**3. Discussion**

In the present study, we showed that the Holstein sirloin samples were characterized by amino acids, amino compounds and nucleic-acid metabolites. We also showed that the relative amount of glutamine, ribose-5-phosphate, uric acid, inosine monophosphate, 5-oxoproline, and glycine in the Holstein sirloin samples was significantly higher than in the Wagyu samples. The abundance of amino acids, amino compounds, and nucleic-acid metabolites clearly reflects the lean meat content of Holstein beef. Previous studies also showed the relationship between metabolomic profiling and beef quality [7–11]. These results indicate that metabolomic analysis is an optimum approach to identify quality-related chemical components of beef. The cooking of meat forms a characteristic taste via

numerous chemical reactions. The Maillard reaction is one of the major chemical reactions of cooked meat that produces many flavoring components [22]. Amino acids and nucleic-acid metabolites are main meat-flavor precursors for the Maillard reaction [23]. Therefore, a higher amount of amino acid and nucleic-acid metabolites in Holstein beef indicates the abundance of a Maillard reaction substrate. These results sugges<sup>t</sup> that breed differences in beef metabolomic profiles affect the taste of cooked meats. The elucidation of metabolomic profile differences between Wagyu and Holstein cooked meat is an important subject for further study.

We showed that the Wagyu sirloin samples were characterized by sugar components and fatty acids. The present study also showed that the relative amount of maltose and xylitol in Wagyu sirloin samples was significantly higher than that in Holstein samples. Ueda et al. reported that the relative amount of malic acid, maltose, trehalose, arabitol, isomaltose, n-acetylserine, and inositol in Wagyu beef was significantly higher than that in Holstein beef [9]. The aging periods of beef affect the meat quality and metabolomic profile [11]. The difference between the results of our study and those of Ueda et al. would be attributed to meat aging conditions. The meat aging period in the present study was at 4 ◦C for 7 days after slaughter. In contrast, the aging in Ueda et al. was at 4 ◦C for 20 days [9]. On the other hand, results showing a higher amount of maltose in Wagyu beef than in Holstein beef were common to both experiments. These results sugges<sup>t</sup> that a higher maltose concentration is a primary feature of Wagyu beef, independent of the aging period. The causative substance of the sweet taste of Wagyu beef remains unclear. Amino acids have different taste properties depending upon their chemical structure [24]. Glycine is categorized as a "sweet" amino acid [24]. The present results showed that glycine was abundant in the Holstein sirloin samples. Therefore, the possibility that amino acids contribute to the sweet taste of Wagyu beef would be excluded. In contrast, the present study showed that maltose and xylitol, categorized as sugar components, are abundant in Wagyu beef. Wagyu is characterized by higher intramuscular fat content than Holstein [12,13]. Previous reports indicated that dietary fat increased the intensity of sweet taste [25,26]. These results sugges<sup>t</sup> that the sweet taste of Wagyu beef is affected by the synergetic interaction between higher sugar components and intramuscular fat. Threshold sweetness concentrations of maltose and xylitol have been reported using sensory test methods [27,28]. Kearsley et al. reported that the threshold sweetness concentration of maltose was 1.07% (w/v), and that of xylitol was 0.51% (w/v) [27]. In molar volumes, Birch et al. showed that the threshold sweetness concentration of maltose was 21.0 mM/l, and that of xylitol was also 21.0 mM/l [28]. However, the effect of dietary fat on the threshold sweetness concentration of sugar components has not been reported. The slaughter age of Wagyu (aged 29–30 months) and Holsteins (aged 21–22 months) in this study was defined in accordance with the commonly applied fattening periods of each breed in Japan. Ueda et al. also analyzed beef samples of Wagyu (aged 31–32 months) and Holsteins (aged 21 months) that were similar to the age of cattle used in this study [9]. Previous studies have shown that the slaughter age affects meat quality and sensory traits of beef [29,30]. To our knowledge, there are no previous studies examining the effects of slaughter age on metabolomic profiling of beef. Therefore, the differences between fattening periods of Wagyu and Holsteins in this study may affect the metabolomic profiling of beef. In addition, there is a possibility that other metabolites, which we could not detect in this study, might affect the sweetness of beef. Further studies are needed to clarify the effects of metabolites on the sweet taste of Wagyu beef.

#### **4. Materials and Methods**
