Effect of Carnosine Supplementation on Warmed-Over Flavor, Volatile Compounds, and Meat Quality in Ground Chicken Breast and Thigh Meat

Abstract

Lipid oxidation produces an unpleasant warmed-over flavor (WOF) in meat. In this study, the antioxidant peptide carnosine (Car) was added to minced chicken breast and thigh meat, and its effect on WOF, volatile compounds, and meat quality during refrigerated storage was assessed. Breast and thigh meat patties were supplemented with four levels of Car (0, 1.0, 2.5, and 15 mg/g meat) and stored under refrigerated conditions for 1, 3, and 7 days. After that, WOF, volatile components, and meat quality factors were analyzed. The sensory attribute ‘oxidized oil odor’, a WOF indicator, was affected by Car supplementation and was lowest in samples supplemented with Car at 15 mg/g meat (Car15). In addition, some volatile aldehydes and thiobarbituric acid reactive substances (TBARS) in cooked meat causing WOF were lowest in Car15. Instead, samples supplemented with Car at 1.0 or 2.5 mg/g meat did not show significant differences in WOF, volatiles, or TBARS compared to controls without Car supplementation. Other meat quality parameters, such as pH, increased in Car15 samples; whereas L* and cooking loss decreased. In summary, supplementation of chicken breast and thigh meat with 15 mg/g Car reduced WOF intensity through inhibition of lipid peroxidation and influenced multiple meat quality parameters.

Introduction

Flavor is a vital sensory trait that directly affects consumers’ meat-purchasing behavior and palatability[1]. Because lipid peroxidation produces an unpleasant warmed-over flavor (WOF) in meat[2,3], direct postmortem supplementation of ground meat with various antioxidants—such as tea catechins[4], vitamin C[4], and grape seed extract[5]—has been explored. Most of these antioxidants are derived from plants or chemically synthesized and are not synthesized de novo or are produced only in limited quantities within animal tissues. Meat supplemented with these compounds is classified as ‘processed meat’ according to Japanese laws[6]. Therefore, strategies aimed at enhancing antioxidant levels through physiological approaches involving feeding or breeding are necessary to reduce WOF in fresh meat. Indeed, Mitsumoto[7] demonstrated that adding antioxidants to feed was more effective than adding them to meat. To enhance the antioxidant content of fresh meat using exogenous compounds, plant extracts, and vitamins must be supplemented continuously as feed additives in the animal diet. In contrast, strategies aimed at enhancing antioxidants that are endogenously synthesized and accumulate within animal tissues may achieve the same aim without the need for continuous dietary supplementation, thereby reducing feed costs and WOF in meat.

Carnosine (β-alanyl-L-histidine, Car) is an endogenous antioxidant found in animal muscle[8]. Supplementing ground meat with Car suppresses the formation of thiobarbituric acid-reactive substances (TBARS), a lipid peroxidation indicator[9,10,11]. Notably, no previous studies have employed sensory evaluation to determine whether Car supplementation in meat alters human perception of WOF. Demonstrating that Car suppresses WOF in meat would provide novel insights into enhancing meat flavor through endogenous antioxidants, independent of exogenous antioxidants.

To determine whether increasing Car content in meat through physiological methods effectively suppresses WOF, it is necessary to investigate the relationship between Car content and WOF. In chickens, Car content in breast meat is approximately 2 mg/g[12]; whereas in thigh meat, it is approximately 1 mg/g[13]. Adjusting the amino acid composition of feed can increase Car content in broiler breast meat by 1.0–2.5 mg/g relative to conventional feed[12,14]. Studies examining the effect of Car supplementation on meat quality have used Car at 5–15 mg/g[9,10,11], which exceeds the levels achievable through feeding. Thus, to assess the practical effectiveness of physiological enhancement strategies, it is necessary to analyze WOF at Car supplementation levels within the range of 1.0–2.5 mg/g muscle.

Furthermore, variations in lipid peroxidation-related parameters among chicken muscle parts may result in differential effects of Car supplementation on meat. First, chicken breast meat has a higher Car content than thigh meat[12,13]. Second, the lipid substrates responsible for WOF formation through peroxidation are more abundant in chicken thighs than in breasts[15]. Third, chicken breast meat exhibits a higher 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging capacity than chicken thigh meat[16]. As no study has compared the effect of Car supplementation between different muscle parts, clarifying whether Car exerts similar effects on WOF in breast and thigh meat would provide useful information for improving meat quality through physiological control of Car content.

This study aimed to determine whether Car supplementation caused significant differences in WOF of chicken meat, as assessed through descriptive sensory analysis. The second objective was to clarify whether WOF could be suppressed at a Car content achievable through physiological approaches. The third objective was to determine whether the effect of Car on WOF differed between chicken breasts and thigh muscles. To achieve these objectives, chicken samples were prepared with no Car supplementation and with Car supplementation at 1.0 and 2.5 mg/g meat, corresponding to reported Car contents achievable through physiological methods. An additional supplementation level of 15 mg/g meat, reported to suppress TBARS in chicken breast meat, was also included. All treatments were applied to chicken breast and thigh meat samples. The samples were analyzed for WOF, TBARS, volatile compounds, and meat quality determinants.

Materials and Methods

Preparation of Car-supplemented meat samples

Broiler chicken (Ross 308 strain) carcasses were purchased (Date Bussan Corporation, Fukushima, Japan), and breast and thigh meat were removed. The meat was trimmed to remove skin, subcutaneous fat, and visible connective tissue, followed by mincing in a meat grinder (Md. KM4000; Delonghi, Treviso, Italy) equipped with a mincer-hole disc with 2-mm holes (Mod. A950, Delonghi). Ground meat from the four carcasses was mixed, but was kept separate in terms of breast or thigh origin. Non-Car-supplemented ground meat served as the control.

The Car supplementation level of experimental samples was established at 1.0 and 2.5 mg/g meat as the Car content achievable within muscle tissue during physiological approaches[12,14] and at 15 mg/g meat, the content reported to inhibit lipid peroxidation in chicken meat[11]. Briefly, meat samples were prepared by supplementing the control with l-carnosine (FUJIFILM Wako Pure Chemical Co., Ltd., Tokyo, Japan) to obtain Car1.0, Car2.5, and Car15 groups, respectively. These samples were formed into circular patties 35 mm in diameter and 10-mm thick, as described by Sasaki et al.[17]. The patties were placed in weighing boats, overwrapped with oxygen-permeable PVC film, and stored in a refrigerator (MPR-721; Panasonic Co., Tokyo, Japan) at 4 °C (refrigerator set temperature) under white light–emitting diode illumination for 1, 3, or 7 days. Each storage trial was performed thrice, with five patty samples prepared for each treatment in each trial. Three experiments were conducted using a new batch of ground meat. TBARS, pH, cooking loss, and color were analyzed immediately after each storage trial. Samples destined for free Car content quantification, sensory evaluation, and volatile compound analysis were vacuum-packed and stored at -30 °C until analysis.

Measurement of free Car content

Free Car content of the control and Car-supplemented patties before storage (day 0) was determined using an L-8900 amino acid analyzer (Hitachi Co., Tokyo, Japan), as described previously[18].

Sample preparation for descriptive sensory analysis

Three meat patties were used for descriptive sensory analysis of each treatment. The stored chicken patties were thawed overnight in a refrigerator set at 4 °C. The patties used in the present study were relatively small (35 mm in diameter and 10 mm in thickness). In a previous study by Sasaki et al.[17], which employed a similar model, cooking conditions were controlled based on the oven set temperature and cooking time. Based on preliminary experiments and methods commonly used in Japan, the patties were cooked at 180 °C for 5 min using a steam convection oven (SSC-10DCNU; Maruzen Co., Ltd., Tokyo, Japan). After cooking, the patties were cooled at room temperature and were cut into 1.0 × 1.0 × 1.0 cm cubes. Each cube was placed in a 50-mL triangular flask capped with a lid. All flasks were labelled with random three-digit codes. The flasks were kept in a warm cabinet (DKN402; Yamato Scientific Co., Ltd., Tokyo, Japan) set at 40 °C until just prior to the descriptive sensory analysis.

Sensory terms describing WOF and their definitions

Table 1 lists the sensory terms employed in this study and their definitions. Because L-carnosine is not considered a food additive, ethical considerations precluded the panelists from consuming Car-containing samples; therefore, only the orthonasal odor characteristics of WOF were evaluated based on four odor terms. The first one was ‘oxidized oil odor’, because lipid oxidation induces WOF[2,3]. The second was ‘fish-like odor,’ coined by Byrne et al.[19] to describe WOF in pork. The third one was a ‘putrid odor’, as described by Konopka et al.[20] in relation to WOF. The fourth term was ‘chicken-like odor’ because WOF onset is accompanied by a decrease in chicken meat-like characteristics[21]. Prior to the analysis, the panelists received a lecture on the definitions of the above four sensory terms employed in this study.

Table 1  List of sensory terms for descriptive sensory analysis.

Classification	Definition
Oxidized oil odor	Odor of oxidized oil
Fish-like odor	Odor of docosahexaenoic acid or eicosapentaenoic acid
Putrid odor	Odor of trimethylamine
Chicken-like odor	Odor of cooked chicken meat

Descriptive sensory analysis

The National Agriculture and Food Research Organization’s (NARO) Ethics Review Committee on Human Subject Research granted ethical approval on November 1, 2022 for the involvement of human participants in this study.

Staff at the Institute of Livestock and Grassland Science, NARO, were screened using discrimination tests for five basic tastes, seasoning concentrations, differences across meat products, and odor, as previously described[22]. Accordingly, 12 persons were selected as panelists. The panelists attended lectures on sensory evaluation methods and the sensory mechanisms of taste, odor, and texture. They also participated in training sessions to learn the sensory expression terms defined in ISO 5492:2008[23] by eating or smelling the reference foods associated with these sensory terms.

Immediately before the descriptive sensory analysis, each panelist was informed of sample safety, evaluation anonymity, and the right to withdraw at any time without providing a reason. All 12 panelists provided written informed consent before the evaluations.

Descriptive sensory sessions were conducted in a sensory test room using individual booths illuminated with red lighting. Room temperature and relative humidity were maintained using an air conditioner set at 22 °C and 50%, respectively. All 12 panelists evaluated all 24 samples (four Car contents × two muscle parts × three storage periods). Because evaluating 24 samples in one session was too much work for the panelists, the evaluation was split into two sessions, with the panelists assessing 12 samples per session. A Latin square design was used to mitigate the effects of evaluation order.

During the evaluation, each panelist was instructed to open the lid of a triangular flask and smell the meat sample. Immediately after smelling, the panelists were instructed to begin their evaluations. Descriptive sensory analysis for the orthonasal odor characteristics listed in Table 1 was conducted using a 150-mm line scale. This line scale was anchored as ‘weak’ 10 mm from the left edge and ‘strong’ 10 mm from the right edge. The panelists drew a line that intersected the line scale perpendicularly, and the length (mm) from the left end of the line scale to the intersection with the line drawn by the panelists provided the sensory odor intensity. The evaluation booth was equipped with a rubber heater set at 40 °C, and panelists evaluated the odor while the heater warmed the flasks. Between each sample test, each panelist took a break of at least 1 min. Although the storage trial of the patties was conducted three times, descriptive sensory analysis was performed solely on patty samples from the first storage trial because of the large number of samples and the considerable burden on the panelists.

Volatile compound analysis

Volatile compounds were analyzed using headspace solid-phase microextraction (HS-SPME), as described by Mariutti et al.[24] and Gkarane et al.[25]. First, chicken patties were cooked in the same manner as in the descriptive sensory analysis and finely chopped using a scalpel. Next, 1.0 g of the meat sample was weighed and transferred into 40-mL glass headspace vials (Supelco, Bellefonte, PA, USA) together with 2 μL of a methanolic solution of 0.02% (v/v) 2-heptanone (Sigma-Aldrich, Munich, Germany) as internal standard. The vials were sealed with a polytetrafluoroethylene-faced silicone septum (Supelco). Meat samples were heated at 60 °C for 5 min in a cool plate (CP-085; SCINICS, Tokyo, Japan) with a block bath for equilibration before exposing the 30/50 μm divinylbenzene/carboxen/polydimethylsiloxane fiber (Supelco) into the headspace, where it was held for 10 min.

Gas chromatography-mass spectrometry analysis of the samples was performed using a GCMS-QP2020 instrument (Shimadzu Co. Ltd., Kyoto, Japan). The conditions for gas chromatography were as indicated by Mariutti et al.[24]. Briefly, after volatile extraction in the headspace, the fiber was removed from the vial and inserted directly into the injection port at 250 °C. The sampling time was 1 min, and the injection port was in the splitless mode. The column (DB-WAX, 30 m × 0.25 mm i.d., 0.25 mm; J&W, Folsom, CA, USA) temperature was raised from 40 to 180 °C at 4 °C/min and remained at 180 °C for 2 min. The flow control mode was a linear velocity set at 47.3 cm/s with He as carrier gas. The column was coupled directly to a quadrupole mass spectrometer and operated in electron ionization mode at 70 eV and 200 °C with an interface temperature of 250 °C. Mass spectra were collected at a scan rate of 1.66 amu/s from m/z 35 to 500 and an interval of 0.3 s. Qualitative data were collected for the total ion chromatogram mode. Compounds were tentatively annotated by comparing the spectral data to the Wiley Registry of Mass Spectral Data (10th edition; John Wiley & Sons, Inc., New York, NY, USA) using a similarity index. The amount of each volatile compound was calculated relative to a peak area for 2-heptanone (internal standard) of 1.0.

Thiobarbituric acid reactive substances (TBARS)

TBARS were determined using a spectrophotometric assay as described previously[26], with some modifications[27]. In meat, lipid peroxidation occurs after cooking, which influences flavor during consumption, as well as during storage, which affects meat shelf-life; both processes play key roles in determining overall quality. To quantify lipid peroxidation in patties after heat treatment and in raw patties immediately after storage, we measured TBARS in patties prepared using the same procedure as that applied for sensory evaluation and the raw patties at the end of storage. Values were expressed as nmol malondialdehyde equivalents per gram of sample. TBARS content was measured in two replicates per patty, and the average value was recorded for each sample.

Cooking loss

The cooked samples used for TBARS measurements were weighed before and after cooking. The weight loss after cooking was calculated and expressed as a percentage of the weight before cooking.

pH

The pH of the raw patties was measured using a pH meter (LAQUA D-71; Horiba, Kyoto, Japan) with a needle-type electrode (6252-10D; Horiba) held directly in the patties. The pH was measured in triplicate for each patty, and the average value was recorded as the pH for each sample.

Color

The surface color of raw patties after storage was measured using a spectrophotometer (CM-700; Konica Minolta, Tokyo,Japan). A spectrophotometer was used with an 8-mm aperture, 10° observer, illuminant D65, and a pulsed xenon lamp as the default light source. L*, a*, and b* values ​​were measured three times at different points in each patty, and average values ​​were used as the respective measured values.

Data analysis

Free Car values were averaged for each control and Car-supplemented patty to confirm adequate Car supplementation.

Descriptive sensory data were analyzed using the lme4 package in R version 4.1.2[28]. ANOVA for the individual factors was performed using a linear mixed model, with Car supplementation level, meat parts, storage period, interaction of these three factors, and order of sample evaluation as fixed effects, and the panelist as a random effect. Data on meat quality factors (volatile compounds, TBARS, cooking loss, pH, and color) were analyzed using a linear mixed model, with Car supplementation level, meat parts, storage period, and the interaction of these three factors as fixed effects, and the replication of the trial as a random effect. To compare the effect of Car supplementation level, storage period, and meat part on the descriptive sensory analysis and meat quality factors in the abovementioned model, the least-square means was calculated for Car supplementation level, meat part, and storage period using the emmeans package in R[29]. To compare the least-square means of the descriptive sensory analysis and meat quality factors between different Car supplementation levels, meat parts, and storage periods, multiple comparisons were performed using the Holm–Sidak test with the multcomp package in R[30].

Results

Free Car content in meat samples

Table 2 reports the free Car content in each meat sample, confirming that the samples were prepared according to theoretical values. Car content in breast meat was 2.08 mg/g meat, which was similar to the value reported by Lackner et al.[12]. Car content in thigh meat was 1.06 mg/g meat, which was similar to the value reported by Cong et al.[13]. Furthermore, the results showed that breast meat had a higher Car content than thigh meat, while Car-supplemented patties exhibited equivalent theoretical values. All samples in this study had the expected Car content.

Table 2   Free carnosine content in meat patties.

	Carnosine supplementation (mg/g meat)	Measured value of carnosine (mg/g meat)	SE1	Difference in carnosine content between control and carnosine-supplemented patties (mg/g meat)
Breast	0 (Control)	2.08	0.27
	1.0	3.14	0.30	1.05
	2.5	5.27	0.14	3.18
	15	17.91	1.08	15.82
Thigh	0 (Control)	1.06	0.02
	1.0	2.06	0.01	1.00
	2.5	3.46	0.38	2.40
	15	16.49	0.79	15.43

¹SE, Standard error.

Descriptive sensory analysis

Descriptive sensory analysis examined the effects of Car supplementation, meat part, and storage period on WOF intensity in chicken meat. Table 3 presents the orthonasal odor intensity results related to WOF in cooked chicken patties. Car supplementation, storage period, and evaluation order significantly affected the ‘oxidized oil odor’ (P < 0.05); whereas meat part and interactions among Car supplementation, storage period, and meat part showed no significant effect. The ‘oxidized oil odor’ was significantly lower in Car15 than in Car2.5 (P < 0.05), indicating that Car15 effectively suppressed WOF. The ‘oxidized oil odor’ was significantly lower after 7 days than after 1 day of storage (P < 0.05). ‘Putrid odor’ and ‘chicken-like odor’ were affected solely by the evaluation order, and ‘fish-like odor’ was not significantly affected by any factors.

Table 3   Effect of carnosine supplementation, storage period, and meat parts on sensory orthonasal odor scores related to WOF in cooked chicken meat patties.

Factor		Oxidized oil odor	Fish-like odor	Putrid odor	Chicken-like odor
Carnosine
	Control	73.9ab	37.4	40.6	51.6
	Car1.0	73.8ab	38.2	40.5	58.2
	Car2.5	75.8a	39.1	43.9	60.3
	Car15	61.8b	32.5	36.1	63.3
	SE1	8.42	6.79	8.05	6.74
Meat parts
	Breast	71.0	35.1	37.4	66.0
	Thigh	71.6	38.5	43.2	61.4
	SE	8.08	6.43	7.63	5.43
Storage period
	1	80.3a	40.6	41.7	60.6
	3	70.9ab	32.1	38.3	62.0
	7	62.7b	37.6	40.8	68.6
	SE	8.25	6.62	7.84	5.69
ANOVA P-value
	Carnosine	0.012	0.441	0.520	0.453
	Meat parts	0.855	0.277	0.117	0.183
	Storage period	< 0.001	0.080	0.740	0.136
	Carnosine × Meat parts	0.526	0.249	0.698	0.273
	Carnosine × Storage period	0.064	0.840	0.235	0.995
	Storage period × Meat parts	0.540	0.151	0.653	0.478
	Carnosine × Storage period × Meat part	0.234	0.146	0.982	0.910
	Evaluation order	0.001	0.300	0.010	0.008

Least-squares means of the sensory odor scores (mm) are shown.

¹SE, Standard error.

^a,b P < 0.05

Volatile compounds

Because the quantity and types of volatile compounds influence WOF, the volatile components in the headspace were analyzed. The 16 compounds, which included six alcohols, six aldehydes, three ketones, and one fatty acid, were annotated using a similarity index based on the Wiley Registry of Mass Spectral Data (Table 4).

Table 4  Effect of carnosine supplementation, storage period, and meat parts on volatile compounds in cooked chicken meat patties.

Factor		1-Butanol	1-Hexanol	2-Ethyl-1-hexanol	1-Octanol	1-Pentanol	1-Octen-3-ol	2-Octenal	Benzaldehyde	Hexanal	Nonanal	Octanal	Pentanal	2,3-Octanedione	2-Butanone	2-Propanone	Hexanoic acid
Carnosine
	Control	0.0054	0.0129	0.0422	0.0074	0.0341A	0.0395a	0.0041a	0.0196b	0.749a	0.0460a	0.0202a	0.0246A	0.0357	0.0023ab	0.0041	0.0060ab
	Car1.0	0.0067	0.0116	0.0511	0.0062	0.0235AB	0.0309ab	0.0017ab	0.0253ab	0.604ab	0.0442ab	0.0180a	0.0228A	0.0285	0.0040a	0.0058	0.0074a
	Car2.5	0.0047	0.013	0.0409	0.0055	0.0232AB	0.0291b	0.0020ab	0.0201b	0.521ab	0.0358bc	0.0145ab	0.0177AB	0.0258	0.0028ab	0.0047	0.0050b
	Car15	0.0063	0.0149	0.0434	0.0058	0.0187B	0.0254b	0.0004b	0.0280a	0.433b	0.0296c	0.0109b	0.0109B	0.0281	0.0012b	0.0054	0.0048b
	SE1	0.0012	0.0037	0.0059	0.0009	0.0097	0.0070	0.0003	0.0028	0.137	0.0040	0.0026	0.0049	0.0080	0.0008	0.0011	0.0015
Meat parts
	Breast	0.0057	0.0081B	0.0433	0.0056	0.0091B	0.0168B	0.0002B	0.0237	0.320B	0.0311B	0.0121B	0.0113B	0.0152B	0.0036A	0.0044	0.0044B
	Thigh	0.0058	0.0181A	0.0455	0.0068	0.0407A	0.0456A	0.0039A	0.0228	0.833A	0.0467A	0.0197A	0.0267A	0.0439A	0.0016B	0.0055	0.0072A
	SE	0.0010	0.0036	0.0050	0.0008	0.0094	0.0067	0.0002	0.0026	0.131	0.0035	0.0024	0.0046	0.0073	0.0006	0.0009	0.0014
Storage period
	1	0.0044b	0.0108B	0.0421	0.0064	0.0278	0.0348a	0.0025A	0.0235	0.751A	0.0425A	0.0191A	0.0269a	0.0313AB	0.0029	0.0061a	0.0069a
	3	0.0051ab	0.0107B	0.0405	0.0066	0.0262	0.0340a	0.0026A	0.0223	0.654A	0.0430A	0.0179A	0.0189a	0.0383A	0.0018	0.0031b	0.0051b
	7	0.0079a	0.0178A	0.0506	0.0057	0.0207	0.0249b	0.0010B	0.0240	0.325B	0.0312B	0.0107B	0.0113b	0.0190B	0.0031	0.0057ab	0.0055ab
	SE	0.0011	0.0036	0.0055	0.0009	0.0096	0.0069	0.0003	0.0027	0.134	0.0037	0.0025	0.0047	0.0077	0.0007	0.0010	0.0014
ANOVA P-value
	Carnosine	0.474	0.489	0.366	0.225	0.006	0.005	< 0.001	0.001	0.002	< 0.001	0.001	0.001	0.407	0.041	0.565	0.010
	Meat parts	0.944	< 0.001	0.619	0.078	< 0.001	< 0.001	< 0.001	0.590	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	0.005	0.225	< 0.001
	Storage period	0.011	< 0.001	0.147	0.556	0.143	0.008	< 0.001	0.652	< 0.001	0.001	< 0.001	< 0.001	0.003	0.214	0.018	0.035
	Carnosine × Meat parts	0.906	0.678	0.831	0.003	0.012	0.002	< 0.001	0.030	0.006	0.001	< 0.001	0.083	0.038	0.779	0.853	0.002
	Carnosine × Storage period	0.394	0.013	0.162	0.010	0.453	0.108	0.035	0.111	0.029	0.124	0.010	0.002	0.459	0.834	0.522	0.085
	Meat parts × Storage period	0.003	0.048	0.232	0.234	0.197	0.056	0.002	0.768	0.099	0.031	0.031	0.286	0.042	0.144	0.938	0.844
	Carnosine × Meat parts × Storage period	0.135	0.302	0.036	0.995	0.350	0.486	0.012	0.491	0.221	0.591	0.752	0.188	0.347	0.254	0.147	0.247

Least-square means relative to the peak area of the internal standard (2-heptanone) are shown.

¹SE, Standard error.

^a-c P < 0.05; ^A,B P < 0.01

Overall, 1-pentanol, 1-octen-3-ol, 2-octenal, hexanal, nonanal, octanal, pentanal, 2-butanone, and hexanoic acid were significantly lower following Car supplementation (P < 0.05); whereas benzaldehyde showed the opposite trend (P < 0.05).

The meat parts affected the content of 1-hexanol, 1-pentanol, 1-octen-3-ol, 2-octenal, hexanal, nonanal, octanal, pentanal, 2,3-octanedione, 2-butanone, and hexanoic acid (P < 0.05). Specifically, these compounds were significantly more abundant in thigh meat than in breast meat, except for 2-butanone (P < 0.05).

The storage period affected the content of 1-butanol, 1-hexanol, 1-octen-3-ol, 2-octenal, hexanal, nonanal, octanal, pentanal, 2,3-octanedione, 2-propanone, and hexanoic acid (P < 0.05). In particular, 1-butanol and 1-hexanol increased with storage time; whereas 1-octen-3-ol, 2-octenal, hexanal, nonanal, octanal, pentanal, and 2,3-octanedione decreased.

The interaction between Car supplementation and meat parts significantly influenced ten volatile compounds (Table 4). For most volatile compounds showing a significant interaction, Car tended to have a greater suppressive effect on volatile compound levels in thigh meat than in breast meat (data not shown)

Six volatile compounds also exhibited a statistically significant interaction between Car supplementation and storage period (Table 4). Specifically, hexanal, octanal, and pentanal exhibited a significant decrease with the storage period in the Car1.0 and Car2.5 groups (data not shown).

The interaction between meat parts and storage period had a significant effect on six kinds of volatile compounds (Table 4). However, no consistent pattern was observed across compounds exhibiting significant interactions (data not shown).

The three-way interaction among Car supplementation, meat parts, and storage period was also significant in two volatile compounds (Table 4), although no consistent associations were identified between them (data not shown).

TBARS, cooking loss, pH, and color

TBARS, cooking loss, pH, and color were also assessed as parameters potentially affected by Car supplementation, meat parts, and storage period.

As reported in Table 5, Car supplementation affected the TBARS content of cooked meat, cooking loss, pH, and L* values (P < 0.05). The cooked meat TBARS of Car15 was significantly lower than that of the other treatments (P < 0.05). The cooking loss of Car15 was significantly lower than that of Car1.0 and Car2.5 (P < 0.05). The pH was significantly higher in Car15 than in all other treatments, while that of Car2.5 was significantly higher than the control’s (P < 0.05). Supplementation with Car significantly decreased L* (P < 0.05).

Table 5   Effects of carnosine supplementation, storage period, and meat parts on TBARS, cooking loss, pH, and color.

Factor		Raw meat TBARS (nmol malondialdehyde equivalent/g meat)	Cooked meat TBARS (nmol malondialdehyde equivalent/g meat)	Cooking loss (%)	Raw meat
					pH	L*	a*	b*
Carnosine
	Control	6.09	35.6a	14.0ab	6.03c	55.9a	3.90	16.0
	Car1.0	5.63	36.2a	15.2a	6.08bc	55.4ab	3.91	16.0
	Car2.5	5.95	34.1a	15.0a	6.17b	54.1b	4.10	16.4
	Car15	5.85	23.7b	12.4b	6.77a	51.1c	4.25	16.0
	SE1	1.04	6.88	0.62	0.06	1.97	0.64	0.38
Meat parts
	Breast	3.56b	23.2b	12.5b	6.09b	51.5b	2.22b	14.1b
	Thigh	8.20a	41.6a	15.9a	6.44a	56.7a	5.86a	18.1a
	SE	0.94	6.63	0.50	0.06	1.94	0.63	0.35
Storage period
	1	5.28	33.7a	14.9a	6.17b	55.8a	4.45a	16.9a
	3	5.92	38.1a	14.7a	6.27a	54.6a	4.48a	16.3a
	7	6.44	25.4b	12.9b	6.34a	52.0b	3.18b	15.0b
	SE	0.99	6.76	0.56	0.06	1.95	0.63	0.36
ANOVA P-value
	Carnosine	0.963	0.004	0.001	< 0.001	< 0.001	0.264	0.501
	Meat parts	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001
	Storage period	0.335	0.001	0.004	< 0.001	< 0.001	< 0.001	< 0.001
	Carnosine × Meat parts	0.785	0.676	0.562	0.023	0.336	< 0.001	0.076
	Carnosine × Storage period	0.846	0.687	0.034	0.837	0.048	0.747	0.595
	Meat parts × Storage period	0.107	0.712	0.448	0.817	0.796	0.983	0.762
	Carnosine × Meat parts × Storage period	0.663	0.994	0.712	0.604	0.953	0.987	0.434

Least-square means are shown.

¹SE, Standard error.

^a-c P < 0.05

TBARS of cooked meat, cooking loss, pH, L*, a*, and b* were affected by the storage period (P < 0.05). TBARS of cooked meat, cooking loss, L*, a*, and b* were significantly lower after 7 days of storage than after 1 and 3 days (P < 0.05). Instead, pH values were significantly higher after 3 and 7 days than after 1 day (P < 0.05).

The effect of meat parts was significant for all items, and was higher in thigh meat than in breast meat (P < 0.05).

Discussion

Effect of Car supplementation level on the suppression of WOF in chicken meat

Supplementation of chicken patties with Car significantly affected the ‘oxidized oil odor’ attribute (Table 3). Car15 samples, whereby Car was above the physiological level, exhibited the lowest intensity of ‘oxidized oil odor’ (Table 3). Therefore, this study demonstrates, for the first time, that WOF in meat can be suppressed by supplementation with Car. The observed relationship between the ‘oxidized oil odor’ (Table 3) and TBARS values of cooked meat (Table 5) is in good agreement with the established correlation between WOF and TBARS in meat[31]. Thus, lipid peroxidation inhibition by Car supplementation caused the difference in WOF that humans could perceive. In addition, Car supplementation affected the levels of some volatile alcohols, aldehydes, and ketones (Table 4). These compounds are secondary lipid oxidation products of meat, and have previously been demonstrated to be key flavor components of WOF in meat[32]. In particular, hexanal correlates positively with the intensity of WOF[33,34]. In this study, hexanal was the lowest in Car15 among all Car treatments (Table 4). Thus, the suppression of secondary lipid oxidation product formation, such as hexanal, by Car supplementation also contributed to reduced WOF intensity.

In the Car1.0 and Car2.5 groups supplemented with Car at concentrations achievable physiologically in chicken muscle, there was no significant effect on ‘oxidized oil odor’ (Table 3), volatile hexanal (Table 4), or TBARS of cooked meat (Table 5) compared to the control group. Thus, this study suggests that Car regulation in muscles through physiological approaches alone may be insufficient to suppress WOF and lipid peroxidation. Lackner et al.[12] reported that adjusting the histidine:lysine ratio in feed given to chickens succeeded in increasing Car content in chicken breast meat by 2.5 mg/g muscle compared to chickens fed feed with an unadjusted histidine:lysine ratio, but TBARS did not vary between muscles with different Car contents. In contrast, Sharula et al.[35] reported that increasing dietary histidine successfully increased Car content in chicken breast meat by 2.0 mg/g muscle, and TBARS were significantly lower in muscle with higher Car content. Sharula et al.[35] reported an increase in both Car and anserine, a methylated form of Car with similar antioxidant properties, in breast muscles; whereas Lackner et al.[12] reported no change in anserine levels in breast muscles. Therefore, integration of the present findings with those of broiler feeding studies indicates how feeding strategies that simultaneously increase both Car and anserine, rather than Car alone, may be more effective at suppressing WOF and lipid peroxidation.

Effect of Car supplementation and meat part interactions on WOF

Multiple physiological parameters related to lipid peroxidation differ between chicken breast and thigh meat, including Car content[12,13], lipid content[15], and radical scavenging activity[16]. Thus, we hypothesized that the effect of Car on lipid peroxidation and WOF might be partially dependent on the meat part. However, results indicated no significant interaction between Car supplementation and meat part for any WOF parameter or TBARS of cooked meat (Tables 3 and 5). Thus, Car supplementation effectively suppressed WOF and lipid peroxidation, regardless of the meat part used. Instead, the interaction between Car supplementation and meat parts significantly affected ten volatile compounds (Table 4). Therefore, Car may affect the production of individual volatile compounds in a muscle part-specific manner.

Comparison of WOF, TBARS, and volatile compounds between breast and thigh meat

In this study, raw and cooked meat TBARS values and the contents of ten volatile compounds were significantly higher in thigh meat than in breast meat (Tables 4 and 5). These differences in TBARS values and volatile compound contents likely reflect the distinct fat content in breast as opposed to thigh meat. Despite these differences, WOF was similar in both meat types (Table 3), suggesting that variations in these factors alone cannot explain the differences in WOF between muscle parts. Odor perception arises from the combined effect of multiple volatile compounds[36]. When two or more odors are mixed, interactions occur, including addition, subtraction, independence, synergy, and masking[37,38]. In the present study, the volatile compounds detected in thigh and breast meat were almost identical, but their amounts and composition differed (Table 4). Therefore, the discrepancy between WOF values and volatile components for breast and thigh meat likely results from interactions among the latter, which requires further exploration.

Comparison of WOF, volatile components, and TBARS between storage periods

TBARS values in raw chicken meat increase with prolonged storage[17]; however, in the present study, TBARS values of raw patties remained unchanged throughout the storage period (Table 5). In Japan, chicken meat is generally produced using feed supplemented with antioxidants, whose type and amount vary substantially between producers. The fact that TBARS values remained unchanged may be due to the high antioxidant status of postmortem muscle. However, chicken meat used in the present study was commercially purchased, and information regarding the antioxidants contained in the feed used for its production could not be obtained.

Similar to raw meat, extended storage duration is associated with increased lipid peroxidation after cooking and elevated WOF and TBARS values[21,39]. Contrary to previous studies, here, the ‘oxidized oil odor’, TBARS of the cooked sample, and seven types of volatile compounds were lowest after a 7-day storage period (Tables 3, 4, and 5). Because the chicken meat used here exhibited low TBARS values even after cooking, fluctuations in aldehydes and ketones resulting from processes not directly related to lipid peroxidation may have contributed to the decrease in TBARS values and WOF observed after storage. Accordingly, clarification of the mechanisms responsible for the time-dependent reduction in WOF-related factors observed in this study requires quantitative analyses of aldehydes and ketones in the meat matrix, along with more sensitive lipid peroxidation assays.

Effect of Car supplementation on meat quality factors other than WOF

Even if Car is effective in suppressing WOF, its suitability for improving overall meat quality requires consideration of its effects on other parameters, such as cooking loss, pH, and color.

Car supplementation reduced cooking loss. Das et al.[9] reported that supplementing buffalo meat with Car at 15 mg/g meat reduced cooking loss while simultaneously increasing water loss by centrifugation. In general, antioxidant supplementation improves the water-holding capacity by stabilizing muscle membrane structures; however, at concentrations exceeding physiological levels, osmotic pressure may exert adverse effects.

In this study, pH increased significantly upon Car supplementation. Das et al.[9] reported that supplementation of buffalo meat with Car at 5–15 mg/g meat increased the pH, which is consistent with the results of the present study. Notably, the pH does not change when Car in muscle is increased from about 0.5 to 2.0 mg/g meat by changing the diet fed to chickens[35,40]. The pH of Car was 7.0[41], which exceeds the chicken meat pH, suggesting that the chicken meat pH increased when large amounts of Car (2.5–15 mg/g meat) were supplemented.

The L* value of chicken meat decreased with increasing Car supplementation. This finding is consistent with the well-established negative correlation between L* and muscle pH[42]. As a result, improving the antioxidant properties of chicken meat using Car may require consideration of its effect on meat color acceptability.

Conclusions

The present study clearly shows that supplementation with 15 mg/g Car, which exceeds the physiological content in chicken meat, suppressed WOF, lipid peroxidation, and volatile compound generation after heating in breast and thigh meat. However, neither WOF nor lipid peroxidation were inhibited when Car was supplemented at 1.0 or 2.5 mg/g meat—the innate level in chicken muscle. Thus, this study revealed that merely increasing Car is insufficient to suppress chicken meat WOF through a physiological approach. Mitsumoto[7] demonstrated that adding antioxidants to feed was more effective for meat antioxidant properties than adding them directly to meat. Thus, future research should employ physiological approaches to analyze the relationship between Car regulation and WOF in chicken meat and determine whether increasing endogenous substances using physiological approaches can effectively improve chicken meat quality. Furthermore, as the SPME procedure measured only volatilized odor compounds, future studies should quantitatively determine WOF-related compounds within the meat matrix to elucidate the impact of Car content on chicken meat.

Acknowledgments

The authors sincerely thank Mr. Takumi Narita, Ms. Yumiko Endo, and Ms. Yuko Kurosawa of the Institute of Livestock and Grassland Science, NARO, for their generous assistance. The authors would like to thank Enago (www.enago.jp) for English language review.

Author Contributions

GW: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft, funding acquisition. SI, KA, MK, HS, SS, MM, AA, IN, and YM: Investigation, Writing, review, and editing. KS: Formal analysis, Investigation, Writing, review, editing, and supervision.

Funding

This study was supported by the Research Program of the Ito Foundation, Tokyo, Japan (2022).

Conflict of Interest

The authors declare no conflict of interest.

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