Analysis and Characterization of the Volatile Flavor Compounds Associated with the Wooden Breast Condition  

Sample collection

A randomized complete block design with 3 replications was implemented for the current study. Thirty normal and SWBs (300 g to 400 g) were collected from 8-wk-old Ross 708 broilers from a commercial poultry plant on 3 separate occasions (n = 3 replications) at 24 h postmortem. The samples were graded by hand palpation based on the degree of woodiness with NBs being flexible and severely woody breasts being extremely rigid and hard throughout the breast by trained graders (Tijare et al., 2016). Chicken patties (approx. 166 g, 30 patties per treatment per replication) were formed from grinding the 30 chicken breasts that were collected from Ross 708 broilers at 30 h postmortem into one homogeneous mixture of ground meat. The breasts were first ground through a 1.27-cm plate (Triumph, Model #103306) followed by a second grind through a 0.48-cm bone extracting plate (Triumph, Model #103060), using a Biro auto feed mixer grinder (Model AFMG, Biro, Marblehead, OH, USA). After grinding, each treatment was emptied into a 136-kg hopper capacity Formax (Model # F-6, Formax, Mokena, IL, USA) to make 166-g patties with 1.27-cm thickness. Patties (n = 30 per treatment) from each replication of both severely wooden and NBs were frozen in their raw form to −62.2°C in a CO₂ cabinet (Model CES-BF-CO2-15x15x21-E, CES Group, Cincinnati, OH, USA). Chicken patties were then stored in a walk-in freezer at −23°C until analysis within 2 mos.

Chemicals

The chemical standards of hexanal, nonanal, and 1-octanol (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) were obtained to verify and quantify gas chromatographic results. An internal standard (chlorobenzene, Supelco, Bellefonte, PA, USA) and an n-alkane mixture of C₈-C₂₀ (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) were used to calculate the linear retention indices of the volatile compounds to validate compounds that were tentatively identified by the gas chromatograph–mass spectrometer. A 5% sodium chloride (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) solution was made using High Performance Liquid Chromatography (HPLC)water (Fisher Scientific Company LLC, Middletown, VA, USA).

Raw color and pH

The lightness (L*), redness (a*), and yellowness (b*) of the chicken patties were measured in triplicate for 10 patties per treatment per replication based on Jarvis et al. (2020a; 2020b) using a HunterLab^® MiniScan EZ spectrophotometer (Model 4500L; Hunter Associates Laboratory Inc., Reston, VA, USA) equipped with D65 illuminant, 10° standard observer angle, a view area of 31.8 mm and calibrated with white and black calibration plates. pH was measured by inserting a temperature-compensated pH probe (Model FlexipHet SS Penetration tip; Cole Palmer, Vernon Hills, IL, USA) attached to an Accumet^™ pH meter (Model Accumet 61; Fisher Scientific, Hampton, NH, USA) after the breast meat was ground and before it was formed into patties. There were 10 pH measurements taken from random locations in the ground meat per treatment for each of the 3 replications. The pH meter was calibrated with standard buffer solutions of pH 4 and 7.

Cooking loss and protein bind

Chicken patty samples (n = 10 per treatment per replication) were analyzed for cooking loss and protein bind (Field et al., 1984; Schilling et al., 2004). The raw samples were weighed before and after cooking to determine cooking loss. These samples were cooked on a tray that was 45 cm × 65 cm × 2.5 cm with 6 patties per tray, at 177°C for 30 min in a convection oven (Model SCVX20E; Hobart, Chattanooga, TN, USA) to an internal temperature of 76°C. Chicken patties were then cooled for 30 min to 20°C prior to measuring protein bind. After cooling, cooked patties were placed one at a time onto a plexiglass stand to hold the sample in place. A steel ball (25.0-mm diameter) was attached to a rod that was secured in a 50-kg load cell with a chuck and used at a crosshead speed of 100 mm/min that was controlled by an Instron Universal Testing Center (Model 3345, Instron, Norwood, MA, USA) to penetrate through the center of each of the 10 chicken patties from each treatment within each replication (Field et al., 1984; Schilling et al., 2004). Protein–protein bind value was reported as the peak force (N) required for the steel ball to penetrate through the chicken patties for each treatment.

Quantitative descriptive analysis

Quantitative descriptive analysis was conducted to evaluate the textural characteristics of the chicken patties, including tenderness, cohesiveness, chewiness, mushiness, initial juiciness, overall juiciness, springiness, and crunchiness, and the potential flavor differences between woody and NB meat samples, including sourness, bitterness, umami, chicken/meaty, cardboardy, metallic, and off-flavors. Chicken patties were placed on an aluminum foil covered tray, covered with aluminum foil, and cooked in a convection oven (Model SCVX20E, Hobart, Chattanooga TN, USA) at 177°C for 22 min to an internal temperature of 76°C. The edges of the chicken patty samples were trimmed, and the remaining sample was cut into twelve 1.27 cm × 1.27-cm cube samples. The panelists (n = 8) were trained for 12 h on the evaluation of texture and flavor attributes of chicken patties on a 0 to 15-cm line scale, in which 0 and 15 are the least and greatest attribute intensity for chicken patty sensory descriptors. Chicken patty texture attributes were based on Civille and Carr (2015), with some slight changes made to accommodate chicken patties. SWBs were used as references for sour, earthy, metallic, and raw notes that have been previously identified and woody breast meat (Jarvis et al. 2020a; Jarvis et al. 2020b). After training, 3 panels were conducted per replication for chicken patty samples. Samples were cooked and prepared as described above, placed into a 28.3-ml plastic cup, labeled with a random 3-digit code, and served to the panelists in a random order that was assigned through Compusense Cloud software (Compusense Cloud, Guelph, Ontario, Canada).

Conditioning of SPME fiber for chromatographic analysis

Fibers were conditioned in the Gas chromatograph (GC) injector port for 30 min at 265°C prior to sampling to desorb contaminants and prevent carryover. An instrument blank (no fiber), a fiber blank (conditioned fiber without volatile extract), and an extraction blank (no sample or standard, only 5% sodium chloride solution) were run to determine the background spectrum. Selected ion chromatograms from these blank spectra were compared to standard and sample spectra for quality control purposes.

Isolation and analysis of volatile compounds

Samples were prepared by adding 5 g of raw chicken patties (6 per treatment per replication) and a 5% NaCl solution in a 2:1 ratio with an internal standard (10 μl, 200 ppm, chlorobenzene, Supelco, Bellefonte, PA, USA) to a 40-mL amber glass vial (O.D. 28 mm × 98 mm H, Supelco, Bellefonte, PA, USA) with an open center propylene screw cap and Teflon faced silicone septum (O.D. 22-mm diameter, Supelco, Bellefonte, PA, USA). A small magnetic octagonal stir bar (8-mm O.D. × 13-mm L, Fisher, Pittsburgh, PA, USA) was also placed in the amber glass vial. Samples were heated on a heating block (Reacti-therm Heating/Stirring Module, Pierce Biotechnology Inc., Rockford, IL, USA) at 80°C for 30 min with constant stirring (Pham et al., 2008). The temperature was verified using a calibrated thermometer inside of a vial in the heating block. A 3-phase Solid phase microextraction (SPME) fiber (2 cm-50/75 um Carboxen^™/polydimethylsiloxane/divenylbenzene [Car/PDMS/DVB], Supelco, Bellefonte, PA, USA) was inserted into the vial through the septum and was exposed to the generated sample headspace for 1 h at 60°C to absorb the volatile aroma compounds. After the extraction of volatile aroma compounds, the volatiles were thermally desorbed from the SPME fiber into the injector port of the gas chromatograph for 5 min at 250°C in splitless mode (Pham et al., 2008).

Gas chromatography-mass spectrometry

An Agilent 7890A gas chromatography system coupled to an Agilent 5975C Mass Spectrometer (Agilent, Santa Clara, CA, USA) was used to separate and determine volatile compounds desorbed from the SPME fiber. The GC was equipped with a split/splitless injector and an HP-5 capillary column (30 m × 0.25 mm × 0.25 μm) with helium as the carrier gas at a flow rate of 1 mL/min. The SPME fiber was inserted into the injector port at 250°C for 5 min in splitless mode. The oven was initially heated at 40°C for 2 min, ramped up to 150°C at 5 °C/min, held for an additional 5 min, and ramped up again to 250°C at 50°C/min, where it was held for 7 min. The Mass spectrometer (MS) source and quadrupole temperatures were 230°C and 150°C, respectively. Volatiles were monitored in a SCAN mode with a m/z range of 35 to 350. The precision of the method was validated by analyzing 3 samples per d on 3 separate days of the same homogenized chicken breast. The accuracy of the method was validated by spiking 3 blanks and 3 chicken samples with nonanal and hexanal at 2 spiking levels of 200 μg/g and 400 μg/g.

The background ions, mainly m/z 207 and 281 from column bleed, were removed using MassHunter Qualitative Analysis Software (Version B.06.00, Agilent Technologies, Santa Clara, CA, USA), and the identity of the compounds was confirmed with a 90% spectrum match probability in the NIST11 Mass Spectral Database. Extracted ion chromatograms of the most characteristic and abundant ions for volatile compounds were used to calculate peak areas (of the extracted ion chromatograms). The peak areas (of extracted ions) were divided by the peak area of m/z 72 (characteristic ion of the internal standard) to obtain the abundance ratio of the volatile compounds, which were then divided by sample weight to produce the abundance ratio per g (ARG). Hexanal, octanol, and nonanal were expressed in ng/g because authentic standards were used. Calibration curves for these compounds were constructed by adding 100 ng of authentic standards in 2.5% NaCl solution with an internal standard (10 μL at 200 ppm, chlorobenzene, Supelco, Bellefonte, PA, USA to similar vials and proceeding with the same extraction procedure (Pham et al., 2008).

Gas chromatography-olfactometry

For identification of the aroma-impact compounds, Osme-Gas chromatography olfactometry (GCO) analyses were conducted using an Agilent 7890A Gas Chromatograph (Agilent, Santa Clara, CA, USA) equipped with a sniffing port (Model ODP2, Gerstel, Linthicum, MD, USA), a flame ionized detector (FID), and an HP-5 column (Agilent, Santa Clara, CA, USA; 30 m × 0.25 mm × 0.25-μm film thickness) was used to separate volatile compounds under the same conditions described previously for GC-MS. The column extended from the oven and was split by a column flow splitter to the FID and the olfactometer (Model ODP2, Gerstel, Linthicum, MD, USA) that was connected to the sniff port. In addition, the sniff port was purged with humidified air at a flow rate of 30 mL/min. The operating conditions were identical to those used for the GC-MS. Three panelists trained in sensory and GCO analyses described the aroma properties of the volatile compounds present in the samples that were separated by the GC (Rousseff et al., 2001). The assessors were trained in GCO analyses for 10 h by sniffing original samples and volatile flavor compounds extracted by SPME from chicken meat samples. The intensity of the perceived aroma was rated by each panelist using a 0–15 potentiometric sliding scale, in which 0 = none and 15 = maximum intensity (McDaniel et al., 1990). Authentic standards and n-alkane series C₅-C₁₈ were separated and quantified by the GC-FID and GC-MS systems under the same operating conditions. Headspace volumes were drawn for the authentic and alkane standards using a gastight digital syringe (1700 Series GASTIGHT^™ Digital Syringe, Hamilton Company, Reno, NV, USA) and immediately introduced into the injector.

Identification of the aroma-impact compounds was based on comparing sample mass spectra with those in the NIST02 Mass Spectral Database (NIST, MD, USA; purchased from Varian Inc.), the linear retention index and aroma quality perceived at the sniffing port with those of authentic standards, and the linear retention index (n-alkanes C₅-C₁₈, Sigma-Aldrich Chemical Co., Milwaukee, WI, USA) and the aroma quality perceived at the sniffing port with those in literature and retention index databases (Acree and Arn, 2023; El-Sayed, 2023).

Statistical analysis

A general linear model was used to analyze the variances of pH, raw color, cook loss, protein bind, sensory descriptive analyses, and volatile compound concentration with treatment as a fixed effect. The rep*trt error term was used to account for the random error between replications and variability in subsamples. When differences occurred among treatments (P ≤ 0.05), Fisher’s Protected Least Significant Difference test was used to separate the treatment means.

pH

The pH of SWB chicken patties (6.05) was greater (P < 0.0001) than that of NB chicken patties (5.94; Table 1). Similar findings have been reported in woody breast meat compared with meat not affected by myopathy (Aguirre, et al., 2018; Jarvis, et al., 2020a; Jarvis, et al., 2020b). Baldi et al., (2020) showed that the mean pH of raw SWB was greater than that of the NB meat, in addition to less lactate, glucose, and glucose-6-phosphate concentrations at 24 h postmortem. In addition, the glycogen content (15 min postmortem) and glycolytic potential of SWB meat were less than that of the unaffected breast meat (Baldi et al., 2020). Overall, metabolites resulting from glycolysis in SWB meat are less prevalent than those of unaffected meat. High pH in muscle has been associated with chronic inflammatory diseases (Alnahhas et al., 2014; Beauclercq et al., 2022; Qiao, et al., 2001) or oxidative stress (Zhang and Barbut, 2005). Chronic oxidative stress in poultry can negatively impact growth performance and subsequent meat quality and can increase the peroxidation of lipid and protein contents in muscle tissues via reactive oxygen species (Nawar and Zhang, 2021; Basiouni et al., 2023).

Instrumental color

There were no differences (P ≥ 0.0686) in lightness (Commission Internationale de l’Éclairage (CIE) L*), redness (CIE a*), or yellowness (CIE b*) between SWB and NB patties (Table 1). Brambila et al. (2017) reported no difference in color between normal and woody breast meat. In contrast, other researchers reported that intact wooden breasts were lighter, redder, and yellower than woody breasts (Cai et al., 2018; Mudalal, et al., 2015). Lightness is increased by the greater moisture content in the woody breasts, which is associated with greater reflectance and a lighter meat color (Soglia, et al., 2016). Additionally, an increase in collagen and fat contents in woody breasts may also affect the appearance of the meat (Soglia et al., 2016). It is evident that homogenization of the patties diluted color differences between SWB and NB.

Cooking loss

SWB patties had greater cooking loss (P = 0.0037) than patties formed with NB meat, with SWB patties yielding 9% less than NB patties (31.4% vs. 22.4 %, Table 1). This observation is consistent with findings by other researchers on wooden breast myopathy (Aguirre et al., 2018; Jarvis et al., 2020a; Jarvis et al., 2020b; Mudalal et al., 2015). The woody breast condition has been linked to physical degradation of the muscle fibers which in turn negatively impacts product quality, particularly cooking loss and texture, due to structural differences such as hypertrophy, greater sarcomere length, reduced fiber quantity, and fibrosis when compared with that of NBs (Baldi et al., 2020; Sihvo et al., 2014; Soglia et al., 2016). These symptoms negatively impact the ability of the myopathic muscle to bind and retain water during processing and storage, resulting in greater cooking loss (Pang et al., 2021). Even though a higher pH is typically associated with greater WHC, SWB patties had less WHC, as indicated by the cooking loss results. Previous studies have also observed the same response, which could be elucidated by further examination of the morphological characteristics of SWB patties (Cai et al., 2018; Soglia et al., 2016; Zotte et al., 2014).

Protein bind

The protein bind value of SWB patties was less than that of NB patties (P < 0.0001; Table 1). This finding agrees with previously conducted research, in which greater protein binding was found for patties formed from normal meat than those formed from myopathic meat in either unprocessed or patty forms (Jarvis et al., 2020a; Jarvis et al., 2020b). Li et al. (2022) reported that breasts affected by woody breast myopathy contained fewer sarcoplasmic and salt-soluble myofibrillar proteins than unaffected breast tissue. Additionally, Wold et al. (2019) reported that SWB contains less protein than unaffected breast, which could potentially impair the normal functioning of the myofibrillar proteins in the muscle tissue, which in turn could result in decreased intermolecular binding postmortem and less protein binding than NB patties (Zhang et al., 2020).

Descriptive sensory analysis

Severe woody breast patties were more tender, juicy, and crunchy than NB patties (P < 0.05, Table 2); however, NB patties were more (P < 0.05) cohesive than SWB patties. No difference existed between SWB and NB patties in chewiness (P = 0.1565), mushiness (P = 0.1992), springiness (P = 0.0946), or stickiness (P = 0.7246; Table 2). Regarding the basic tastes, SWB patties had more intense (P < 0.05) bitter and umami notes than NB patties. There was no difference in the sour taste (P = 0.4525) among SWB and NB patties. Regarding the flavor profiles of the 2 samples, SWB patties were more bitter, umami, and metallic, and had more intense off-flavor (P ≤ 0.0221) than NB patties. There was no difference (P = 0.4083) in the chicken/meaty flavor characteristics. The woody breast condition has been associated with a heightened presence of off-flavors, characterized by descriptors such as sour, bitter, tangy, unclean, and earthy (Jarvis et al., 2020a). Severe woody breast meat has been reported as less tender, chewier, and crunchier than NB meat (Aguirre et al., 2018; Nawar and Zhang, 2021; Caldas-Cueva et al., 2021b; Jarvis et al., 2020a; Von Staden et al., 2019; Sun et al., 2022; Zhuang et al., 2018). However, only a few studies specifically discussed the sensory quality of patties that were formulated with woody breast meat (Aguirre et al., 2018; Caldas-Cueva et al., 2021a; Caldas-Cueva et al., 2021b; Jarvis et al., 2020b; Sun et al., 2021; Sun et al., 2022). In these studies, the authors agree that chewiness, cohesiveness, springiness, tenderness, and juiciness are sensory descriptors that describe chicken patties. Out of the 6 studies that investigated the descriptive sensory attributes of patties made from woody breast meat, only one, also from the current research group, comprehensively characterized flavor as was done in the present study (Jarvis et al., 2020b). Therefore, when interpolating from the currently available data, evidence suggests that broiler breast (pectoralis major) patties formed from SWB meat are likely to be more bitter, umami, and metallic, and have more off-flavor notes than NB.

Gas chromatography–mass spectrometry

Forty-eight volatile compounds were identified from the chicken patties, including hydrocarbons, alcohols, aldehydes, ketones, phenols, pyridines, furans, esters, and terpenes (Table 3). Aldehydes were the predominant class of compounds in the samples, followed by alcohols and hydrocarbons. For the aldehydes, 23 were detected, all of which differed in relative abundance between SWB and NB patties (P ≤ 0.031) except for pentanal, tridecanal, tetradecanal, and hexadecanal (P ≥ 0.062). Primary lipid oxidation products, such as hexanal, heptanal, 2-heptenal, 2-octenal, nonanal, 2-decenal, (E,E) 2,4-decadienal, and 2-undecenal were greater (P ≤ 0.003) in SWB than in NB. Six alcohol compounds were identified in the cooked patties (Table 3). Pentanol (P < 0.0001), heptanol (P = 0.0001), 1-octen-3-ol (P = 0.0003; mushroom), 2-octen-1-ol (P = 0.0002; blue cheese, sweaty), octanol (P = 0.0003; smoky, rusty, sulfur), and 1-nonanol (P = 0.055; grassy, herb, sweet, woody) were greater in SWB patties than in NB patties. Ten hydrocarbons were identified in SWB and NB patty samples (Table 4). Cyclooctane (P < 0.0001; floral, woody, pine sol), cyclodecane (P = 0.020), 3-ethyl-2-methyl-1,3-hexadiene (P < 0.0001), and 1-tridecene (P = 0.0404) all were greater in SWB patties. Two ketones, 2-heptanone and 3-octen-2-one, were identified in the NB and SWB patty samples. These ketones were more abundant (P ≤ 0.006) in the SWB samples than in the NB patty samples. A single furan, 2-pentyl furan, was identified in the samples and was greater (P = 0.006; floral, citrus) in SWB patties than in NB patties. Two pyridine compounds, 3-methyl pyridine and 2-propyl pyridine, with 2-propyl pyridine exhibiting significantly higher levels in SWB patties compared to NB counterparts, contributed to sensory attributes such as savory, brothy, and fatty notes (P < 0.0001).

Lipid oxidation in meats is either caused by thermal oxidation (such as cooking) or autoxidation during storage (Dinh et al., 2021). Although these 2 mechanisms of meat oxidation are similar in process, they differ in their resultant sensory properties that can be attributed to the variable presence and abundance of compounds. These compounds contribute distinct sensory attributes to the patties, including grassy, earthy, and sour notes, which were more intense in SWB than NB patties and substantiated by the descriptive analysis results. Due to the innate conditions of thermal oxidation, a greater abundance of compounds considered desirable meat flavor characteristics is produced compared with autoxidation (Nawar, 1984; Song et al., 2011). Additionally, autoxidation primarily results in the formation of aldehydes from the degradation of hydroperoxides, the most abundant classification of chemicals found in the current study (Esterbauer et al., 1991). The SWB patties contained a greater abundance of compounds that are negatively associated with meat quality. These data indicate that a greater rate of phospholipid oxidation occurs in woody breast meat when compared to that of NB meat (Aguirre et al., 2018; Nawar and Zhang, 2021; Von Staden et al., 2019; Zhuang, et al., 2018). This likely happens for 2 reasons. First, there are 30%–40% more phospholipids and fatty acids in SWB than in NB, even though their percentage of specific fatty acids are similar (Villegas-Cayllahua et al., 2024). Second, mitochondrial oxidation of fatty acids and oxidative stress are increased in SWB in comparison with NB meat (Zhang et al., 2020).

Hexanal, a key indicator of meat lipid oxidation was more prevalent in SWB patties than in normal patties (Domínguez et al., 2019). The identified aldehydes that are produced through oxidation likely contributed to the off-flavors observed in descriptive sensory analysis. For example, the grassy (hexanal, nonanal, 2,4-nonadienal), citrus (heptanal, [Z]-4-decenal, decanal, 2-decenal, [Z,Z]2,4-decadienal), sour (heptanal), earthy (heptanal, nonanal), onion (2-heptenal), and fatty (2,4-nonadienal, [E,E]2,4-decadienal, 2-undecenal) odors from these compounds were also detected in descriptive sensory analysis. Undesirable flavor compounds from SWB patties may also be increased during grinding and patty formation (Sohaib et al., 2017). Hexanol, heptanol, octanol, and nonanol are derived from oleic acid, and butanol and pentanol are derived from linoleic acid (Domínguez et al., 2019; Sohaib et al., 2017). Alcohols can also be formed from the degradation of amino acids through protein oxidation (Sohaib et al., 2017), which may have also occurred more readily in the SWB patties.

Gas chromatography–olfactometry

Twenty-five different aroma-impact compounds (Table 4) were identified. The identities of the aroma compounds were verified by comparing the perceived aromas and retention indices of the aromas with established aroma databases and the GC-MS results. The most intense aroma compounds were hexanal (grassy), 1-octen-3-ol (mushroom), 2-pentyl furan (floral, citrus), 2-ethylhexyl acetate (citrus, earthy, green, onion), and nonanol (grassy, herb, sweet, woody), with aroma scores between 5 and 6 on a 15-cm scale. The compounds 1-nonanol (P = 0.0049; grassy, herb, sweet, woody) and cyclooctane (P = 0.0197; floral, woody, pine sol) had greater intensities in SWB patties than in NB patties.

Both 1-nonanol and cyclooctane are the products of autooxidation, as are many other compounds such as hexanal, 1-octen-3-ol, and 2-pentyl furan. Of these compounds, hexanal, 1-octen-3-ol, 2-pentyl furan, and 1-nonanol are lipid oxidation products, contributing to the earthy, unclean off-flavor in the SWB meat. The grassy (hexanal and 1-nonanol), floral (2-pentyl furan and cyclooctane), woody (1-nonanol and cyclooctane), and mushroom (1-octen-3-ol) aromas from these compounds might also contribute to the tangy, unclean, and earthy off-flavor that was detected in SWB patties in sensory analysis. Severe woody breast meat was described as more bitter and metallic, with more off-flavor during descriptive analysis, which is likely related to 1-octen-3-ol concentration, with a low threshold of 1 ppb and associated with metallic notes in fatty meats (Pham et al., 2008).

The differences in composition between SWB and NB meat were primarily attributed to undesirable volatile flavor compounds produced from the greater rate of phospholipid and protein oxidation in woody breast meat (Li et al., 2022). Increased prevalence of oxidative stress is associated with the wooden breast condition, which has been hypothesized to stem from environmental factors such as heat (Nawar and Zhang, 2021), mitochondrial misfunction, feeding practices (Jia et al., 2024), and genetic predisposition to the myopathy (Zhang et al., 2020).