Comparison of Tastes-Related Components and Eating Quality between Hanwoo Steer and Cow Longissimus thoracis Muscles

The Longissimus thoracis (LT) muscles (10^th–13^th rib) with same quality grade (1⁺ grade) from left carcass sides of Hanwoo steers (31–32 months old, average carcass weight of 391 kg, marbling score of 6.8, n=5) and cows (47–53 months old, average carcass weight of 383 kg, marbling score of 6.0, n=5) collected at 24 h postmortem from commercial abattoirs (Jeonju, Korea) were used in the present investigation. After collecting, the LT muscles were vacuum-packaged and then transported to the meat laboratory of animal product division for analysis. Following trimming of visual fats, the muscle samples were cut into sub-sample sizes starting from the cranial end of the muscles depending on the type of analysis in the following order: proximate composition (10^th rib); fatty acids, FAAs, metabolites and nucleotides (11^th rib); sensory evaluation (12^th rib); pH, color, Warner-Bratzler shear force (WBSF) and water holding capacity (WHC) and cooking loss (13^th rib). The analyses of proximate composition, pH, color, WHC, cooking loss and WBSF were performed on fresh samples (48 h postmortem), while the fatty acids, FAAs, metabolites, nucleotides and sensory evaluation samples were vacuum-packed and frozen at −20°C until use.

The pH values of the beef samples were measured in triplicate by inserting a stainless steel probe of pH*K 21 meter (NWK-Technology GmbH, Kaufering, Germany) deeply into the muscle tissues. Prior to use, the pH meter was calibrated with standard solutions (pH 4.0 and 7.0).

The protein, moisture, fat and collagen contents were determined using a Food Scan^TM Lab 78810 (Foss Tecator, DK) according to the association of official analytical chemists methods (AOAC, 2007).

The color of the meat samples was measured using a Minolta Chroma Meter CR-400 with a D65 illuminant*1 and 2° observer (Minolta Camera, Osaka, Japan) as described in our previous study (Cho et al., 2020), and expressed as CIE L* (lightness), CIE a* (redness), CIE b* (yellowness), Chroma and hue angle (h°).

The cooking loss and WBSF of the beef samples were measured using the protocols as described in our previous (Cho et al., 2017). Briefly, each sample was made into a 2.5-cm thick steak that was placed into plastic bags and cooked in a pre-heated water bath until their internal temperature reached 80°C. After cooling for 30 min under running water (18°C–20°C), the cooked samples were absorbed with wiping papers to remove the surface water and then their weights were recorded. The cooking loss (%) of each the sample was calculated as the difference between the pre-cooked and post-cooked weights divided by the initial weight. After the cooking loss measurement, the cooked samples were used for the WBSF analysis. Eight cores (1.27 cm in diameter) were prepared parallel to the muscle fiber direction of each the sample using a 0.5-inch metal corer. The WBSF values were obtained by determining the force (kilogram force, kgf) required to cut the cores using a V-shaped shear blade of Instron Universal Testing Machine (Model 4465, Instron, High Wycombe, UK) using a crosshead speed of 400 mm/min and a 40 N load cell.

The WHC of the samples during storage was determined using centrifugation-based method developed by Kristensen and Purslow (2001). Each sample was analyzed in duplicate and the WHC percentage was calculated as a ratio of moisture to the water loss.

The FAA content in the samples was analyzed following the procedure as described by Dashdorj et al. (2013) with suitable modifications. Briefly, 2.5 g of each sample was homogenized with 5 mL distilled water at 1,200×g for 1 min. After filtering through Whatman filter paper (No. 4, Whatman, Clifton, NJ, USA), 100 μL filtrate taken and mixed with 900 μL methanol containing 0.1% formic acid. After centrifuging at 13,000×g for 10 min at 4°C, the supernatant was collected and then filtered through a 0.45-μm filter membrane (Millipore, Cork, Ireland). The filtrate was derivatized using AccQ-Tag^TM (Waters, Milford, MA, USA) according to the manufacturer’s instrument. Finally, 5 μL of each derivatized sample was injected into a Waters ACQUITY UPLC (model: Xevo TQ-S, Waters) connected to an Intrada Amino Acid column: 2×50 mm, 3μm (Imtaka, Uphur St, Suite A, Portland). The eluents and conditions used for separation of FAAs were same as those used in the above-cited reference (Dashdorj et al., 2013). For identification and quantification of the FAAs, the external amino acid standards (Sigma-Aldrich, St. Louis, MO, USA) were used, and the detected FAAs were then expressed as milligram per 100 g meat (mg/100 g meat).

Nucleotides were analyzed using the method of Jung et al. (2011) with suitable modifications. For separation, 10 μL of each sample was injected into Waters ACQUITY UPLC connected to a C18-reverse phase column: 4.6×150 mm, 3 μm (Cadenza). The mobile phases used were A: 40% tetrabutylammonium hydroxide solution-TBA-oH (25 mL), 85% H₂PO₄ solution (500 μL) and distilled water (1,000 mL), and B: 40% TBA-oH (25 mL), 85% H₂PO₄ solution (500 μL) and methanol (1,000 mL). The separation of nucleotides was carried out at 40°C, the detection was monitored at a wavelength of 260 nm and flowing rate of 0.8 mL/min. The solvent gradient set was: initial 100% A, linear change to 95% A for 1 min, linear change to 100% B for 11 min and then 95% A for additional 8 min. The nucleotides were identified by comparing their retention times with those of external standards, and then quantified using internal standard (Purine) (Sigma-Aldrich).

The lipid content in the muscle samples was extracted using a solvent mixture of chloroform: methanol (2:1, v/v) as described by Folch et al. (1957). For the extraction, 10 g of sample was homogenized with 150 mL of the solvent mixture at 300×g for 3 min using a homogenizer (Polytron, PT-MRC 2100, Switzerland). After filtration through Whatman filter paper, the filtrate was added with approximately 20 g of Na₂SO₄, thoroughly mixed for 1 min, and then the upper lipid layer was transferred into an Erlenmeyer flask. After drying at 55°C using a rotary evaporator, the lipids layer was reconstituted with 1 mL tricosanoic acid and 1 mL of 0.5N NaOH. Finally, the lipid was converted to fatty acid methyl esters following the procedure of Morrison and Smith (1964). The fatty acids were analyzed by a gas chromatography (GC, Model Star 3600, Varian Technologies, Palo Alto, CA, USA) connected with a capillary column (30 m×2 mm×0.25 μm film thickness). The conditions used for separation, and identification of fatty acids were same as those described in our previous study (Cho et al., 2020). The results were expressed as relative percent (%) of total fatty acids based on total peak area.

The sensorial characteristics of both the beef types were evaluated using the procedure as described in our previous study (Cho et al., 2020). The procedure used for the sensory evaluation was approved by the Institutional Review Board of National Institute of Animal Science (No.11-1390744-000007-01). Briefly, the panel consisted of 7 trained members (at 24 to 40 years old) who were the institution’s staffs. For evaluation, seven representative strips (50×75×4 mm) were prepared from each sample using a meat slicer. Two sessions per day were carried out, and each session had 7 panelists in which each the panelist evaluated 7 samples. The slices were cooked on an open tin-coated grill at around 160°C–170°C for approximately 2 min and turned at the start of browning. Immediately after cooking, the cooked samples were placed on individual paper dishes and served to the panelists. The panelists evaluated for tenderness, flavor, roasty flavor, taste (intensity of taste after chewing), juiciness, tenderness and overall acceptability using a 6-point scale. The panelists were asked to refresh their palate with drinking water and unsalted crackers between samples. The sensory evaluation was performed in the sensory panel booth room equipped with white lighting.

The cooked LT muscle samples (the samples were cooked under the identical conditions as those used in the sensory evaluation) were used for the metabolites analysis. The analysis of metabolites was performed following the protocol as described by Lin et al. (2007). Briefly, after grinding each the sample (20 mg) was extracted with acetonitrile/water (1:1, v/v) mixture on ice for 10 min. After centrifuging at 3,000×g for 10 min at 4°C, the supernatant was transferred to a new tube which was then freeze-dried. The lyophilized samples were dissolved in 700 μL of deuterated water containing 2 mM 3-trimethylsilyl-2,2,3,3-tetradeuteropropionic acid-d4 (TSP-d4, Sigma-Aldrich) as an internal standard, and then was transferred into a 5 mm NMR tube for analysis.

¹H-NMR spectra were acquired on a 600 MHz Agilent NMR spectrometer (Agilent Technologies, Palo Alto, CA, USA) equipped with 600 MHz 4-mm gHX NanoProbe (Agilent Technologies, Santa Clara, CA, USA) at a ¹H frequency of 599.93 MHz. The ¹H-NMR conditions were set as follows: spinning rate at 2,050 Hz and 25°C; number of scans, 126; acquisition time, 3 s; relaxation delay, 3 s; 90° pulse, 6.35 μs; total acquisition time per sample, 13. 9 min. The acquired spectra were phased and then the baseline was corrected and referenced to the TSP-d4 peak using a Vnmrj (version 4.2, Agilent Technologies, Palo Alto, CA, USA).

Metabolites in the ¹H-NMR spectra of the cooked beef samples were tentatively identified using Chenomx 600 MHz library database and Chenomx NMR Suite 7.1 professional. Concentration of each the identified metabolite was calculated by its corresponding peak area divided by the internal standard’s peak area multiplied by the known concentration of international standard.

The Statistical Analysis System (SAS) package (SAS Institute, Cary, NC, USA, 2015) was applied for analysis of the data. The data were analyzed by using the General Linear Model procedure of the SAS, and the beef type (gender) was considered and fixed as the main effect while the quality traits examined were considered random in the model. Means were compared using Duncan’s multiple range test. Significance was set at p<0.05.

The proximate composition in the LT muscles from the two sex types (cows and steers) is presented in Table 1. The protein, moisture, fat and collagen content were: 18.66% vs 19.49%, 61.30% vs 61.45%, 17.82% vs 16.69% and 2.19% vs 2.12% for the steers and cow meats, respectively. No significant (p>0.05) differences occurred in all of these contents between the two beef types. This could be due to the LT samples which were in the same quality grade (1⁺ grade). Studies have reported that the proximate composition (e.g., fat and protein) in beef muscles differ depending on the quality grades (Gajaweera et al., 2020; Jung et al., 2013; Kim and Lee, 2003). Compared with our data, those of Gajaweera et al. (2020) found similar protein, fat, moisture and collagen contents in longissimus thoracis muscles from Hanwoo steers with same 1⁺ grade. However, compared with fat content (2.5%–5.5%) and marbling scores (2.3–3.7) reported for Hereford cows slaughtered at 4–12 years old by Galli et al. (2008), or cull Danish Friesian cows (Vestergaard et al., 2007), the cow meat in the present study had several times greater level. This fact could be attributed to the differences in genetics and feeding diet used among the studies.

Table 2 shows the technological quality and instrument color traits of the LT muscles from both sex types. There were no significant differences occurring in the cooking loss, WBSF, WHC as well as color traits between the two beef types (p>0.05). In general, the cooking loss level, shear force values and WHC percent of both the beef types in the present study were almost similar to those reported for Hanwoo steer beef muscles with the same quality grade in literature (Cho et al., 2020; Gajaweera et al., 2020). Interestingly, compared to shear force values (5–6 kg) reported for the same sex type such as Hereford cows slaughtered at 4–12 years (Galli et al., 2008) and cull Danish Friesian cows (Vestergaard et al., 2007), the cow meat in the present study had approximately a half lower value. This could be attributed to the higher IMF content in the Hanwoo cow meat compared with those in the meats from other cattle breeds used in these studies. Regarding this, a lot of studies have found a negative correlation between the IMF and shear force in beef; the higher the fat content the lower the force required to shear and cut the meat (Gajaweera et al., 2018; Park et al., 2000; Wood et al., 2008). The shear force value is considered as an indicator reflecting the tenderness of meats. Compared with WBSF values reported by Obuz et al. (2004) for beef muscles from high USDA quality grade (e.g., choice and select), the WBSF values obtained on the both beef types in the present study were lower. Researchers have found that the sex, mainly by hormonal status, is a vital factor affecting the physicochemical quality and color traits of beef muscles (Panjono et al., 2009; Picard et al., 2019; Sauerwein and Meyer, 1989). Furthermore, research conducted to examine the effect of animal age on meat quality traits of Hanwoo female cattle has shown that increasing the slaughter age resulted in increased shear force, and decreased WHC and lightness of beef longissimus muscle (Cho et al., 2017). Based on the findings reported in these studies, it may be said that sex and slaughter age both have the important effects on the beef quality. In the present study, though the cows were slaughtered at a mature age (47–53 months), and the steers were slaughtered at younger age (31–32 months), no differences occurred in all the technological quality traits examined between these two beef types. This may be explained due to: (i), the beef samples in both the sex types were in the same quality grade (1⁺ grade) group because beef cuts within a same quality grade usually are uniform in quality; (ii), the steers produced after castration (castrated male cattle) usually are changed in their hormonal profiles (Plouzek and Trenkle, 1991) which reduced the action and aggressive behavior whereas, increased the fat deposition in muscle tissues (Destefanis et al., 2003). Other researchers have also shown an increased IMF content and improved meat quality in castrated male cattle (steers) compared to intact male cattle (bulls) (Silva et al., 2019). Till now, no reports comparing the technological quality traits between the steer and cow meats are available. Corresponding to the present findings, however, Choat et al. (2006) and Mueller et al. (2019) also reported no differences in shear force and color traits such as; lightness and redness between steer and female (heifers) meats.

The concentrations of FAAs and nucleotides found in the LT muscles of both sex types are presented in Table 3. FAAs are mainly responsible for the tastes of meats, especially sweetness and umami (Dashdorj et al., 2013; Jayasena et al., 2013; Mateo et al., 1996). Based on their similar taste quality, Kato et al. (1989) and Sforza et al. (2001) categorized the FAAs into several classes for instances; glycine, alanine, serine, proline and glutamic acid are associated with sweet taste; glutamic acid, aspartic acid, alanine, serine, lysine and methionine are associated with umami taste; aspartic acid, glutamic acid and histidine are associated with salty taste; valine, leucine, isoleucine, phenylalanine, arginine, proline, tryptophan and methionine are associated with bitter taste. Results show that the most predominant FAAs found in the both beef types were alanine and glutamine which are responsible for sweet taste. Supporting the present results, Cho et al. (2007) and Jayasena et al. (2015) reported similar trend for these amino acids in Hanwoo longissimus muscles. Noticeably, no significant differences occurred in these two major amino acids between the two beef types (p>0.05). Other some FAAs associated with umami (e.g., glutamic acid and lysine), sweetness (e.g., proline and glutamic acid) and saltiness (e.g., histidine and glutamic acid), were significantly (p<0.05) higher in the cows compared to the steers. Supporting the present findings, Lee et al. (2019) reported higher levels of glutamic acid, leucine, valine and threonine in Hanwoo female longissimus muscles compared to the castrated male cattle.

Regarding the nucleotides, inosine-5′-phosphate (IMP) is known as the major breakdown product of adenosine 5-triphosphate (ATP), which is then degraded into hypoxanthine and inosine in meats after slaughter (Nishimura et al., 1988). These breakdown products of 5’-nucelotides are important components contributing and enhancing the taste especially umami of cooked meats (Jayasena et al., 2013; Ichimura et al., 2017; Nishimura et al., 1988). Nucleotides such as IMP is mainly responsible for the tastes of meat, especially sweetness and umami (Dashdorj et al., 2013; Mateo et al., 1996). Results showed that there were no differences in all the detected nucleotides between the cow and steer meats (p>0.05), implying a similar conversion rate of ATP to the IMP and others between the two sex types. Compared with our data, those of Dashdorj et al. (2013) found higher amounts of IMP and hypoxanthine in Hanwoo heifer longissimus muscles after 7 and 14 days of chiller ageing. These contrasting results could be attributed to the different sampling time between the studies because increasing post-mortem ageing usually results in increased amounts of IMP and hypoxanthine in fresh meats (Tikk et al., 2006).

By using the NMR spectroscopy technique, 27 metabolites were detected and identified in the cooked LT muscles from both the beef types (Table 4). In general, the NMR profiles displayed a broad range of compounds comprising amino acids, peptides, carbohydrates and nucleotides, etc. Among them, alanine, glutamine, glycine, leucine, methionine, tyrosine and valine have also been found in beef longissimus lumborum muscles (Frank et al., 2020). All of these amino acids have also found in the raw LT muscle of both the sex types (Table 3). Out of the amino acids, however, only glycine and tyrosine showed a significant (p<0.05) difference between the two sex types. Furthermore, the most predominant amino acid found was alanine whose amount was similar in both the beef types (p>0.05). Alanine is associated with sweet taste of cooked meat (Kato et al., 1989). AMP and inosine were two unique nucleotides found in both the beef types. They are known as the products degraded from ATP by endogenous enzymes in muscle tissues after slaughter as mentioned above. The amount of inosine was about 0.13 mmol/kg greater in the cow meat compared to that in the steer meat. The other metabolites identified such as; acetate, betaine, lactate, carnosine, creatine and creatinine have previously been reported in raw beef muscles (Frank et al., 2020; Kim et al., 2016). Of which, carnosine (an antioxidant dipeptide) was found at an abundant level but no differences occurred in its amount between the two beef types (p>0.05). Carnosine is a metabolite generated from the proteolysis by muscle enzymes (Frank et al., 2020), and contributes significantly to red meat taste (Cambero et al., 2000). Creatine, a lipid metabolism-derived compound, was found at a high level in both the beef types, which agrees with finding of Frank et al. (2020). While, creatinine is known as a breakdown product of creatine from the muscle protein metabolism. Results show that the concentrations of creatine and creatinine were greater in the cow meat compared to that of the steer meat (p<0.05). However, the role of these two metabolites in development of cooked meat taste has not been characterized. In general, the results indicating the variations in levels of some metabolites could be partly related to the sex difference that affects the protein and lipid metabolisms in muscle tissues as well as the proteolysis/breakdown process in meat after slaughter. However, the differences in concentrations of metabolites between the two beef types apparently are negligible.

The proportion of fatty acids in the LT muscles from both sex types are presented in Table 5. Fatty acids not only reflect the nutritional value but also considerably affect the development of cooked meat flavor (Mottram, 1998). Excepting palmitic acid (C16:0), vaccenic acid (C18:1n-7) and oleic acid (C18:1n-9), all the other remaining fatty acids, total saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) did not differ between the two sex types of beef (p>0.05). Studies have found that castrated cattle (steers) are usually changed in their hormonal profiles which directly affects body fat deposition (Plouzek and Trenkle, 1991) and distribution of fatty acids in muscle tissues as well (Lee et al., 2009). This may be the main reason explaining why most of the fatty acids in the steer meat were not different from those of the cow meat. The current findings would agree with those of Mueller et al. (2019), who did not find differences in most of fatty acids between steer and heifer meats whereas, the significant differences only were found between bull and heifer or bull and steer meats.

In general, it was observed that both the sex types of beef had the fatty acid profile characteristic of high concentrate-fed animals, being indicated by a high level of MUFAs such as C18:1n9 and n-6/n-3 fatty acids ratio, and a lower level of n-3 PUFAs such as linolenic acid (C18:3n-3) (De Smet et al., 2000). Our results align with those of Jayasena et al. (2015), Lee et al. (2019) who reported similar trends for most of the fatty acids in beef muscles from Hanwoo steers and heifers. Likewise, compared to pastured Australian Angus beef, concentrates-fed Hanwoo beef is lower in C18:3n-3 content and higher in n-6/n-3 fatty acid ratio (Cho et al., 2005). Out of the fatty acids identified, C18:1n-9 was the most abundant fatty acid present in both the beef types, followed by palmitic acid (C16:0), which agrees with those of Jayasena et al. (2015) and Cho et al. (2020). Interestingly, the level of C18:1n-9 was significantly higher in the steer meat (47.86%) compared to that in cow meat (41.48%) (p<0.05). Our results are in accordance with those of Lee et al. (2019): C18:1n-9 content was higher in Hanwoo steer meat compared with that in female cattle meat. Previous studies have found that production system (feeding diet and regimes, etc.) significantly affects the fat deposition and fatty acid profiles in beef muscles (Schor et al., 2008; Wood et al., 2008). Therefore, the result indicating the variations in the fatty acids between the two beef types may be mainly attributed to these factor’s effects. Unfortunately, the information regarding the diets and feeding regimes for the both sex types of beef in the present study was not recorded.

Sensory evaluation of meat is of utmost important to ascertain the virtual merits in the meat-producing practices as well as the impacts of chemical constituents on the precise sensorial attributes of meat. The results of sensory evaluation for the two beef types are presented in Table 6. On the 6-points scale, the tenderness, juiciness, taste and overall acceptability scores given by the panelists were: 4.22 vs 4.36, 3.78 vs 3.93, 3.56 vs 3.62 and 4.02 vs 4.06 for the steer and cow meat, respectively. There were no statistical differences occurring in all of these sensory traits between the two sex types of beef (p>0.05). Similar to our results, those of Choat et al. (2006) showed that cattle sex (steer and heifer) had no effects on juiciness or flavor, tenderness and overall tenderness scores of longissimus muscles. Likewise, research conducted to examine the effects of sex on eating properties of Angus cattle beef has found no differences in flavor, aroma, juiciness and overall acceptability between steer and heifer (Mueller et al., 2019). The result indicating no differences in the taste score could be related to the tastes-related components (FAAs and metabolites) whose variations in amount might not be large enough for the consumers to discriminate these two beef types. On the other hand, the results indicating no differences in the other eating quality traits such as tenderness, juiciness and roasty flavor, etc. could be attributed to the IMF content that was similar in both the sex types of beef (Table 1). Supporting the present findings, Mueller et al. (2019) also found no differences in eating quality traits between steer and heifer longissimus thoracis muscles that contained a same IMF level. Till now, it is well known that marbling degree or IMF content is the vital factor determining the eating quality of beef since the level of IMF has been found positively correlated to all the eating attributes (Chung et al., 2018; Jo et al., 2012; Joo et al., 2017). In contrast to the current findings, Gajaweera et al. (2018) found higher tenderness, juiciness and overall-liking scores for meat of steers compared to cow meat. These authors also found a positive correlation between the IMF content with the sensory attribute scores in that the significantly higher eating quality scores in the steer meat was due to its double greater IMF level compared to that of cow meat. From the results/observations of our investigation it could be said that the cattle sex apparently had no effects on the eating quality when the quality grade (e.g., marbling or IMF) was adjusted to a common level.