigure aim 1. of Chemical this study structures was toof investigateastaxanthin isomers: the eﬀ (ectsa) (all- ofE)-astaxanthin; feeding laying (b) (all- hensE)-adonirubin; with diets containing P. carotinifaciens(c) (all-E)-adonixanthin;powder of (d di) (9ﬀZerent)-astaxanthin; particle (e sizes) (15Z)-astaxanthin. on the concentrations of astaxanthin, adonirubin, and adonixanthin in egg yolk and the yolk color. We also evaluated the impact of the feeding on some The aim of this study was to investigate the effects of feeding laying hens with diets containing egg qualities such as egg weight, yolk weight, albumen height, and the Haugh unit. Moreover, we P. carotinifaciens powder of different particle sizes on the concentrations of astaxanthin, adonirubin, investigatedand adonixanthin the E /inZ -isomeregg yolk and ratios the of yolk astaxanthin, color. We also adonirubin, evaluated the and impact adonixanthin of the feeding in on egg some yolk using anegg improved qualities high-performancesuch as egg weight, liquidyolk weight, chromatography albumen height, (HPLC) and the system. Haugh Apartunit. Moreover, from some we notable exceptions,investigated the the carotenoid E/Z-isomer ratios all-E-isomer of astaxanthin, is the adonirubin most predominant, and adonixanthin geometric in egg isomer yolk using in plants an and microorganismsimproved high-performance such as P. carotinifaciens liquid chromatograp(Figure1a–c)hy (HPLC) [ 16,21– 23system.]. In contrast,Apart fromZ-isomers some notable of carotenoids existexceptions, in considerable the carotenoid quantities all-E in-isomer humans is the and most other predominant animals (Figure geometric1d,e) isomer [ 21,24 –in26 plants]. Several and studies havemicroorganisms reported that suchZ-isomers as P. ofcarotinifaciens carotenoids (Figure are found 1a–c) in [16,21–23]. egg yolk [2In,6 ,27contrast,,28]. For Z-isomers example, of we have recentlycarotenoids reported exist that in considerable even though quantities most dietary in humans lycopene and represents other animals the all-(FigureE-isomer, 1d,e) [21,24 more− than26]. 65% of Several studies have reported that Z-isomers of carotenoids are found in egg yolk [2,6,27,28]. For lycopene in egg yolk is present as the Z-isomers [27]. Walker et al. reported that when laying hens were

Symmetry 2020, 12, 923 3 of 13 fed with astaxanthin-rich diets, several types of astaxanthin Z-isomers could be detected [6]. However, they did not provide the exact value of the Z-isomer ratio, likely because of the diﬃculty in accurately analyzing carotenoid isomers in egg yolk. Recently, several studies have reported that astaxanthin Z-isomers had a higher bioavailability and showed stronger bioactivities, such as antioxidant and anti-inﬂammatory activities, than those by the all-E-isomer [29–31]. Hence, the evaluation of the carotenoid E/Z-isomer ratio in foods, especially that of astaxanthin, is very important.

2. Materials and Methods

2.1. Reagents Two forms of carotenoid-rich dried cell powders from the P. carotinifaciens ASB-57 strain [14], Panaferd-AX (average particle diameter, approximately 100 µm; astaxanthin content, 2.0%; adonirubin content, 0.61%; adonixanthin content, 0.22%) and Panaferd-P (average particle diameter, approximately 10 µm; astaxanthin content, 1.9%; adonirubin content, 0.63%; adonixanthin content, 0.20%) were obtained from JXTG Nippon Oil & Energy Corporation (Tokyo, Japan) [8,14,15]. Panaferd-P was obtained by ﬁnely pulverizing Panaferd-AX [8]. High-purity (all-E)-astaxanthin was purchased from Sigma-Aldrich Co. Ltd. (Dorset, United Kingdom), and (all-E)-adonirubin and (all-E)-adonixanthin were obtained from JXTG Nippon Oil & Energy Corporation. HPLC-grade acetone, ethyl acetate, hexane, and dichloromethane (CH2Cl2), and analytical-grade acetone were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). The basal diet used in this study was purchased from Kameya Syoji Co., Ltd. (Ginan, Japan), and its ingredients and chemical composition are shown in Table1. The chemical composition of the feed was analyzed as a mixed condition. Although the amino acid composition of the feed was not analyzed in this study, the feed was mixed by the feed company so as to meet the amino acid requirements for layers producing 56 g of egg mass a day. The amino acid requirements were based on the Japanese Feeding Standard for Poultry (2011), edited by the National Agriculture and Food Research Organization.

Table 1. Ingredients and nutrient composition of the basal diet.

Ingredient g/kg Corn 566.0 Soybean meal 174.0 Limestone 93.4 Corn gluten meal 50.0 Rice bran 30.0 Vegetable oil 27.9 Fish meal 25.0 Corn gluten feed 20.7 Calcium phosphate 8.60 Sodium chloride 4.40 Chemical Composition (Dry Matter Basis) g/kg Metabolizable energy (kcal/kg) 2720 Dry matter 898.4 Organic matter 850.3 Crude protein 183.4 Ether extract 69.2 Crude fiber 39.3 Neutral detergent fiber 797.2 Acid detergent fiber 297.6 Nitrogen free extract 558.4 Crude ash 149.7 Calcium 15.5 Phosphorus 9.6 Symmetry 2020, 12, 923 4 of 13

2.2. Feeding Experiments Lohmann Julia Lite hens (24 weeks old) housed in individual cages (19.5 39.0 39.5–43.0 cm) were × × used in this study. The laying hens were randomly assigned into three groups: basal diet alone, basal diet with Panaferd-AX, or basal diet with Panaferd-P (astaxanthin content: 8 mg/kg diet). The dietary astaxanthin content used in this study was determined to provide suﬃcient egg yolk pigmentation in accordance with previous studies [12,13]. After moving the hens to their individual cages, they were fed with the basal diet for a week, followed by the experimental diets for 21 days. During the experiment, the laying hens were allowed free access to water and each diet. The evaluation of the egg quality was conducted at 4, 7, 14, and 21 days from the start of experimental feeding. The concentrations of astaxanthin, adonirubin, and adonixanthin, and their Z-isomer ratios in yolk were determined using normal-phase HPLC as described below (Section 2.3). The egg yolk color, assessed by the yolk color fan score and lightness (L*), redness (a*), and yellowness (b*) values, as well as the other egg qualities such as egg weight, yolk weight, albumen height, and the Haugh unit, were evaluated using an Egg Multi Tester (EMT-7300; Robotmation Co., Ltd., Tokyo, Japan) and a color spectrophotometer (CM-700d; Konica Minolta, Inc., Tokyo, Japan). These experiments were approved and carried out in accordance with the Institutional Animal Care and Use Committee of Meijo University.

2.3. HPLC Analysis The determination of the carotenoid (astaxanthin, adonirubin, and adonixanthin) concentrations and their E/Z-isomer ratios in egg yolk were performed using normal-phase HPLC with a Phenomenex silica gel column (Luna 5 µm Silica (2), 150 mm 4.6 mm, 100 Å) [16,32,33]. Several studies successfully × separated astaxanthin, adonirubin, and adonixanthin isomers by normal-phase HPLC with the Phenomenex silica gel column using hexane/acetone (83:17, v/v) [32] and hexane/ethyl acetate/acetone (70:20:10, v/v/v) solvent systems [16,33]. However, these solvent systems cannot differentiate the peaks of the carotenoid isomer and the peaks derived from components originally contained in the egg yolk. Hence, to clearly separate them, a new hexane/ethyl acetate/acetone (75:23:2, v/v/v) solvent system was used in this study. The flow rate and column temperature were set at 1.2 mL/min and 40 ◦C, respectively. The detection and quantification of the carotenoid isomers were performed by peak area integration at 470 nm using a photodiode array detector (SPD-M20A; Shimadzu, Kyoto, Japan). The peaks of astaxanthin, adonirubin, and adonixanthin isomers were identified according to HPLC retention times, visible spectral data (absorption maxima and shape of spectrum), and relative intensities of the Z-peak (350–370 nm) compared to the main absorption peak of the isomer (Figure S1; Q-ratio) [16,32–34]. The carotenoid Z-isomer ratio (%) was estimated as the amount of total Z-isomers to the amount of total carotenoid isomers including the all-E-isomer.

2.4. Carotenoid Extraction from Egg Yolk The extraction of astaxanthin, adonirubin, and adonixanthin isomers from egg yolk was performed using acetone, following previously established protocols [22,27]. All procedures were performed at room temperature unless otherwise indicated, and light exposure was kept to a minimum throughout the extraction. Approximately 1 g of egg yolk was weighed into a 100-mL screw-capped glass bottle, and 50 mL of acetone was added to the bottle. Carotenoid extraction was performed using ultrasonic treatment (CPX1800H-J; Yamato Scientific Co., Ltd.) at 80 W and 38 kHz for 15 min on ice (approximately 5 ◦C). The residue was removed with a 0.22-µm polytetrafluoroethylene (PTFE) membrane filter (Osaka Chemical Co., Ltd., Osaka, Japan), and the filtrate containing carotenoid isomers was dried under reduced pressure at 35 ◦C for 5 min. The obtained extract was dissolved in a defined amount of ethyl acetate and filtered through a 0.22-µm PTFE membrane filter for HPLC analysis. Symmetry 2020, 12, 923 5 of 13

2.5. Statistical Analysis Data were statistically analyzed by analysis of variance (ANOVA) and the Tukey's multiple comparison tests using a commercially available computer program (Excel Statistics; SSRI Co., Ltd.). Diﬀerences were considered statistically signiﬁcant when p < 0.05.

3. Results and Discussion

3.1. Proﬁle of Carotenoid Isomers in Egg Yolk

The use of reversed-phase HPLC with C18 or C30 columns and normal-phase HPLC with a silica gel column to separate astaxanthin isomers has been well documented [16,32–38]. However, ample studies have shown that normal-phase HPLC exhibits a greater separation ability than that by reversed-phase HPLC when separating carotenoid isomers [16,32–38]. In addition, a sufficient separation of adonirubin and adonixanthin isomers can be obtained in normal-phase HPLC [16,32–34]. Thus, this study used normal-phase HPLC with a Phenomenex silica gel column to separate astaxanthin, adonirubin, and adonixanthin isomers. However, previously reported solvent systems using hexane/acetone (83:17, v/v) [32] and hexane/ethyl acetate/acetone (70:20:10, v/v/v) [16,33] do not clearly separate peaks corresponding to the carotenoid isomer and components originally contained in the egg yolk. Hence, we used an improved solvent system using hexane/ethyl acetate/acetone (75:23:2, v/v/v). On the other hand, we also evaluated other solvent systems, such as hexane/ethyl acetate (82:12, v/v), hexane/acetone (82:12. v/v), and hexane/ethyl acetate/acetone (76:16:8, 75:15:10, and 75:20:5, v/v/v), but their good separation could not be obtained. The typical chromatograms of a mixture of astaxanthin, adonirubin, and adonixanthin isomers, obtained by the thermally Z-isomerized treatment of their all-E-isomer standards at 80 ◦C for 3 h in CH2Cl2 [38–40], as well as extracts from Panaferd-AX, Panaferd-P, and egg yolks before and after the 21-day feeding of the experimental diets, are shown in Figure2. This improved HPLC method could not only clearly separate astaxanthin, adonirubin, and adonixanthin isomers (peaks 1–10; peaks 5 and 6 were tentatively identified as (9Z)-astaxanthin and a mixture of (13Z)- and (15Z)-astaxanthin according to previous studies [16,32–34]; Table S1), but also their isomers and egg yolk-derived peaks (denoted by asterisks). In Panaferd-AX and Panaferd-P dried cell powders, large peaks from (all-E)-astaxanthin, (all-E)-adonirubin, and (all-E)-adonixanthin were observed (Figure2b,c), as in previous studies [ 16,41], and minor peaks from the Z-isomers of astaxanthin and adonirubin were detected. Before the feeding with the experimental diets, the astaxanthin, adonirubin, and adonixanthin isomer peaks were not detected in egg yolk, but large peaks from lutein and zeaxanthin were observed (Figure2d). After 21 days of feeding diets containing Panaferd-AX and Panaferd-P, the peaks from astaxanthin, adonirubin, and adonixanthin isomers could be detected (Figure2e,f). To the best of our knowledge, this is the first study to show that feeding laying hens with valuable rare carotenoids such as adonirubin and adonixanthin causes their accumulation in the hens’ egg yolks. Furthermore, it is interesting that the egg yolk contained an abundance of astaxanthin Z-isomers despite the hens being fed with diets rich in (all-E)-astaxanthin. This data contributes to improving the nutritional value of egg yolk by using P. carotinifaciens for pigmentation. Symmetry 2020, 12, 923 6 of 13 Symmetry 2020, 12, x FOR PEER REVIEW 6 of 12

FigureFigure 2. Normal-phase 2. Normal-phase HPLC HPLC chromatograms chromatograms ofof ( a)) a a standard standard mixture mixture of (all- of (all-E)-astaxanthinE)-astaxanthin (AST), (AST),

(all-E(all-)-adonirubinE)-adonirubin (ADR), (ADR), and and (all- (all-E)-adonixanthinE)-adonixanthin (ADR), which which were were treated treated with with CH CH2Cl2 2atCl 802 at°C 80 ◦C for 3 hfor in 3 orderh in order to increase to increase their theirZ -isomers,Z-isomers, extracts extracts from from ( (bb) )Panaferd-AX, Panaferd-AX, (c) (Panaferd-P,c) Panaferd-P, (d) egg (d) yolk egg yolk beforebefore feeding feeding the the experimental experimental diets, diets, and and egg egg yolksyolks after feeding feeding diets diets containing containing (e) Panaferd-AX (e) Panaferd-AX and (f) Panaferd-P. Peak identifications: 1, 2 = adonirubin Z-isomers, 3, 4 = astaxanthin Z-isomers, 5 = and (f) Panaferd-P. Peak identifications: 1, 2 = adonirubin Z-isomers, 3, 4 = astaxanthin Z-isomers, (9Z)-astaxanthin, 6 = (13Z + 15Z)-astaxanthin, 7−10 = adonixanthin Z-isomers, LUT = lutein, ZEA = 5 = (9Z)-astaxanthin, 6 = (13Z + 15Z)-astaxanthin, 7–10 = adonixanthin Z-isomers, LUT = lutein, zeaxanthin [16,32−34]. Asterisk symbols (*) show several egg yolk-derived compounds. Several peaks ZEA = zeaxanthin [16,32–34]. Asterisk symbols (*) show several egg yolk-derived compounds. Several (1–10) were tentatively identified, as shown in Table S1. peaks (1–10) were tentatively identified, as shown in Table S1. 3.2. Evaluation of Carotenoid Concentration and Z-isomer Ratio in Egg Yolk 3.2. Evaluation of Carotenoid Concentration and Z-isomer Ratio in Egg Yolk The effects of feeding laying hens diets containing P. carotinifaciens of different particle sizes The(Panaferd-AX effects of and feeding Panaferd-P) laying on hens the concentrations diets containing of astaxanthin,P. carotinifaciens adonirubin,of di andfferent adonixanthin particle sizes (Panaferd-AXand their total and Z Panaferd-P)-isomer ratios on in the egg concentrations yolk are shown ofin astaxanthin,Figures 3−5. Astaxanthin, adonirubin, adonirubin, and adonixanthin and and theiradonixanthin total Z-isomer could be ratios detected in egg in yolkegg yolk are shownfrom the in fourth Figures day3– 5of. Astaxanthin,feeding, and the adonirubin, carotenoid and adonixanthinconcentrations could reached be detected their maximum in egg levels yolk around from the 14−21 fourth days in day both of Panaferd-AX- feeding, and and the Panaferd- carotenoid concentrationsP-containing reached diets. When their hens maximum were fed levels with around a diet 14–21containing days Panaferd-P, in bothPanaferd-AX- the egg yolk and Panaferd-P-containingconcentrations of astaxanthin, diets. When adonirubin, hens were and adon fed withixanthin a diet were containing significantly Panaferd-P, higher thanthe those egg in yolk concentrationshens fed with of a astaxanthin, diet containing adonirubin, Panaferd-AX. and This adonixanthin is likely due to were the smaller significantly particle highersize of Panaferd- than those in P. Several studies have shown that carotenoids’ bioavailability depends on their particle size, such hens fed with a diet containing Panaferd-AX. This is likely due to the smaller particle size of Panaferd-P. that the micronization of carotenoids effectively enhances their bioavailabilities [42,43]. In addition, Severalsince studies Panafed-P have shownwas obtained that carotenoids’ by pulverizing bioavailability Panafed-AX, depends the release on theirof carotenoids particle size,from such the that the micronizationmicroorganism of cell carotenoids by pulverization effectively treatment enhances would their also improve bioavailabilities their bioaccessibilities [42,43]. In addition, [44]. This since Panafed-Ptheory was is supported obtained by Li pulverizing et al. [19]; that Panafed-AX, is, the breakup the release of the ofcell carotenoids wall by pulverization from the microorganism treatment cell bywould pulverization increase the treatment ability of would hens to also access improve the caro theirtenoids bioaccessibilities in the feeds, facilitating [44]. This their theory uptake is supported and by Liincorporation et al. [19]; that into is, the breakupegg yolk. of These the cell improvem wall byents pulverization to carotenoid treatment bioaccessibility would increaseand/or the abilitybioavailability of hens to access would the carotenoidsresult in increased in the feeds, concentrations facilitating theirof astaxanthin, uptake and incorporationadonirubin, and into the egg yolk.adonixanthin These improvements in the egg yolk of to hens carotenoid fed with bioaccessibility Panafed-P. When and fed/ orwith bioavailability diets containing would Panaferd- result in increasedAX and concentrations Panaferd-P (astaxanthin of astaxanthin, content: adonirubin, 8 mg/kg diet) and for 21 adonixanthin days, the astaxanthin in the egg concentration yolk of hens in fed with Panafed-P. When fed with diets containing Panaferd-AX and Panaferd-P (astaxanthin content: 8 mg/kg diet) for 21 days, the astaxanthin concentration in egg yolk reached 1.21 µg/g and 1.85 µg/g, respectively. Akiba et al. [12] reported similar concentrations of astaxanthin in egg yolk upon feeding Symmetry 2020, 12, x FOR PEER REVIEW 7 of 12 Symmetry 2020, 12, x FOR PEER REVIEW 7 of 12 Symmetryegg yolk2020 reached, 12, 923 1.21 μg/g and 1.85 μg/g, respectively. Akiba et al. [12] reported similar7 of 13 eggconcentrations yolk reached of astaxanthin 1.21 μg/g inand egg 1.85yolk μupong/g, feedingrespectively. a diet Akibacontaining et al. P. rhodozyma[12] reported, a yeast similar that concentrations of astaxanthin in egg yolk upon feeding a diet containing P. rhodozyma, a yeast that ahas diet been containing commerciallyP. rhodozyma used for, apigmenting yeast that hens’ has been egg yolk. commercially They found used that for laying pigmenting hens fed hens’ with eggthis has been commercially used for pigmenting hens’ egg yolk. They found that laying hens fed with this yolk.diet (astaxanthin They found thatcontent: laying 8 hens mg/kg fed withdiet) thisfor diet28 (astaxanthindays produced content: eggs 8 mgwith/kg yolk diet) astaxanthin for 28 days diet (astaxanthin content: 8 mg/kg diet) for 28 days produced eggs with yolk astaxanthin producedconcentrations eggs withof 1.39 yolk μg/g. astaxanthin These results concentrations indicate ofthat, 1.39 similarµg/g. Theseto P. rhodozyma results indicate, P. carotinifaciens that, similar tois concentrations of 1.39 μg/g. These results indicate that, similar to P. rhodozyma, P. carotinifaciens is P.commercially rhodozyma, P. suitable carotinifaciens for pigmentingis commercially a hen’s suitableegg yolk. for pigmenting a hen’s egg yolk. commercially suitable for pigmenting a hen’s egg yolk. TheThe totaltotal ZZ-isomer-isomer ratiosratios ofof astaxanthin,astaxanthin, adonirubin,adonirubin, andand adonixanthinadonixanthin inin Panaferd-AXPanaferd-AX werewere The total Z-isomer ratios of astaxanthin, adonirubin, and adonixanthin in Panaferd-AX were 3.9%,3.9%, 11.3%,11.3%, and and 5.0%, 5.0%, respectively, respectively, and thoseand inthose Panaferd-P in Panaferd-P were 4.2%, were 11.6%, 4.2%, and 11.6%, 5.3%, respectively.and 5.3%, 3.9%, 11.3%, and 5.0%, respectively, and those in Panaferd-P were 4.2%, 11.6%, and 5.3%, Inrespectively. contrast, in bothIn contrast, diets, the inZ -isomerboth diets, ratios the of astaxanthin, Z-isomer adonirubin,ratios of astaxanthin, and adonixanthin adonirubin, in egg yolkand respectively. In contrast, in both diets, the Z-isomer ratios of astaxanthin, adonirubin, and wereadonixanthin approximately in egg 25%, yolk 20%, were and approximately 7%, respectively 25%, (Figure 20%, 3andb, Figure 7%, respectively4b, and Figure (Figures5b). These 3b, results4b, and adonixanthin in egg yolk were approximately 25%, 20%, and 7%, respectively (Figures 3b, 4b, and suggest5b). These that results the carotenoid suggest Zthat-isomer the carotenoid ratio in egg yolkZ-isomer differs ratio depending in egg onyolk the differs types ofdepending carotenoids. on Forthe 5b). These results suggest that the carotenoid Z-isomer ratio in egg yolk differs depending on the example,types of carotenoids. astaxanthin andFor lycopeneexample, (reported astaxanthin in our and previous lycopene study (repor [26ted]) had in higherour previousZ-isomer study contents. [26]) types of carotenoids. For example, astaxanthin and lycopene (reported in our previous study [26]) Thishad higher may be Z related-isomer to contents. the fact thatThis the mayZ-isomers be related of to astaxanthin the fact that and the lycopene Z-isomers are of more astaxanthin bioavailable and had higher Z-isomer contents. This may be related to the fact that the Z-isomers of astaxanthin and thanlycopene the all- areE -isomersmore bioa [24vailable,28]. Since than the the mechanisms all-E-isomers underlying [24,28]. Since the di thefferences mechanisms in carotenoid underlyingZ-isomer the lycopene are more bioavailable than the all-E-isomers [24,28]. Since the mechanisms underlying the ratiosdifferences in egg in yolk carotenoid remain unknown, Z-isomer futureratios studiesin egg shouldyolk remain be conducted unknown, on thisfuture topic. studies In any should case, thebe differences in carotenoid Z-isomer ratios in egg yolk remain unknown, future studies should be factconducted that high on amounts this topic. of astaxanthinIn any case, are the present fact that as Z -isomershigh amounts (over 25%)of astaxanthin in egg yolks are of present hens fed as with Z- conducted on this topic. In any case, the fact that high amounts of astaxanthin are present as Z- astaxanthin-richisomers (over 25%) sources in (Panaferd-AXegg yolks of andhens Panaferd-P) fed with isastaxanthin-rich a very important sources finding (Panaferd-AX in relation to theirand isomers (over 25%) in egg yolks of hens fed with astaxanthin-rich sources (Panaferd-AX and nutritionalPanaferd-P) value. is a very important finding in relation to their nutritional value. Panaferd-P) is a very important finding in relation to their nutritional value.

Figure 3. Effects of feeding laying hens with diets containing P. carotinifaciens of different particle sizes Figure 3. Effects of feeding laying hens with diets containing P. carotinifaciens of different particle sizes Figure(Panaferd-AX 3. Effects and of feedingPanaferd-P) laying for he 21ns days with on diets the containing (a) astaxanthin P. carotinifaciens concentration of differentand (b) total particle Z-isomer sizes (Panaferd-AX and Panaferd-P) for 21 days on the (a) astaxanthin concentration and (b) total Z-isomer (Panaferd-AXratio in egg yolk. and Error Panaferd-P) bars depict for 21 the days standard on the deviation (a) astaxanthin (n = 8). concentration Means with differentand (b) total letters Z-isomer within ratio in egg yolk. Error bars depict the standard deviation (n = 8). Means with different letters within ratioeach inday egg are yolk. significantly Error bars different depict the (p < standard 0.05), whereas deviation means (n = with 8). Means similar with letters different are not letters different. within p each day are significantlysignificantly didifferentfferent ((p << 0.05), whereas means with similar letters are not didifferent.fferent.

Figure 4. Effects of feeding laying hens with diets containing P. carotinifaciens of different particle sizes Figure 4. Effects of feeding laying hens with diets containing P. carotinifaciens of different particle sizes (Panaferd-AX and Panaferd-P) for 21 days on the (a) adonirubin concentration and (b) total Z-isomer Figure(Panaferd-AX 4. Effects and of feedingPanaferd-P) laying for he 21ns days with on diets the containing (a) adonirubin P. carotinifaciens concentration of differentand (b) total particle Z-isomer sizes ratio in egg yolk. Error bars depict the standard deviation (n = 8). Means with different letters within (Panaferd-AXratio in egg yolk. and Error Panaferd-P) bars depict for 21 the days standard on the deviation (a) adonirubin (n = 8). concentration Means with differentand (b) total letters Z-isomer within each day are significantly different (p < 0.05), whereas means with similar letters are not different. ratioeach inday egg are yolk. significantly Error bars different depict the (p < standard 0.05), whereas deviation means (n = with 8). Means similar with letters different are not letters different. within each day are significantly different (p < 0.05), whereas means with similar letters are not different.

Symmetry 2020, 12, x FOR PEER REVIEW 8 of 12 Symmetry 2020, 12, 923 8 of 13 Symmetry 2020, 12, x FOR PEER REVIEW 8 of 12

Figure 5. Effects of feeding laying hens with diets containing P. carotinifaciens of different particle sizes Figure 5. Effects of feeding laying hens with diets containing P. carotinifaciens of different particle sizes (Panaferd-AXFigure 5. Effects and of feedingPanaferd-P) laying for he 21ns withdays dietson the containing (a) adonixanthin P. carotinifaciens concentration of different and particle (b) total sizes Z- (Panaferd-AX and Panaferd-P) for 21 days on the (a) adonixanthin concentration and (b) total Z-isomer isomer(Panaferd-AX ratio in andegg yolk.Panaferd-P) Error bars for depict21 days the on standard the (a) deviationadonixanthin (n = 8).concentration Means with and different (b) total letters Z- ratio in egg yolk. Error bars depict the standard deviation (n = 8). Means with different letters within withinisomer ratioeach inday egg are yolk. significantly Error bars differentdepict the (p standard < 0.05), deviationwhereas means(n = 8). withMeans similar with differentletters are letters not each day are significantly different (p < 0.05), whereas means with similar letters are not different. different.within each day are significantly different (p < 0.05), whereas means with similar letters are not 3.3. Evaluationdifferent. of Egg Yolk Pigmentation 3.3. Evaluation of Egg Yolk Pigmentation 3.3. EvaluationThe evaluation of Egg Yolk of the Pigmentation egg yolk pigmentation was performed by examining the appearances (FigureThe6), evaluation the yolk color of the fan egg score, yolk and pigmentation L*, a*, and was b* values performed of the by egg examining yolk (Figure the7 appearances). Feeding (FigurehensThe with 6), evaluation dietsthe yolk containing color of the fan Panaferd-AXegg score, yolk and pigmentation L*, and a*, and Panaferd-P b* was values performed markedly of the egg by improved yolk examining (Figure the yolkthe7). Feedingappearances appearance hens with(Figureas well diets 6), as containingthe the yolk color color fanPanaferd-AX fan score; score, the and score L*,Panaferd-P increaseda*, and b* markedly values by a value of improved the of egg two yolk the or (Figureyolk more appearance compared 7). Feeding as to henswell the aswithcontrol the diets color group. containing fan score; When Panaferd-AXthe comparing score increased Panaferd-AX-and Panaferd-P by a valu and emarkedly of Panaferd-P-containing two or improved more compared the yolk diets, to appearance the the control latter asgroup. had well a Whenassignificantly the comparingcolor fan higher score; Panaferd-AX- yolk the color score fan increasedand score Panaferd-P-contain than by the a valu former.e of twoing These diets, or resultsmore the comparedlatter are correlated had ato significantly the with control the egg group.higher yolk yolkWhenconcentrations color comparing fan score of astaxanthin,Panaferd-AX- than the former. adonirubin, and Panaferd-P-containThese andresults adonixanthin. are correlateding diets, In thiswith thestudy, latter the egg had when yolk a significantly hens concentrations were fed higher with of astaxanthin,yolkdiets color containing fan adonirubin, scoreP. carotinifaciens than andthe former. adonixanthin.(astaxanthin These results In concentration:this arestudy, correlated when 8 mghens with/kg), were the the egg fed yolk yolk with color concentrations diets fan containing score was of P.astaxanthin,over carotinifaciens 14. In contrast,adonirubin, (astaxanthin when and fed concentration: adonixanthin. with diets rich 8 In mg/kg), this in lutein study, the suchyolk when color as hens alfalfa fan were score and fed marigoldwas with over diets 14. flower Incontaining contrast, (lutein whenP.concentration: carotinifaciens fed with approximately (astaxanthindiets rich in concentration: 18–30lutein mg such/kg) 8as[ mg/kg),2 ]alfalfa or diets the and rich yolk marigold in color lycopene fan flower score such was as(lutein tomatoesover 14.concentration: In (lycopene contrast, approximatelywhenconcentration: fed with 18–30 100 diets mg mg/kg) /richkg) [in27 [2] ],lutein theor diets yolk such rich color as in fanalfalfalycopene scores and such were marigold as around tomatoes flower 8–12. (lycopene This(lutein suggests concentration: that the 100pigmentationapproximately mg/kg) [27], e ffi18–30 ciencythe yolkmg/kg) of color astaxanthin [2] fanor diets scores in rich egg were in yolk ly arcopene isound higher 8–12.such than asThis fortomatoes othersuggests carotenoids (lycopene that the concentration: suchpigmentation as lutein efficiency100and mg/kg) lycopene. of [27],astaxanthin Furthermore, the yolk in coloregg feeding yolk fan isscores hens higher withwere than Panaferd-AX ar foround other 8–12. carotenoids and This Panaferd-P suggests such asthat increased lutein the andpigmentation the lycopene. a* value Furthermore,efficiencyand decreased of astaxanthin feeding the L* and hens in b* eggwith values. yolk Panaferd-AX Theis higher appearances than and forPanaferd-P of other egg yolkscarotenoids increased showed such the these a*as trends valuelutein and welland decreased (Figurelycopene.6); theFurthermore,that L* is, and the feedingb* values.feeding of theThehens astaxanthin-rich appearances with Panaferd-AX of dietsegg yolksand contributed Panaferd-P showed to these a increased redness trends enhancement thewell a* (Figure value ofand 6); the thatdecreased egg is, yolk. the feedingthe L* and of theb* values.astaxanthin-rich The appearances diets contributed of egg yolks to a showed redness theseenhancement trends well of the (Figure egg yolk. 6); that is, the feeding of the astaxanthin-rich diets contributed to a redness enhancement of the egg yolk.

Figure 6. AppearancesAppearances of of egg egg yolks yolks ( (aa)) before before and and after after 21 21 days days of of f feedingeeding hens with diets containing P.Figure carotinifacienscarotinifaciens 6. Appearances powderspowders of ofofegg didifferent ﬀyolkserent (a particleparticle) before sizes: sizes:and after ((bb)) Panaferd-AX Panaferd-AX21 days of feeding andand ((c chens)) Panaferd-P.Panaferd-P. with diets containing P. carotinifaciens powders of different particle sizes: (b) Panaferd-AX and (c) Panaferd-P.

Symmetry 2020, 12, 923 9 of 13 Symmetry 2020, 12, x FOR PEER REVIEW 9 of 12

Figure 7. Effects of feeding laying hens with diets containing P. carotinifaciens of different particle sizes Figure(Panaferd-AX 7. Effects and of feeding Panaferd-P) laying for he 21ns days with ondiets the containing (a) yolk color P. carotinifaciens fan score and of ( bdifferent) L*, (c) a*,particle and (sizesd) b* (Panaferd-AXvalues of egg yolk. and Panaferd-P) Error bars depict for 21 the days standard on the deviation(a) yolk color (n = 8).fan Meansscore and with (b di) ffL*,erent (c) a*, letters andwithin (d) b* valueseach day of egg are significantlyyolk. Error bars diff depicterent ( pthe< 0.05),standard whereas deviation means (n with= 8). Means similar with letters different are not letters different. within each day are significantly different (p < 0.05), whereas means with similar letters are not different. 3.4. Evaluation of Other Egg Qualities 3.4. EvaluationThe effects of ofother feeding Egg Qualities hens with diets containing Panaferd-AX- and Panaferd-P on egg qualities (eggThe weight, effects yolk of weight,feeding albumenhens with height, diets containing and Haugh Panaferd-AX- unit) have been and shown Panaferd-P in Table on2 .egg There qualities were (eggno significant weight, yolk diff erencesweight, inalbumen egg weight, height, yolk and weight, Haugh albumenunit) have height, been shown and the in Haugh Table 2. unit There between were nothe significant different diets differences (with and in egg without weight, adding yolk P.we carotinifaciensight, albumen). height, Furthermore, and the the Haugh particle unit size between of the thedried differentP. carotinifaciens diets (withcell and powder without did notadding affect P. the carotinifaciens above-mentioned). Furthermore, egg qualities. the particle Yang et size al. did of the not driedobserve P. significantcarotinifaciens diff erencescell powder in egg did weight, not affect yolk the height, above-me and thentioned Haugh egg unit qualities. when feeding Yang 1.3et al. mg did/kg notastaxanthin observe tosignificant Hy-Line Browndifferences laying in hensegg weight, [45]. Walker yolk etheight, al. also and did the not Haugh observe unit significant when feeding effects 1.3 on mg/kgthe yolk astaxanthin weight and to the Hy-Line Haugh Brown unit when laying Hy-Line hens W-36[45]. Walk henser were et al. fed also up todid 400 not mg observe/kg of astaxanthinsignificant effectsin their on diets the [ 6yolk]. Our weight results and are the consistent Haugh unit with when these Hy-Line previous W-36 studies hens in that were they fed suggest up to 400 that mg/kg feeding of astaxanthinhens with astaxanthin in their diets does [6]. not Our have results significant are cons effectsistent on with the egg these quality previous other studies than the in carotenoid that they suggestconcentration that feeding and egg hens color. with astaxanthin does not have significant effects on the egg quality other than the carotenoid concentration and egg color.

Table 2. Effects of feeding laying hens with diets containing P. carotinifaciens powders of different particle sizes (Panaferd-AX and Panaferd-P) on egg quality (1Feeding diets without P. carotinifaciens; 2Calculated as 100 × log (H + 7.6 – 1.7 × W0.37), where H is the albumen height (mm) and W is the egg weight (g)).

Diet Items Days Control1 Panaferd-AX Panaferd-P SEM p-Value Egg weight (g) 0 57.3 57.7 55.3 4.8 0.576 4 58.0 55.8 59.2 5.8 0.517 7 58.8 58.4 59.0 3.6 0.947 14 60.4 58.6 59.9 3.0 0.503

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Table 2. Eﬀects of feeding laying hens with diets containing P. carotinifaciens powders of diﬀerent particle sizes (Panaferd-AX and Panaferd-P) on egg quality (1 Feeding diets without P. carotinifaciens; 2 Calculated as 100 log (H + 7.6 1.7 W0.37), where H is the albumen height (mm) and W is the egg × − × weight (g)).

Diet Items Days Control 1 Panaferd-AX Panaferd-P SEM p-Value 0 57.3 57.7 55.3 4.8 0.576 4 58.0 55.8 59.2 5.8 0.517 Egg weight 7 58.8 58.4 59.0 3.6 0.947 (g) 14 60.4 58.6 59.9 3.0 0.503 21 59.8 59.7 61.0 3.6 0.701 0 13.8 14.6 13.0 2.6 0.459 4 14.2 13.7 15.2 2.8 0.578 Yolk weight 7 14.9 14.5 14.6 1.1 0.794 (g) 14 15.5 14.9 15.3 1.0 0.442 21 16.0 15.7 15.6 0.8 0.626 0 7.0 7.1 7.9 1.0 0.121 4 7.7 8.1 7.4 1.6 0.706 Albumen 7 7.4 7.7 7.7 1.2 0.885 height (mm) 14 8.3 7.6 8.5 1.2 0.250 21 7.1 7.5 6.8 1.3 0.598 0 84.7 84.3 89.9 10.1 0.468 4 91.6 93.4 86.2 10.6 0.384 Haugh unit 2 7 86.6 91.5 89.1 7.5 0.441 14 94.6 90.4 95.7 6.4 0.240 21 83.2 87.0 82.8 10.1 0.664

4. Conclusions Feeding hens with dried P. carotinifaciens cell powders (Panaferd-AX and Panaferd-P) increased the concentrations of valuable carotenoids (astaxanthin, adonirubin, and adonixanthin) in their egg yolk and enhanced the egg yolk pigmentation. Their pigmentation eﬃciencies were improved by the pulverization of the P. carotinifaciens powder. Moreover, this study accurately evaluated the total Z-isomer ratios of astaxanthin, adonirubin, and adonixanthin in egg yolk for the ﬁrst time, using normal-phase HPLC with an improved solvent system. The all-E-isomers of carotenoids are the most predominant geometric isomers in P. carotinifaciens. However, large amounts of astaxanthin and adonirubin Z-isomers were present (over 25% and 20% of the total Z-isomer ratio, respectively) in the egg yolk. Since several types of carotenoid Z-isomers, including astaxanthin, have a higher bioavailability and bioactivity than the all-E-isomers, the information produced by this study is valuable for enhancing the nutritional value of hens’ egg yolk through feeding hens astaxanthin-rich diets.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-8994/12/6/923/s1, Figure S1: Calculation of Q-ratio as an indicator of the Z-peak intensity, Table S1: Absorption maxima and relative intensities of the Z-peaks for adonirubin, astaxanthin, and adonixanthin isomers. Author Contributions: Conceptualization, M.H., Y.K., and Y.H.; analysis, M.H., Y.K., and Y.H.; investigation, M.H., Y.K., S.J. and Y.H.; resources, Y.K., K.H., and T.U.; writing—original draft preparation, M.H. and Y.H.; writing—review and editing, Y.K., K.H., T.U., and S.J.; project administration, K.H. and T.U; All authors have read and agreed to the published version of the manuscript. Funding: This work was partly supported by JSPS KAKENHI Grant Number 19K15779. Acknowledgments: We thank our laboratory members for their helpful discussions. Conﬂicts of Interest: The authors declare no conﬂict of interest. Symmetry 2020, 12, 923 11 of 13

References

1. Nimalaratne, C.; Wu, J. Hen egg as an antioxidant food commodity: A review. Nutrients 2015, 7, 8274–8293. [CrossRef][PubMed] 2. Karadas, F.; Grammenidis, E.; Surai, P.F.; Acamovic, T.; Sparks, N.H.C. Effects of carotenoids from lucerne, marigold and tomato on egg yolk pigmentation and carotenoid composition. Br. Poult. Sci. 2006, 47, 561–566. [CrossRef][PubMed] 3. Karadas, F.; Surai, P.F.; Sparks, N.H.; Grammenidis, E. Effects of maternal dietary supplementation with three sources of carotenoids on the retinyl esters of egg yolk and developing quail liver. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2005, 140, 430–435. [CrossRef][PubMed] 4. Shin, H.S.; Kim, J.W.; Kim, J.H.; Lee, D.G.; Lee, S.; Kil, D.Y. Effect of feeding duration of diets containing corn distillers dried grains with solubles on productive performance, egg quality, and lutein and zeaxanthin concentrations of egg yolk in laying hens. Poult. Sci. 2016, 95, 2366–2371. [CrossRef] 5. Skˇrivan,M.; Englmaierová, M.; Skˇrivanová, E.; Bubancová, I. Increase in lutein and zeaxanthin content in the eggs of hens fed marigold flower extract. Czech J. Anim. Sci. 2015, 60, 89–96. [CrossRef] 6. Walker, L.A.; Wang, T.; Xin, H.; Dolde, D. Supplementation of laying-hen feed with palm tocos and algae astaxanthin for egg yolk nutrient enrichment. J. Agric. Food Chem. 2012, 60, 1989–1999. [CrossRef] 7. Magnuson, A.D.; Sun, T.; Yin, R.; Liu, G.; Tolba, S.; Shinde, S.; Lei, X.G. Supplemental microalgal astaxanthin produced coordinated changes in intrinsic antioxidant systems of layer hens exposed to heat stress. Algal Res. 2018, 33, 84–90. [CrossRef] 8. Sato, W.; Nagai, H.; Kawashima, Y.; Ikarashi, M.; Sakai, Y. Formula Feed for Poultry. U.S. Patent 15,575,965, 31 May 2018. 9. Higuera-Ciapara, I.; Felix-Valenzuela, L.; Goycoolea, F.M. Astaxanthin: A review of its chemistry and applications. Crit. Rev. Food Sci. Nutr. 2006, 46, 185–196. [CrossRef] 10. Guerin, M.; Huntley, M.E.; Olaizola, M. Haematococcus astaxanthin: Applications for human health and nutrition. Trends Biotechnol. 2003, 21, 210–216. [CrossRef] 11. Fakhri, S.; Abbaszadeh, F.; Dargahi, L.; Jorjani, M. Astaxanthin: A mechanistic review on its biological activities and health benefits. Pharm. Res. 2018, 136, 1–20. [CrossRef] 12. Akiba, Y.; Sato, K.; Takahashi, K.; Takahashi, Y.; Furuki, A.; Konashi, S.; Nishida, H.; Tsunekawa, H.; Hayasaka, Y.; Nagao, H. Pigmentation of egg yolk with yeast Phaffia rhodozyma containing high concentration of astaxanthin in laying hens fed on a low-carotenoid diet. Jpn. Poult. Sci. 2000, 37, 77–85. [CrossRef] 13. Akiba, Y.; Sato, K.; Takahashi, K.; Toyomizu, M.; Takahashi, Y.; Konashi, S.; Nishida, H.; Tsunekawa, H.; Hayasaka, Y.; Nagao, H. Improved pigmentation of egg yolk by feeding of yeast Phaffia rhodozyma containing high concentration of astaxanthin in laying hens. Jpn. Poult. Sci. 2000, 37, 162–170. [CrossRef] 14. Nagai, H.; Sato, W.; Takahashi, T.; Sato, H. Carotenoid Production Method. European Patent 3,441,464, 13 February 2019. 15. Hirasawa, K.; Satoh, H.; Yoneda, H.; Yata, T.; Azuma, M. Method for Producing Astaxanthin by Fermentation. United States Patent 10,240,215, 26 March 2019. 16. Honda, M.; Kageyama, H.; Hibino, T.; Sowa, T.; Kawashima, Y. Efficient and environmentally friendly method for carotenoid extraction from Paracoccus carotinifaciens utilizing naturally occurring Z-isomerization-accelerating catalysts. Proc. Biochem. 2020, 89, 146–154. [CrossRef] 17. Maoka, T.; Yasui, H.; Ohmori, A.; Tokuda, H.; Suzuki, N.; Osawa, A.; Shindo, K.; Ishibashi, T. Anti-oxidative, anti-tumor-promoting, and anti-carcinogenic activities of adonirubin and adonixanthin. J. Oleo Sci. 2013, 62, 181–186. [CrossRef][PubMed] 18. Hirasawa, K.; Tsubokura, A. Method for Separating Carotenoid. United States Patent 8,853,460, 7 October 2014. 19. Li, H.; Jin, L.; Wu, F.; Thacker, P.; Li, X.; You, J.; Wang, X.; Liu, S.; Li, S.; Xu, Y. Effect of red pepper (Capsicum frutescens) powder or red pepper pigment on the performance and egg yolk color of laying hens. Asian Australas. J. Anim. Sci. 2012, 25, 1605–1610. [CrossRef] 20. Hayasaka, Y.; Tsunekawa, H. Color-Improving Feed for Laying Hen. Japan Patent 07-115915, 9 May 1995. 21. Yu, W.; Liu, J. Astaxanthin isomers: Selective distribution and isomerization in aquatic animals. Aquaculture 2020, 520, 734915. [CrossRef] Symmetry 2020, 12, 923 12 of 13

22. Honda, M.; Murakami, K.; Watanabe, Y.; Higashiura, T.; Fukaya, T.; Kanda, H.; Goto, M. The E/Z isomer ratio of lycopene in foods and effect of heating with edible oils and fats on isomerization of (all-E)-lycopene. Eur. J. Lipid Sci. Technol. 2017, 119, 1600389. [CrossRef] 23. Humphries, J.M.; Khachik, F. Distribution of lutein, zeaxanthin, and related geometrical isomers in fruit, vegetables, wheat, and pasta products. J. Agric. Food Chem. 2003, 51, 1322–1327. [CrossRef] 24. Boileau, T.W.M.; Boileau, A.C.; Erdman, J.W., Jr. Bioavailability of all-trans and cis-isomers of lycopene. Exp. Biol. Med. 2002, 227, 914–919. [CrossRef] 25. Honda, M.; Nakayama, Y.; Nishikawa, S.; Tsuda, T. Z-Isomers of lycopene exhibit greater liver accumulation than the all-E-isomer in mice. Biosci. Biotechnol. Biochem. 2020, 84, 428–431. [CrossRef] 26. Coral-Hinostroza, G.N.; Ytrestøyl, T.; Ruyter, B.; Bjerkeng, B. Plasma appearance of unesterified astaxanthin geometrical E/Z and optical R/S isomers in men given single doses of a mixture of optical 3 and 3’ R/S isomers of astaxanthin fatty acyl diesters. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2004, 139, 99–110. [CrossRef] [PubMed] 27. Honda, M.; Ishikawa, H.; Hayashi, Y. Alterations in lycopene concentration and Z-isomer content in egg yolk of hens fed all-E-isomer-rich and Z-isomer-rich lycopene. Anim. Sci. J. 2019, 90, 1261–1269. [CrossRef] [PubMed] 28. Kang, D.K.; Kim, S.I.; Cho, C.H.; Yim, Y.H.; Kim, H.S. Use of lycopene, an antioxidant carotinoid, in laying hens for egg yolk pigmentation. Asian Australas. J. Anim. Sci. 2003, 16, 1799–1803. [CrossRef] 29. Yang, C.; Zhang, H.; Liu, R.; Zhu, H.; Zhang, L.; Tsao, R. Bioaccessibility, cellular uptake, and transport of astaxanthin isomers and their antioxidative effects in human intestinal epithelial Caco-2 cells. J. Agric. Food Chem. 2017, 65, 10223–10232. [CrossRef][PubMed] 30. Yang, C.; Zhang, L.; Zhang, H.; Sun, Q.; Liu, R.; Li, J.; Wu, L.; Tsao, R. Rapid and efficient conversion of all-E-astaxanthin to 9Z-and 13Z-isomers and assessment of their stability and antioxidant activities. J. Agric. Food Chem. 2017, 65, 818–826. [CrossRef] 31. Yang, C.; Hassan, Y.I.; Liu, R.; Zhang, H.; Chen, Y.; Zhang, L.; Tsao, R. Anti-inflammatory effects of different astaxanthin isomers and the roles of lipid transporters in the cellular transport of astaxanthin isomers in Caco-2 cell monolayers. J. Agric. Food Chem. 2019, 67, 6222–6231. [CrossRef] 32. Su, F.; Xu, H.; Yang, N.; Liu, W.; Liu, J. Hydrolytic efficiency and isomerization during de-esterification of natural astaxanthin esters by saponification and enzymolysis. Elec. J. Biotechnol. 2018, 34, 37–42. [CrossRef] 33. Honda, M.; Sowa, T.; Kawashima, Y. Thermal-and photo-induced isomerization of all-E-and Z-isomer-rich xanthophylls: Astaxanthin and its structurally related xanthophylls, adonirubin and adonixanthin. Eur. J. Lipid Sci. Technol. 2020, 122, 1900462. [CrossRef] 34. LaFountain, A.M.; Frank, H.A.; Prum, R.O. Carotenoids from the crimson and maroon plumages of Old World orioles (Oriolidae). Arch. Biochem. Biophys. 2013, 539, 126–132. [CrossRef] 35. Kaga, K.; Honda, M.; Adachi, T.; Honjo, M.; Kanda, H.; Goto, M. Nanoparticle formation of PVP/astaxanthin inclusion complex by solution-enhanced dispersion by supercritical fluids (SEDS): Effect of PVP and astaxanthin Z-isomer content. J. Supercrit. Fluids 2018, 136, 44–51. [CrossRef] 36. Zhao, L.; Chen, F.; Zhao, G.; Wang, Z.; Liao, X.; Hu, X. Isomerization of trans-astaxanthin induced by copper (II) ion in ethanol. J. Agric. Food Chem. 2005, 53, 9620–9623. [CrossRef][PubMed] 37. Qiu, D.; Wu, Y.C.; Zhu, W.L.; Yin, H.; Yi, L.T. Identification of geometrical isomers and comparison of different isomeric samples of astaxanthin. J. Food Sci. 2012, 77, C934–C940. [CrossRef][PubMed] 38. Yuan, J.P.; Chen, F. Isomerization of trans-astaxanthin to cis-isomers in organic solvents. J. Agric. Food Chem. 1999, 47, 365–3660. [CrossRef][PubMed] 39. Honda, M.; Kodama, T.; Kageyama, H.; Hibino, T.; Kanda, H.; Goto, M. Enhanced solubility and reduced crystallinity of carotenoids, β-carotene and astaxanthin, by Z-isomerization. Eur. J. Lipid Sci. Technol. 2018, 120, 1800191. [CrossRef] 40. Honda, M.; Kageyama, H.; Hibino, T.; Ichihashi, K.; Takada, W.; Goto, M. Isomerization of commercially important carotenoids (lycopene, β-carotene, astaxanthin) by natural catalysts: Isothiocyanates and polysulfides. J. Agric. Food Chem. 2020, 68, 3228–3237. [CrossRef][PubMed] 41. Mitsuhashi, K.; Someya, T.; Hayashi, M.; Yamada, M.; Uchizawa, S.; Hirasawa, K.; Kawashima, Y. Method for producing carotenoid-containing composition, and carotenoid-containing composition. U.S. Patent 9,687,422, 27 June 2017. 42. Vishwanathan, R.; Wilson, T.A.; Nicolosi, R.J. Bioavailability of a nanoemulsion of lutein is greater than a lutein supplement. Nano. Biomed. Eng. 2009, 1, 38–49. [CrossRef] Symmetry 2020, 12, 923 13 of 13

43. Affandi, M.M.M.; Julianto, T.; Majeed, A.B.A. Enhanced oral bioavailability of astaxanthin with droplet size reduction. Food Sci. Technol. Res. 2012, 18, 549–554. [CrossRef] 44. Yonekura, L.; Nagao, A. Intestinal absorption of dietary carotenoids. Mol. Nutr. Food Res. 2007, 51, 107–115. [CrossRef] 45. Yang, Y.X.; Kim, Y.J.; Jin, Z.; Lohakare, J.D.; Kim, C.H.; Ohh, S.H.; Lee, S.H.; Choi, J.Y.; Chae, B.J. Effects of dietary supplementation of astaxanthin on production performance, egg quality in layers and meat quality in finishing pigs. Asia Australas. J. Anim. Sci. 2006, 19, 1019–1025. [CrossRef]

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