Correlation between skeletal muscle fiber type and free amino acid levels in Japanese Black steers

Abstract Free amino acids are important components of tastants and flavor precursors in meat. To clarify the correlation between muscle fiber type and free amino acids, we determined the concentrations of various free amino acids and dipeptides in samples of different muscle tissues (n = 21), collected from 26‐month‐old Japanese Black steers (n = 3) at 2 days postmortem. The proportions of the myosin heavy chain (MyHC), slow (MyHC1) and fast (MyHC2) isoforms were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The contents of free amino acids and dipeptides were measured by high performance liquid chromatography (HPLC). The MyHC isoform composition varied among the tissue samples. The MyHC1 proportion ranged from 6.9% ± 3.9% to 83.3% ± 16.7%. We confirmed that there was a strong positive correlation between MyHC1 composition and total free amino acid concentrations, including those for two dipeptides. Among the 31 measured free amino acids and dipeptides, 11 showed significant positive correlations and five showed significant negative correlations with MyHC1 composition. These results suggest that a high MyHC1 content induces high free amino acid contents in bovine muscles possibly because of greater oxidative metabolism. This high level of free amino acids could contribute to the intense flavor of meat that is rich in slow‐twitch fibers.


| INTRODUC TI ON
Skeletal muscle tissues are composed of slow-twitch (type 1) and fast-twitch (type 2) muscle fibers. Metabolically, slow-twitch fibers have abundant mitochondria and myoglobin and rely on oxidative metabolism, whereas fast-twitch fibers have less mitochondria and myoglobin and mainly rely on the glycolytic pathway. Myosin heavy chain (MyHC) is a predominant and key component of skeletal muscle proteins and is used as a marker protein of muscle fiber type.
Muscle fiber composition is thought to affect the color, pH, water holding capacity, tenderness, and nutritional value of meat (Choi & Kim, 2009), but the relationship between the free amino acid content and muscle fiber composition of meat is not fully understood. Free amino acids contained in meat are important flavor precursors (Toldrá, Flores, & Sanz, 1997). For instance, amino acids are converted to amines by decarboxylation (Hernández-Jover, Izquierdo-Pulido, Veciana-Nogués, & Vidal-Carou, 1996) and are essential for Maillard reactions that produce numerous volatile products (Mottram, 1998). Moreover, the free amino acid level is important for taste (Kato, Rhue, & Nishimura, 1989;Nishimura, Ra Rhue, Okitani, & Kato, 1988). If the free amino acid level of meat can be determined, it could be used to assess meat quality.
We hypothesized that slow-twitch fibers would contain more free amino acids than fast-twitch fibers because of the slow-twitch fiber mitochondria content. Enzymes for the tricarboxylic acid (TCA) cycle are abundant in mitochondria and many free amino acids are metabolized from substrates in this cycle. The aim of this study was to determine the relationship between muscle fiber type and free amino acid content, using 21 samples of different skeletal muscle tissues from Japanese Black steers.

| Animals and tissue collection
Three Japanese Black steers (26 months old) raised in Kuju Agricultural Research Center, Kyushu University were used. The steers were cared for and slaughtered according to Guidelines for Animal Experiments in the Faculty of Agriculture of Kyushu University and to laws of the Japanese Government (Law No. 105,Notification No. 6). The steers were raised in a pen with group feeding using the slightly modified standard feeding system for the production of marbled beef (Albrecht et al., 2011;Gotoh et al., 2009).
The steers were slaughtered in an approved slaughterhouse and the carcasses were kept at 0°C for 48 hr. Then, they were transported to a meat processing facility at room temperature for 2 hr and processed within 4 hr. We collected samples of 21 different muscle tissues from all over the carcass (  to high-resolution SDS-PAGE to assess the MyHC isoform composition, as described in detail previously (Mizunoya, Wakamatsu, Tatsumi, & Ikeuchi, 2008

| Measurements of amino acids
Free amino acids in the muscle samples were determined by HPLC (Pico-Tag™; Waters) in accordance with a previous method (Rubio, 2003). The tissue samples were homogenized in an ice-cold 0.2 M perchloric acid solution, containing 0.01 mM EDTA and left for deproteinization on ice for 30 min. Then, the tissue homogenates were centrifuged at 20,000 × g for 15 min at 0°C. The supernatants were filtered through 0.20μm filters (Millipore, Bedford, USA), and each 20μl muscle sample solution was dried under reduced pressure.

| Statistics
Results are expressed as means ± SD. Pearson correlation coefficients were calculated using Excel 2004 (Microsoft) to determine the relationship between MyHC1 composition and free amino acid content. We used a two-tailed t test, and significance was set at p < 0.05.

| RE SULTS AND D ISCUSS I ON
We measured the muscle fiber type compositions in 21 steers' muscle tissue samples ( Figure 1 and  (Kirchofer, Calkins, & Gwartney, 2002) and pigs (Suzuki, Watanabe, Konno, & Ohwada, 1999). However, the serratus ventralis muscle has previously been classified as an intermediate muscle, which means that its slow-and fast-twitch fiber composition is balanced (Kirchofer et al., 2002;Robe & Xiong, 1994). Because the fiber-type composition can differ even within the same muscle tissue according to the muscle portion (e.g., cranial, middle, or caudal) (Suzuki et al., 1999), the serratus ventralis muscle could have a high slow-twitch fiber composition in some portions. In our experiments, the MyHC1 composition of the proximal portion of the biceps femoris muscle was about three times that in the distal portion (Table 1).
We investigated the correlation between the proportion of MyHC1 and the total free amino acid and dipeptide contents and found a strong positive correlation (p < 0.00001) (Figure 2 and Table 2). This indicates that an increase in slow-twitch fiber content induces an increase in the total free amino acid content. This correlation could be related to meat flavor through the effects of amino acids as taste enhancers or precursors of aroma compounds (Toldrá et al., 1997). In fact, in a tasting panel evaluation of lamb, redder meat, which is rich in slow-twitch fibers, was classed as having a more intense flavor than whiter meat, which is rich in fast-twitch fibers (Valin, Touraille, Vigneron, & Ashmore, 1982).
The correlations between the MyHC1 proportion and the free amino acids are summarized in Table 2. Among the 31 free amino acids and dipeptides, 11 showed significant positive correlations and five showed significant negative correlations with MyHC1 composition.
Marked positive correlations were observed for alanine, β-alanine, glutamine, 3-methylhistidine, hydroxyproline, ornithine, and tryptophan (p < 0.01). Glutamine is a key precursor of α-ketoglutarate, which is an intermediate in the TCA cycle. Furthermore, histidine, which positively correlated with MyHC1 (p < 0.05), enhances metabolic reaction rates in the TCA cycle (Shimizu, 2007). This correlation is consistent with the high oxidative metabolism expected in slowtwitch fibers. Contrastingly, marked negative correlations were observed for phosphoserine, anserine, and carnosine (p < 0.01), which suggests that these compounds are abundant in fast-twitch fibers.
We expected to find negative correlations for anserine and carnosine because white muscles, which are rich in fast-twitch fibers, generally have greater anserine and carnosine concentrations than red muscles, which are rich in slow-twitch fibers. For example, the concentrations of these dipeptides in chicken breast muscle (white muscle) are six times those in chicken thigh muscle (red muscle) (Crush, 1970). Anserine and carnosine concentrations in porcine longissimus dorsi (white muscle) are 1.5 times those in vastus intermedius (red muscle) (Mei, Cromwell, Crum, & Decker, 1998).
Most free amino acids stimulate taste and can modify the palatability of foods depending on the concentrations at which they are present (Schiffman, Hornack, & Reilly, 1979;Schiffman, Sennewald, & Gagnon, 1981). Thus, it is important to know whether the concentrations of free amino acids exceed the taste detection thresholds. In Table 2, we have referred the taste thresholds in the right-hand column. Concentrations of free amino acids in meat are expressed in micromoles per gram of wet tissue, and solution-based concentrations of these substances could not be determined without water content information. Here, we simply replaced the wet tissue mass with water volume. The concentrations of glutamic acid in bovine muscles exceeded the threshold in all samples, even at the minimum value; however, this amino acid did not show a significant correlation to fiber type. The samples with the maximum contents of aspartic acid and lysine exceeded the taste thresholds. Though lysine did not show a significant correlation with muscle fiber type, aspartic acid, which tastes flat, sour, and slightly bitter, showed a significant negative correlation with the MyHC1 proportion and may contribute to the taste of meat with high fast-twitch fiber content. Overall, the contents of most of the free amino acids in meat were below the taste thresholds.
It should be noted that the actual taste response will be more complicated because of the effects of taste enhancers. Interestingly, purine nucleotides, such as inosine monophosphate (IMP), considerably potentiate the taste responses of free amino acids. Nelson et al. showed that stimulation of mouse fungiform papillae with various amino acids and IMP enhanced the responses of the chorda tympani induced by the amino acids (Nelson et al., 2002). Therefore, purine nucleotide contents in meat should be examined in future.
During postmortem meat aging, adenosine triphosphate-related compounds degrade and generate products such as IMP and hypoxanthine. It has been suggested that the contents of purine nucleotides and their metabolites differ according to the muscle fiber composition in porcine muscles (Chikuni et al., 2013), and high levels of IMP and adenosine triphosphate degradation products have been found in fast-twitch fiber predominant muscle tissues. Therefore, F I G U R E 2 Correlation between total free amino acid contents and the proportion of MyHC1 in samples from 21 different muscle tissues in Japanese Black steers. Different symbols indicate different animals (n = 3, labeled as Nos. 1-3) the relationship between nucleotide levels and muscle fiber types should be investigated in detail in various meats in the future.
In conclusion, we found that there was a strong positive cor-