Effects of Astaxanthin-rich Dried Cell Powder from Paracoccus carotinifaciens (Panaferd^®-AX) on Fatty Acid Composition, Carotenoids, Free Amino Acids, and Imidazole Dipeptides in Broiler Chickens

Abstract

This study aimed to evaluate the effect of supplementation with Panaferd^®-AX, an astaxanthin-rich dried cell powder obtained from the carotenoid-producing bacterium Paracoccus carotinifaciens, on the muscle concentration of carotenoids, fatty acids, free amino acids, and imidazole dipeptides in broiler chickens. Thirty male broiler chickens (Ross 308) were allocated to three groups at 14 days of age. Until 28 days of age, the control group was fed a basal diet; whereas the two test groups were fed a basal diet supplemented with Panaferd^®-AX at 0.025% or 0.15%, corresponding to 5 ppm or 30 ppm astaxanthin, respectively. At the end of the experiment, body weight, body weight gain, feed intake, feed conversion rate, and tissue weight did not differ between the groups. Feeding Panaferd^®-AX increased muscle astaxanthin, as well as plasma zeaxanthin, and lutein concentrations, but did not affect fatty acid composition. In the pectoralis major muscle, it decreased lipid peroxidation and drip loss; while increasing carnosine content. In summary, Panaferd^®-AX increased muscle antioxidant content (i.e., carotenoids and carnosine), which consequently reduced lipid peroxidation and drip loss in the skeletal muscle of broiler chickens.

Introduction

The appearance and flavor of poultry meat (e.g., color, firmness, odor) are crucial factors guiding acceptance by consumers[1,2]. As chicken muscle has a higher polyunsaturated fatty acid content than other meats, freshness is quickly lost because of lipid peroxidation[3], leading to a rancid odor and poor flavor[4].

As a result of lipid peroxidation, oxidative stress induces drip loss, nutritional value loss, and flavor degradation[4,5,6]. Lipid peroxidation in chicken skeletal muscles can be counteracted in part by supplementation with feed rich in antioxidants[7,8,9,10].

Astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) is a xanthophyll carotenoid widely distributed in nature[11,12]. Because of its red color, astaxanthin is used as a pigment in aquaculture[11,12,13] and in the poultry industry[14]. In addition, astaxanthin has 10-times and 100-times higher antioxidant activity against singlet oxygen and free radicals than β-carotene or α-tocopherol[15]. Dietary supplementation with astaxanthin has been shown to alleviate lipid peroxidation in the skeletal muscles of broiler chickens[16,17].

The present study examined the effect of dietary supplementation with astaxanthin on muscle carotenoids, free fatty acids, free amino acids, and imidazole dipeptides in broiler chickens. Currently, commercially available food-grade astaxanthin is produced by bacteria of the genus Paracoccus[18,19]. This study used an astaxanthin‐rich dried cell powder (Panaferd^®‐AX) obtained from carotenoid‐producing Paracoccus carotinifaciens as a dietary supplement.

Materials and Methods

Animals and experimental design

All animal procedures were reviewed and approved by the Animal Care and Use Committee of Kagoshima University (approval number: A22012). Thirty male 0-day-old Ross 308 broiler chicks were provided by a commercial hatchery (Kumiai Hina Center, Kagoshima, Japan). The chicks were housed in an electrically heated battery brooder inside a temperature-controlled room. The starting temperature of 30°C was lowered by 1°C every 2 days until it reached 25°C, whereupon it was maintained constant. A continuous 20-h light and 4-h dark photoperiod was used. The chicks were given ad libitum water and a semi-purified corn/soybean meal diet (starter diet) until 10 days of age, followed by water and a basal diet (grower diet) (Table 1). At 14 days of age, chicks of similar body mass were selected and housed individually in wire-bottom aluminum cages (40 × 50 × 60 cm) inside a temperature-controlled room at 25°C. The chicks were randomly allocated to one of three diets (10 birds per diet): i) basal diet (control group), ii) basal diet supplemented with Panaferd^®-AX (ENEOS Techno Materials Corporation, Kawasaki, Japan) at 0.025% (5 ppm astaxanthin), and iii) basal diet supplemented with Panaferd^®-AX at 0.15% (30 ppm astaxanthin). The carotenoid contents of Panaferd^®-AX and each experimental diet are summarized in Table S1. The fatty acid and constituent amino acid compositions are reported in Tables S2 and S3. Diets were formulated to meet the nutritional requirements of the broilers[20]. Body weight and feed intake were measured to calculate the feed conversion ratio. At 28 days of age, the chickens were weighed and euthanized by cervical dislocation under carbon dioxide anesthesia. The right half of fillets (the pectoralis major muscle) was used for the determination of drip loss, while a portion of the remaining half was snap-frozen in liquid nitrogen and stored at −80°C.

Table 1.  Basal diet composition and analysis.

Ingredients (g/100 g)		Starter diet		Grower diet
	Corn meal	48.85		53.10
	Soybean meal	42.6		37.78
	Corn oil	4.45		5.20
	CaCO3	1.00		1.00
	CaHPO4	1.50		1.30
	NaCl	0.50		0.50
	DL-Methionine	0.25		0.32
	L-Lysine monohydrochloride	0.20		0.15
	L-Threonine	0.10		0.10
	L-Valine	0.05		0.05
	Mineral and vitamin premix1	0.50		0.50
Calculated analysis
	Crude protein (%)	23.30		21.50
	Metabolizable energy (Mcal/kg)	3.00		3.11

¹Content per kg of the vitamin and mineral premix: vitamin A (90 mg), vitamin D3 (1 mg), DL-alpha-tocopherol acetate (2000 mg), vitamin K3 (229 mg), thiamin nitrate (444 mg), riboflavin (720 mg), calcium d-pantothenate (2174 mg), nicotinamide (7000 mg), pyridoxine hydrochloride (700 mg), biotin (30 mg), folic acid (110 mg), cyanocobalamin (2 mg), calcium iodinate (108 mg), MgO (198,991 mg), MnSO₄ (32,985 mg), ZnSO₄ (19,753 mg), FeSO₄ (43,523 mg), CuSO₄ (4019 mg), and choline chloride (299,608 mg).

Carotenoid concentration

Identification and determination of individual carotenoids in plasma and pectoralis major muscle was carried out using ultraviolet–visible spectroscopy and liquid chromatography tandem mass spectrometry, as described by Maoka et al.[21]. Analysis was outsourced to the Research Institute for Production Development (Kyoto, Japan).

Fatty acid concentration

The pectoralis major muscles of broiler chickens were lyophilized, and then 0.02 g of that was extracted and methylated using a fatty acid methylation kit (06482-04; Nacalai Tesque, Kyoto, Japan), followed by purification with the Fatty Acid Methyl Ester Purification Kit (06483-94; Nacalai Tesque), according to the manufacturer’s instructions.

The fatty acid concentration in skeletal muscle of chickens was determined using a GC-2014 gas chromatographer (Shimadzu Co., Kyoto, Japan) equipped with a flame ionization detector. The temperature of the injector and detector was set at 250°C. Helium was used as the carrier gas at a flow rate of 14.2 mL/min. The injection volume was 1 µL and the split ratio was set at 10:1 for total fatty acids. A DB-23 capillary column (60 m × 0.25 mm, 0.15 µm, 50% cyanopropylmethylpolysiloxane; Agilent Technologies, Santa Clara, CA, USA) was used. The oven temperature was kept at 50°C for 1 min, increased to 175°C at a rate of 25°C/min, further increased to 230°C at a rate of 4°C/min, and held at 230°C for 5 min. A mixture of fatty acid methyl esters (F.A.M.E. Mix C4–C24, Supelco; Sigma-Aldrich, St. Louis, MO, USA) was used as a standard. The composition of each fatty acid in the pectoral muscle is expressed as a percentage.

Muscle malondialdehyde (MDA) concentration

To evaluate lipid peroxidation in the skeletal muscle of chickens, the MDA concentration was determined colorimetrically as a 2-thiobarbituric acid-reactive substance according to the method described by Ohkawa et al.[22]. Briefly, 0.3 g of the pectoralis major muscle was homogenized in 1 mL of 1.15% KCl and centrifuged at 20,000 × g for 5 min. Then, 80 μL of each supernatant was mixed with 80 μL of 8.1% sodium dodecyl sulfate, 220 μL of 20% acetic acid (pH 3.5), and 300 μL of 0.8% 2-thiobarbituric acid. After vortexing, the samples were incubated at 95°C for 1 h and then transferred to ice. After adding 1 mL of butanol-pyridine 15:1 (v/v), they were vortexed and centrifuged at 20,000 × g for 5 min. Absorbance of the supernatant, which contained the butanol-pyridine layer, was measured at excitation and emission wavelengths of 535 and 585 nm, respectively.

Determination of skeletal muscle drip loss

Drip loss was measured using the method described by Berri et al.[23]. The pectoralis major muscle was weighed immediately after dissection, placed in a plastic bag, stored at 4°C for 48 h, and wiped and weighed again. The difference in mass corresponded to drip loss, which was expressed as a percentage of the initial muscle mass.

Free amino acid and imidazole dipeptide concentrations

The concentration of free muscle amino acids and imidazole dipeptides was determined according to previously reported methods[24]. One gram of frozen pectoralis major muscle was weighed and homogenized in 10 mL ice-cold 0.1 mol/L HCl with 100 μmol/L d-norvaline (FUJIFILM Wako Chemicals, Osaka, Japan) as internal standard. Hexane (10 mL) was added, mixed by vortexing for 1 min, and the solution was centrifuged at 22,000 × g for 5 min. Next, 400 μL of the lower layer was mixed with 1,200 µL of water-acetonitrile (1:2 v/v) by vortexing for 1 min, and centrifuged again at 20,000 × g for 5 min. The supernatant was filtered using a 0.2-µm pore size filter and analyzed by high-performance liquid chromatography on a NexeraX2 system (Shimadzu Co., Ltd., Kyoto, Japan) with a Kinetex 2.6 µm column (EVO C18, 100 × 3.0 mm; Phenomenex, Torrance, CA, USA). Amino acids were separated as described by the Shimadzu Corporation (2019) and their content was expressed as mg/100 g of tissue.

Statistical analysis

All statistical analyses were performed using R version 4.4.3[25]. Comparisons were performed using Tukey’s multiple comparison test. Because a portion of carotenoids was not detected in plasma and the pectoralis major muscle of control chickens, their comparison in test samples was performed using Student’s t-test. Statistical significance was set at p < 0.05, and data are expressed as the means ± standard error of the mean.

Results and Discussion

Final body weight, body weight gain, and feed intake increased in broiler chickens fed Panaferd^®-AX at 5 ppm and 30 ppm astaxanthin, but the difference was not statistically significant compared to controls (Table 2). Similarly, dietary supplementation with Panaferd^®-AX did not improve feed conversion ratios or weight of the pectoralis major muscle, thigh muscles, liver, heart, and abdominal fat tissue (Table 2). These results were consistent with those of our previous study on dietary administration of Panaferd^®-P[16], another supplement containing ground products found in Panaferd^®-AX. Hence, Panaferd^®-AX affected neither growth performance nor muscle yield in broiler chickens. In contrast, Jeong and Kim[26] and Awadh and Zangana[27] reported that dietary supplementation with astaxanthin improved weight gain and feed conversion ratios in broiler chickens. This discrepancy may be ascribed to the use of different astaxanthin sources (i.e., Phaffia rhodozyma, Haematococcus pluvialis, and P. carotinifaciens), rather than widely different doses (2–40 ppm). Unlike yeast (P. rhodozyma) and algae (H. pluvialis), P. carotinifaciens are gram-positive bacteria, whose cell wall contains mannan and its hydrolyzed form, mannan oligosaccharides[28]. These compounds act as prebiotics to regulate gut microbiota[29,30]. In particular, when given as a feed additive, mannan oligosaccharides promote growth in chickens[31]. These results suggest that differences in astaxanthin sources may affect growth performance in broiler chickens.

Table 2.  Effect of dietary supplementation with Panaferd^®-AX on growth performance and tissue weight of broiler chickens.

							Panaferd®-AX
			Control				5 ppm astaxanthin				30 ppm astaxanthin
Growth performance
	Final body weight (g)		1064.81	±	70.03		1215.53	±	45.17		1245.10	±	62.79
	Body weight gain (g)		703.73	±	68.78		852.84	±	42.39		882.53	±	59.77
	Feed intake (g)		1023.67	±	109.70		1225.11	±	80.45		1276.94	±	63.97
	Feed conversion ratio		1.46	±	0.09		1.48	±	0.14		1.49	±	0.11
Tissue weights
	Pectoralis major muscle (g)		192.76	±	14.39		223.97	±	10.49		230.15	±	14.20
	Thigh muscles (g)		200.18	±	13.75		226.69	±	9.43		232.99	±	10.80
	Liver (g)		22.52	±	2.05		27.15	±	1.11		26.65	±	1.49
	Heart (g)		5.30	±	0.46		6.42	±	0.25		6.49	±	0.42
	Abdominal fat tissue (g)		3.24	±	0.90		4.42	±	1.14		4.75	±	1.10

Results are expressed as means ± standard error of the mean (n = 10).

Table 3 shows the carotenoid composition of plasma and pectoralis major muscles in chickens. P. carotinifaciens-derived pigments (i.e., astaxanthin, adonixanthin, canthaxanthin, and adonirubin) were detected in the plasma and pectoralis major muscle of chickens fed the Panaferd^®-AX-supplemented diet, but not in those fed the basal diet. Although astaxanthin accounted for more than 60% of carotenoids in Panaferd^®-AX, its content in the plasma and pectoralis major muscle was akin to that of other carotenoids. This phenomenon might be due to differences in carotenoid absorption by chickens[32,33]. Interestingly, although corn-derived yellow xanthophylls (i.e., lutein and zeaxanthin) were reported to be undetected in Panaferd^®-AX[34], they accumulated in the plasma of broiler chickens fed Panaferd^®-AX-supplemented diets. In contrast, β-carotene was detected neither in the plasma nor in the pectoralis major muscle of broiler chickens fed Panaferd^®-AX (data not shown). Dietary supplementation with 30 ppm Panaferd^®-AX increased lutein oxidation products in the pectoralis major muscle. Although animals cannot biosynthesize carotenoids, they absorb feed-derived carotenoids and convert them into various derivatives in vivo[35]. Salmonids metabolize astaxanthin to zeaxanthin or lutein[36]. The reductive degradation of astaxanthin has been demonstrated in chickens[37]. Therefore, astaxanthin degradation might be one of the reasons for an astaxanthin content akin to that of other carotenoids, but a higher plasma zeaxanthin and lutein concentration in broiler chickens fed Panaferd^®-AX.

Table 3.  Effect of dietary supplementation with Panaferd^®-AX on plasma and muscle concentrations of carotenoids in broiler chickens.

	Plasma (ng/mL)										Pectoralis major muscle (ng/g tissue)
	Control			Panaferd®-AX							Control			Panaferd®-AX
				5 ppm astaxanthin			30 ppm astaxanthin							5 ppm astaxanthin			30 ppm astaxanthin
β-Cryptoxanthin	102.11	±	23.71ab	91.19	±	11.15b	157.55	±	10.00a		13.95	±	4.64	23.34	±	13.35	17.75	±	4.14
Lutein oxidation products	63.41	±	6.82	76.25	±	6.34	230.76	±	79.71		25.50	±	4.19b	39.81	±	7.2ab	48.74	±	4.6a
Lutein	479.80	±	52.10c	703.64	±	60.92b	918.73	±	13.94a		368.83	±	70.97	267.40	±	13.24	272.98	±	19.75
Zeaxanthin	429.10	±	86.95c	716.97	±	79.80b	1018.43	±	28.24a		140.73	±	19.78	171.67	±	9.70	163.08	±	14.58
Canthaxanthin	n.d.			101.15	±	17.66	560.86	±	40.23*		n.d.			29.61	±	4.18	120.07	±	11.29*
Adonirubin	n.d.			171.53	±	24.54	742.23	±	89.21*		n.d.			22.15	±	3.48	79.93	±	6.88*
Astaxanthin	n.d.			145.58	±	24.85	632.18	±	31.61*		n.d.			45.95	±	5.20	119.61	±	16.50*
Adonixanthin	n.d.			75.53	±	8.37	489.44	±	32.98*		n.d.			12.87	±	2.46	53.89	±	4.27*

Results are expressed as means ± standard error of the mean (n = 10). Different superscript letters in plasma and the pectoralis major muscle indicate significant differences at the 5% level. *, P < 0.05 vs. Panaferd^®-AX Low. (Student's t-test).

Even though dietary supplementation with 5 ppm Panaferd^®-AX did not affect the color of the pectoralis major muscle in broiler chickens, 30 ppm Panaferd^®-AX increased muscle redness (a*) and yellowness (b*) after 48 h of storage (Table S4). These color changes may be explained by carotenoid accumulation following Panaferd^®-AX consumption or alterations to the chemical state of myoglobin. Post-slaughter muscle color changes gradually, reflecting the oxidation state of the iron in myoglobin. Specifically, myoglobin changes to metmyoglobin, whereby the iron becomes oxidized and myoglobin-containing tissues assume an unappealing brown discoloration[37]. Because antioxidants such as carotenoids retard oxidation of muscle myoglobin[38], dietary supplementation with Panaferd^®-AX might improve myoglobin stability and, consequently, increase the redness of the pectoralis major muscle.

Because chicken muscle is rich in polyunsaturated fatty acids, it is more sensitive to oxidative deterioration during storage[3]. Dietary supplementation with Panaferd^®-AX did not affect fatty acid composition in the pectoralis major muscle of broiler chickens after 48 h of storage (Table 4). However, the muscle content of several unsaturated fatty acids (e.g., oleic, linoleic, and alpha-linolenic acid) increased in chickens fed Panaferd^®-AX, albeit not significantly. Because astaxanthin and other carotenoids in Panaferd^®-AX can successfully quench singlet oxygen[39,40,41], their accumulation in muscles might protect saturated and unsaturated fatty acids from oxidative deterioration during storage. Additionally, dietary supplementation with 5 ppm and 30 ppm Panaferd^®-AX reduced significantly muscle MDA concentration and drip loss during storage for 48 h (Table 5). This result is consistent with previous studies[16,26]. Astaxanthin is found between the phospholipid layers of cell membranes, forming hydrogen bonds and neutralizing lipid peroxyl radicals or reactive oxygen species[42]. Our results suggest that astaxanthin and other carotenoids in Panaferd^®-AX protect fatty acids from lipoperoxidation in the pectoralis major muscle of broiler chickens.

Table 4.  Effect of dietary supplementation with Panaferd^®-AX on composition of muscle fatty acids in broiler chickens.

	Pectorals major muscle (%)
	Control			Panaferd®-AX
				5 ppm astaxanthin			30 ppm astaxanthin
Myristic acid	0.18	±	0.03	0.21	±	0.03	0.21	±	0.02
Palmitic acid	20.22	±	0.46	20.70	±	0.50	20.66	±	0.64
Palmitoleic acid	0.74	±	0.04	0.95	±	0.20	1.11	±	0.18
Heptadecenoic acid	0.93	±	0.14	0.84	±	0.11	0.69	±	0.10
Stearic acid	13.36	±	0.73	12.83	±	1.18	11.54	±	0.96
Oleic acid	22.26	±	0.43	22.93	±	2.17	24.15	±	1.65
Linoleic acid	30.26	±	1.01	30.32	±	1.21	31.84	±	1.61
γ-Linolenic acid	0.21	±	0.04	0.18	±	0.03	0.22	±	0.03
α-Linolenic acid	0.46	±	0.08	0.46	±	0.08	0.52	±	0.03
Arachidonic acid	8.65	±	0.08	8.07	±	1.37	6.99	±	1.16

Results are expressed as mean ± standard error of the mean (SEM) (n = 10).

Table 5.  Effect of dietary supplementation with Panaferd^®-AX on MDA concentration and drip loss in the pectoralis major muscle of broiler chickens.

					Panaferd®-AX
		Control			5 ppm astaxanthin			30 ppm astaxanthin
Muscle MDA concentration (nmol MDA / g tissue)		277.95	±	23.18a	188.18	±	18.60b	198.45	±	23.03b
Drip Loss (%)		4.18	±	0.27a	3.17	±	0.08b	3.49	±	0.18b

Results are expressed as means ± standard error of the mean (n = 10). MDA, malondialdehyde. Different superscript letters indicate significant differences at the 5% level.

Perenlei et al.[43] reported that dietary supplementation with astaxanthin-producing P. rhodozyma increased total free amino acid content in the pectoralis major muscle after 120 h of storage. In addition, chicken muscle (especially pectoralis major muscle) has been known to be rich in carnosine and anserine[44], which act as antioxidants[45] and buffer agents[46]. Therefore, we measured free amino acid, carnosine, and anserine contents in the pectoralis major muscle. Although dietary supplementation with Panaferd^®-AX did not affect muscle free amino acid content (except for proline) after 48 h of storage, it increased carnosine levels (Table 6). Anserine tended to increase the astaxanthin concentration in a dose-dependent manner; although neither “the total anserine + carnosine content” nor “the ratio of carnosine to anserine” were affected by dietary supplementation with Panaferd^®-AX (Table 6). Because astaxanthin possesses antioxidant properties in vivo[47,48], we believe that astaxanthin contained in Panaferd^®-AX, rather than carnosine or anserine, might serve as antioxidants in the pectoralis major muscle of broiler chickens. Moreover, β-alanine is the rate-limiting factor for carnosine and anserine production, and their level in skeletal muscle depends on circulating β-alanine concentrations[49]. β-alanine is synthesized in the liver via degradation of uracil, a pyrimidine nucleotide[50]. Our previous study showed that dietary supplementation with orotic acid, a pyrimidine precursor, altered pyrimidine metabolism and increased plasma β-alanine in broiler chickens[51]. Because Panaferd^®-AX is a dried powder of P. carotinifaciens, nucleic acids in Panaferd^®-AX might be used in the salvage pathway for pyrimidine[52], thereby boosting β-alanine availability.

Table 6.  Effect of dietary supplementation with Panaferd®-AX on muscle free amino acids and imidazole dipeptide contents in the pectoralis major muscle of broiler chickens.

				Panaferd®-AX
	Control			5 ppm astaxanthin			30 ppm astaxanthin
Aspartic acid	12.08	±	1.29	14.41	±	1.70	13.54	±	1.20
Glutamic acid	26.83	±	3.92	29.94	±	2.49	30.19	±	2.70
Asparagine	4.27	±	0.26	3.81	±	0.48	3.55	±	0.30
Serine	23.42	±	1.78	27.14	±	4.61	22.54	±	1.24
Glutamine	14.67	±	1.43	14.17	±	1.07	15.81	±	1.05
Histidine	4.11	±	0.41	4.75	±	0.74	4.26	±	0.35
Glycine	23.20	±	3.51	27.42	±	3.94	32.32	±	3.51
Threonine	24.06	±	1.78	23.13	±	2.00	24.16	±	2.68
Arginine	13.26	±	1.21	15.15	±	1.31	15.45	±	0.88
Alanine	23.85	±	1.71	22.09	±	3.13	21.23	±	1.47
Tyrosine	16.25	±	0.95	17.66	±	1.74	16.83	±	0.63
Valine	9.95	±	0.79	11.82	±	1.06	10.99	±	0.69
Methionine	9.52	±	0.94	9.05	±	0.76	11.72	±	3.83
Cystine	42.57	±	2.66	40.18	±	3.91	45.17	±	3.22
Tryptophan	17.28	±	2.08	23.75	±	3.17	15.58	±	2.27
Phenylalanine	7.03	±	0.49	7.99	±	0.72	7.29	±	0.34
Isoleucine	5.68	±	0.43	7.28	±	0.67	6.15	±	0.38
Leucine	16.00	±	1.27	18.82	±	1.59	16.20	±	1.05
Lysine	20.03	±	0.98	20.25	±	1.59	18.55	±	1.68
Proline	22.97	±	1.43ab	27.84	±	2.72a	20.78	±	1.70b
β-alanine	5.69	±	0.85	8.33	±	2.07	6.22	±	1.30
Carnosine	252.66	±	21.62b	326.28	±	28.53a	247.26	±	29.58ab
Anserine	879.23	±	34.16	953.16	±	54.46	1026.40	±	59.88
Carnosine + Anserine	1131.89	±	55.7	1279.44	±	82.99	1273.66	±	89.46
The ratio of Carnosine to Anserine	0.29	±	0.09	0.36	±	0.16	0.24	±	0.08

Results are expressed as mean ± standard error of the mean (n = 10). Different superscript letters indicate significant difference at the 5% level.

Dietary supplementation with Panaferd^®-AX increased free proline content in the pectoralis major muscle of broiler chickens (Table 6). In skeletal muscle, collagen plays important functional roles, such as providing tissue elasticity and transmitting contractile forces from myofibrillar proteins to tendons and bones[53]. Proline is a proteinogenic amino acid involved in collagen biosynthesis[54], and collagen is degraded by matrix metalloproteinases[53,55]. Interestingly, matrix metalloproteinases are activated by reactive oxygen species[56]. Owing to the strong antioxidant activity of astaxanthin in vivo[47,48], Panaferd^®-AX may have suppressed collagen degradation in the skeletal muscle of chickens.

In conclusion, dietary supplementation with Panaferd^®-AX, an astaxanthin-rich dried cell powder from P. carotinifaciens, increased the carotenoid concentration and affected the color of skeletal muscles in broiler chickens. Additionally, Panaferd^®-AX decreased the MDA content and drip loss, while increasing carnosine levels.

Acknowledgments

We thank Kagoshima Chicken Foods Company, Ltd. (Kagoshima, Japan) for supplying the chicks used in this study. We also thank Traci Raley, MS and ELS(D) from Edanz (https://jp.edanz.com/ac) for editing a draft of the manuscript.

Author Contributions

Yoshinao Kume: formal analysis, investigation, validation, original draft preparation. Mizuki Kamegawa: formal analysis, investigation, validation, original draft preparation. Miori Shintaku: formal analysis, investigation. Ayumi Katafuchi: formal analysis, investigation, validation. Saki Shimamoto: methodology, formal analysis, validation, manuscript review and editing. Miyu Kamimura: methodology, formal analysis, manuscript review and editing. Daichi Kuwahara: conceptualization, manuscript review and editing. Yukiko Osawa: conceptualization, manuscript review and editing. Shinya Ishihara: statistical analysis, manuscript review and editing. Akira Ohtsuka: conceptualization, methodology, manuscript review and editing, supervision. Daichi Ijiri: conceptualization, methodology, validation, statistical analysis, original draft preparation, manuscript review and editing, project administration. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

This work was funded by a joint research effort with ENEOS Techno Materials Corporation.

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