Differences in toughness and aging potential of longissimus lumborum muscles between Hanwoo cow, bull and steer

Thirty Hanwoo cattle including bull (n = 10), cow (n = 10), and steer (n = 10) were obtained from the commercial meat processing plant. They were slaughtered at different ages (bull slaughtered average age at 26 months, steer slaughtered average age at 31.2 months, cow slaughtered average age at 46 months). The following day after slaughter, their carcasses were evaluated and graded by an official grader for carcass traits (carcass weight, ribeye area, back fat thickness and yield grade, etc.) according to the beef carcass grading [18].

Longissimus lumborum (LL) muscles were collected from the left sides of carcasses. Muscles were sealed with vacuum-packaged and aged at 4°C for 3 and 21 days. Each gender contained 10 samples for each aging time. When the aging was completed, they were prepared into sub-sample size depending on analyses. Except for the samples used for the share force, tensile extension, color, and cooking loss, the rests were vacuum-packaged and store at −80°C until use.

The pH values of these samples were determined using a Meat pH meter (HI99163, Hanna instrument, Padovana, Veneto, Italy). Each sample was measured 4 times.

The color was determined using a Konica Minolta Spectrophotometer (CM-2500d, Konica Minolta, Tokyo, Japan), the machine contained an 8-mm measuring port AT, D65 illuminant, and 10° observer (Sinodevices, Tokyo, Japan). Each sample was blooming at 4°C for 30 min, then measured at three different locations on the surface. The samples were measured for L*, a*, and b*. L* means the lightness of meat, a* means the redness of meat, and b* means the yellowness of meat.

Cooking loss, and texture analysis (Warner Bratzler shear force [WBSF], TPA, and tensile tests) were determined on the samples (3 cm thick steak with the weight of 300 g). Particularly, the samples were immediately placed in plastic bags and cooked in a water bath until their core temperature had increased to 70°C. After cooking the samples cooled in circulating water for 30 min. The weight of samples was recorded before and after cooking, then, using the following equation to measure cooking loss:

After cooking loss measurement, the samples were measured for WBSF, TPA, and tensile tests using an Instron Universal Testing Machine (Model 3342, Instron, Norwood, MA, USA). Every sample was cut into more than 6 trips which are parallel to the muscle fiber direction. The WBSF was evaluated with 1.5 cm diameter samples and sheared at a crosshead speed of 400 mm/min, using a 40 kgf load cell. Tensile testing was conducted with 70 × 10 × 10 mm thick per sample. Stretching was performed at 50 mm/min. TPA (hardness) was done on 3 cuts in a rectangular trapezoid shape with 10 × 10 × 40 × 30 mm per sample. Each sample underwent 2 cycles of 60% compression at a constant speed of 50 mm/min.

The sensory evaluation was followed by our lab’s previously established protocols [19]. The panel consisted of eight faculty members. Every sample was cut into 4 cm (length) × 3 cm (height) × 0.5 cm (thick) size to be tested. Three sessions were held for every sample at different times. The panelists evaluated contained tenderness, juiciness, flavor, overall acceptability, and overall rating. Each panelist assessed the cooked beef meat samples in a randomized order, and everyone needs to give a value from 1 to 100 (ie: from denotes unacceptable to extremely acceptable) after the test. Each panelist was asked to use distilled water (DW) to refresh their mouths in between samples.

A procedure developed by Rule [20] was used to detect the composition of fatty acid. The samples were made into thin slices which were then freeze-dried for 48 h. About 500 mg of each dried sample was placed in a 20 mL vial with 2 mL of 14% boron-trifluoride in methanol and 2 mL of high performance liquid chromatography (HPLC) grade methanol. The vials were sealed with a scrimp cap, then they were placed in a heating block set at 80°C, and vortex mixing every 5 min, for maintained 2 h. Thereafter, 3 mL DW and hexane were added, respectively, and followed by centrifuging at 1,000 g force for 5 min. Each sample was infused with about 1 mL of hexane and sealed in a vial. The fatty acids composition was determined using an Agilent Gas Chromatography-Mass Spectrometer system (GC-MS) (GC 7890B, MS 5977B Agilent Technologies, Santa Clara, CA, USA) with an auto-sampler. The injection temperature was set at 250°C, the carrier gas with a speed 45 cm/s with a split ratio of 50:1. Fatty acid methyl esters were separated with a 1.0 mL/min helium flow which is on a WCOT-fused silica capillary column (30 m × 0.25 mm × 0.25 μm). The oven was programmed as follows: 150°C/2 min, 150°C to 230°C at 10°C/min, 230°C/15 min. The fatty acids were identified by comparison with the retention time with those of fatty acid standards (F.A.M.E. Mix., CRM 18918, 47015-U, Sigma-Aldrich Supelco, Bellefonte, PA, USA). The proportion of the fatty acid calculated use the peak area of each identified fatty acid against total identified peak area.

The oxidative stability was determined using the procedure developed by Buege and Aust [21], which was detected the values of TBARS to measure oxidative stability. Briefly, an Ultra Turrax T25 homogenizer (IKA Labortechnik, Selangor, Malaysia) was used 2.5 g meat samples with the solution for 15 s at 11,000 rpm. The solution contained 7.5 mL DW, 25 µL BHA (butyl hydroxyanisole) and 10 mL thiobarbituric acid solution and trichloroacetic acid solution (TBA/TCA). After homogenizing, the sample was immediately placed in ice, and added TBA/TCA solution to homogenate until the volume to 30 mL. The samples were heated at 90°C in a water bath for 15 min. Then, taken out and placed in ice to cool for 20 min. The absorbance of the sample is determined at 531 nm against a blank that contains all the reagents minus the lipid on an Ultrospec 2000 spectrophotometer (Pharmacia Biotech, Cambridge,UK). Multiply the absorbance reading by 5.88 (mg/kg) to calculate the malondialdehyde (MDA) concentration in the sample.

All data were analyzed using the General Linear Model Procedure of the SAS version 9.3 program (SAS Institute, Cary, NC, USA) [22]. The breed and aging were considered as the fixed factors while the carcass traits, quality attributes, etc. were considered as the variables. Means were compared using Duncan’s Multiple Range Test. The significance level was set at p < 0.05.

The carcass traits as affected by gender are presented in Table 1. It was observed that gender affected carcass weight, back fat thickness and, the rib eye area (p < 0.01). Cow showed the lowest carcass weight, probably due to the estrous cycle effect and its specific feeding diet. Studies have shown that compared with bulls, steers gain weight at a significantly slower rate and with less efficiency [4,5]. In our study, steers and bulls had no difference in carcass weight, this could be attributed to the slaughter age difference. The back-fat thickness was in the following order: steer > cow > bull. The previous study also reported that castrated animals are easier to deposit fat than non-castrates [23]. Bulls had lower back fat thickness compared with cows, this is due to the influence of testicular hormones that cause a significantly higher proportion of lean and a lower proportion of fat [24]. The yield index was also affected by gender (p < 0.05), due to yield index, back fat thickness, ribeye area, and carcass weight has the following relation: Yield index = [68.184 − {0.625 × back fat thickness (mm)}] + {0.130 × rib eye area (cm²)} − {0.024 × carcass weight (kg)} + 3.23. Steers had the highest marbling and the lowest firmness meanwhile bulls had the lowest marbling and the highest firmness (p < 0.001). This is due to testosterone can inhibit fat development in bulls. The marbling and firmness values showed no difference between steers and cows. This can be attributed to the marbling of Hanwoo steers significantly increasing between 12 and 27 months [25], and higher marbling indicating higher softness [26].

Gender showed no influence on color parameters (a* and b*) at 3 d aging (Table 2). However, significantly lower L* and higher pH values were observed in bull meat compared with steers or cow meat (Table 2). L* values difference can be attributed to the different genders contained different fatness which can influence muscle lightness [27]. Result of the pH values is in agreement with that color values has a negative correlation with muscle pH values [28], meanwhile, Jeremiah et al. [29] found that steers had the lowest ultimate pH values and bulls had the highest compared with steers, bulls and heifers. Regarding aging time, color values increased with increased aging time, especially in bulls and cows (p < 0.05). The aging resulted in an increase in lightness, redness, and yellowness values (p < 0.05). Previous studies also reported that Bruce et al. [30] and Vitale et al. [31] also showed an increase in L*, a*, and b* values of beef Longissimus thoracis after 14d aging. This may be explained due to the higher blooming ability of vacuum-aged meat.

Although, there are no significant differences in cooking loss among genders in Table 2. Bull meat had the highest cooking loss (%) (19.5%) compared with steers (16.8%) and cows (16.6%) in both the aging times (3d or 21d), indicating that the meat of cow and steer had a better water holding capacity. This could be related to the chemical composition differences among the beef breeds. The finding is consistent with those of Pogorzelska-Przybyek et al. [4]. Ozawa et al. [32] reported a lower cooking loss in higher marbling Japanese black steer meat. Cooking loss (%) gradually increased from 3 d to 21 d aging for all genders. Similar results were found in the studies of Boakye and Mittal [33] who showed an increased cooking loss in beef longissimus dorsi (LD) muscle with increased aging time from day 4 to 16. This may be attributed to an increase in protein denaturation which led to the loss of water holding of the muscle tissues.

The TBARS content is often measured and used as an indicator of lipid oxidation levels in foods including meats and meat products [34]. The TBARS concentration is related to the levels of MDA which is the secondary lipid oxidation compound. TBARS values were significantly influenced by aging time (p < 0.01) (Table 2). At the initial measurement, the meat of the cow showed a lower TBARS content compared to the bulls and steers. This could be related to the fatty acid composition differences among the cattle genders [35]. At 21 d of aging, the TBARS increased in all the samples. These may be explained by the activity of endogenous or microbiological enzymes. Lipid oxidation produces off-flavors, rancidity, and deterioration in meat and meat products [36]. For example, TBARS of values 2.0 mg MDA/kg were considered to be the lower limit for acceptance of oxidized beef by Campo et al. [37], McKenna et al. [38] adopted 1.0 mg MDA/kg as an arbitrary threshold, and Hughes et al. [39] found that TBARS levels between 2.60 and 3.11 mg MDA/kg were considered acceptable to consumers in long term aged beef striploin. In our study, the highest TBARS value is 0.45 mg MDA/kg which was much lower than the values reported by these authors.

The mean values of TPA, tensile tests, and WBSF are shown in Table 3. The WBSF, tensile tests, and TPA have been recommended as a tenderness standard by the American Meat Science Association [40] As expected, the WBSF, tensile tests, and TPA values decreased as increasing the aging time in all the samples (Table 3). As increasing the aging time from 3 to 21 d, the bulls, cows, and steers reduced shear force from 4.81, 3.97, and 3.67 kgf to 2.29, 2.08, and 2.26 kgf, respectively. In the same aging time, bulls had the highest WBSF values compared to cows or steers. These differences may be attributed to chemical composition differences such as intermuscular fat (IMF) and subcutaneous fat among the genders [41]. With extending aging time, all the meat samples reduced WBSF values, but the bull meat showed the highest percentage (52.39%) of tenderness improvement compared to the cows (47.61%) and steer meat (38.4%).

At the initial measurement, tensile tests values were significantly affected by aging and gender (p < 0.01). Although all the tensile tests values showed a decrease with aging time from 3 d to 21 d, bulls still had the highest tensile maximum force values in all the aging times. This can be attributed to adipocytes’ excessive development caused disorganization of the perimysial connective tissue [42]. Whilst, tensile strain, tensile extension and tensile maximum force values showed no significant difference among the genders (p > 0.05) at 21d aging time. Results can be attributed to the increase myofibrillar fragmentation index with increase aging time [43], and myofibrillar fragmentation index could shows the advancement of myofibrillar degradation.

Excepting hardness, all the TPA including, springiness (mm), chewiness (N), hardness (N), and gumminess (N), were not affected by aging. When increasing the aging time up to 21 d, the hardness decreased in all the genders, however, bull meat exhibited the highest hardness values in both aging times. This is consistent with that reported by Lepper-Blilie et al. [44]. Thus, it may be said that aging could improve the tenderness of meat from all cattle genders.

The effects of gender and aging on sensory attributes such as tenderness, juiciness, flavor, and acceptability are presented in Table 4. There were no differences in the tenderness, juiciness, flavor, and acceptability scores between steer and cow meat. Compared to the meat of these two genders, bull meat had a significant difference for all the sensory traits at 3d aging time (p < 0.01). Although, there was no difference for all the sensory traits among the genders at 21d aging time, bull meat had the lowest scores. These differences can be attributed to the IMF content differences (Table 1). Similarly, previous studies have shown that beef with higher marbling is tender, flavorful, and juicier [4,5,45]. Extending the aging up to 21 d, no differences in the tenderness, juiciness, and flavor scores occurred among the genders. This means that the eating quality of the meat from all genders was improved during aging. The mechanisms underlying this phenomenon could be related to the breakdown of myofibrils into smaller peptides by the endogenous enzymes, which improved the tenderness and flavor characteristics [46].

The fatty acid profiles of beef from the three gender are presented in Table 5. The fatty acids composition in muscle tissues play an important role in cooked flavor development [47]. A total of 14 fatty acids were identified in which the most predominant fatty acids being oleic acid (C18:1), palmitic acid (C16:0), and stearic acid (C18:0). Our results are consistent with those reported in the previous studies on Hanwoo cattle [48], or American Angus [49], and in Japanese Wagyu [50].

Total saturated fatty acids (SFAs) content was similar in all genders. The monounsaturated fatty acids (MUFA) content was lower and the polyunsaturated fatty acids (PUFAs) content was higher in bull meat compared to the other remaining genders (p < 0.01). Moreover, bull meat had significantly higher levels of C18:2 and C18:0, and had lower levels of C16:1 and C18:1 than either steers or cows (p < 0.01). Our results are in accordance with those of Legako et al. [51] who reported a higher MUFA and lower PUFA content in beef with higher marbling. It has also been reported that the C18:1 and MUFA are positively associated with beef flavor. Thus, the higher flavor score in the steer beef (Table 4) could be due to its higher oleic acid content. Contrastingly, the PUFAs content such as C18:2 has been reported to negatively affect the beef flavor [52]. In our study, we found that the C18:2 and MUFA were the highest in the bull beef (p < 0.01).