*4.2. Animal Feeding*

Feeding conditions, one of the environmental factors for animals, have grea<sup>t</sup> influences on animal physiology, including skeletal muscle growth and maturation, and the final meat yield and quality. As the feeding factors, the nutritional condition and stress originating from the farming system are considered as the most important ones [82,83], due to their influences on downstream meat production. The influence of feeding on animals are expected to be observed as metabolome changes via gene expression and physiological alteration in tissues. For these reasons, metabolomics approaches have attempted to capture metabolite signatures of feeding systems aiming at the improvement of meat production.

In beef cattle, to address an increasing demand by consumers for beef produced in a sustainable farming system, the e ffect of di fferences in feeding on the muscle metabolome was assessed by metabolomic approaches with GC–MS and LC–MS/MS [20]. Between grass- and grain-fed Angus steers, significant variations between the cattle groups was observed in a variety of lipids, including polyunsaturated fatty acid (PUFA) in the *latissimus dorsi* muscle, showing higher ω3 and lower ω6 fatty acids in grass-fed cattle than in the grain-fed. The PCA classified these samples by the diet, and RF analysis based on the metabolites resulted in a predictive accuracy of 100% between the dietary conditions, in which lipids and amino acids were potential biomarkers discriminating the two groups of cattle. Intriguingly, the cortisol level was lower in grass-fed cattle, suggesting they might experience less stress than the grain-fed [20]. These results indicated that metabolite signatures could be utilized as indices not only in feeding-originated beef quality but also in animal welfare and authentication. In other studies, the e ffects of dietary amino acids and mate extract administration on beef were also investigated by NMR approaches followed by PCA [21,22]. Biopsy *semitendinosus* muscle samples of dairy calves fed protein-based milk replacer at 7 weeks of age were classified by amino acid supplementation [21]. Additionally, in a study of feedlot Nellore cattle, the dietary addition of mate extract, one of the antioxidant additives administered to broiler chickens, resulted in increased content of IMP, creatine, carnosine, and conjugated linoleic acid in the LT muscle, and some of these were in a dose-dependent manner [22]. Coupled with redox status analysis, the results further indicated that the beef of mate extract had increased oxidative stability, tenderness, and consumer acceptability. Thus, not only the global feeding conditions but also nutrients and ingredients in the animal feed cause

changes in muscle and meat metabolomes, which can be assessed by MEATabolomics. In such studies, metabolites can be screened and be identified as potential biomarkers of the feeding.

Regarding pork, a GC–MS approach was applied to the screening of pigs fed with Clenbuterol, a β-adrenergic agonist [23]. The *biceps femoris* muscles of pigs treated with Clenbuterol were discriminated from the counterparts in the subsequent PLS-DA by compounds associated with fatty acids and amino acids. Recently, a similar study was conducted using a REIMS approach [26], in which carcasses of pigs fed with ractopamine, another β-agonist, were discriminated by the lipid profiles of REIMS with high accuracy of classification in three di fferent types of muscle. Due to the high-throughput and accurate performance, this methodology can be widely applied to fields of practical meat research.

Such nutrimetabolomic approaches have also been attempted in chicken, of which growth performance, breast yield, and meat quality are improved with dietary lysine supplementation [12]. Aiming at an increase in flavor compounds in chicken, the e ffect of dietary lysine supplementation at a 1.5-fold level of a standard requirement on the breast muscle was investigated by a CE–MS approach. Supplementation resulted in increased levels of lysine degradation products, such as saccharopine, α-amino adipic acid, and glutamate, the latter being one of the most important taste-improving amino acids in meat [24]. On the other hand, in a study investigating the e ffect of duck aging on the meat metabolome, duck meat samples of di fferent ages were classified in PCA and PLS–DA by NMR metabolite data, showing increased levels of lactate and anserine and decreased levels of fumarate, betaine, taurine, and inosine with increasing age [25].

## *4.3. Muscle Type*

The type of skeletal muscle of pig and cattle is associated with meat quality (WHC, meat color, and tenderness) [84,85] and sensory traits [86,87]. This is due to the di fferences in physiological characteristics [88,89] and compositions of proteins [90,91] and metabolites [14,54,92,93] between di fferent types of muscles that have distinct compositions of fast and slow type muscle fibers. Due to the di fferent physiological properties of the muscle fibers, muscles with di fferent fast/slow fiber type composition undergo di fferent postmortem aging processes, as shown in protein [94,95] and metabolite [14,16] degradation, which could be the cause of the intermuscular di fference in meat quality. Contents of flavor-associated metabolites such as amino acids, sugar, and nucleotides di ffered between porcine LL and *vastus intermedius* (VI) muscles in a CE–MS metabolomics study [14], and between bovine muscles in an LC–MS [15] or a UPLC–MS/MS study [16]. These results indicate the association of fast and slow type fiber composition with contents of flavor-associated metabolites in aged meat [14,15]. It is noticeable that the rate and extent of the postmortem metabolite changes including glycolysis, metabolism of amino acids and nucleotides are di fferent between the muscle types, as shown in di fferent accumulation of lactate in glycolysis, hypoxanthine in purine metabolism, and aliphatic amino acids and methionine in amino acid metabolism [14]. During postmortem aging of pork, thiamine is accumulated, while thiamine triphosphate level declines, which is another di fference between muscle types [54]. In addition, the metabolomic di fference between *longissimus* and *semitendinosus* muscles of four cattle breeds was investigated by an HR–MAS NMR approach, which resulted in a good classification of the two muscles in Chianina and Bu ffalo, but not in Holstein Friesian and Maremmana cattle [39]. Such intermuscular di fferences may be associated with di fferences in meat quality and sensory traits between those muscles.

#### *4.4. Postmortem Aging and Storage*

Aging and storage of postmortem muscle have grea<sup>t</sup> influences on meat quality, as well as animal genetic background and feeding conditions. Even when an animal has muscles of high meat quality potential, the final meat quality could be lowered by inappropriate management, resulting in meat deterioration such as spoilage and discoloration in parallel with abnormal metabolome changes. Inversely, the development of metabolites in meat during postmortem aging under the appropriate condition has a beneficial influence on the final meat quality.

MEATabolomics has contributed to postmortem meat aging studies to account for influences of postmortem aging on meat quality in terms of metabolites. In pork, postmortem changes in the LL and VI muscles were investigated by a CE–MS approach [14]. The result of this study indicated that a variety of postmortem muscle metabolisms were activated, especially within 24 h after slaughter. The PCA classified the samples primarily into LL and VI muscles, and secondarily to at-slaughter and day 14 (168 h) aging points (Figure 1). In the LL samples, intermediate glycolytic products, glucose 6-phosphate and fructose 6-phosphate (F-6P), increased until 24 h postmortem, while the downstream compounds such as fructose 1,6-bisphosphate (F-1,6P) and phosphoenolpyruvate decreased with being almost exhausted at 24 h postmortem, indicating the rate-determining activity of phosphofructokinase (F-6P → F-1,6P) in postmortem LL muscle glycolysis. Additionally, in the LL muscle, most of the amino acids and identified dipeptides increased after day 1 during the aging, suggesting that protein degradation began to be dominant after day 1. Regarding ATP degradation, marked di fferences in compound levels were observed in the pathways of ADP → AMP → IMP → inosine → hypoxanthine between the LL and VI muscles, suggesting the intermuscular di fferences in enzymatic activities in the pathways [14]. These pathways in pig LL and VI muscles were overrepresented in the analysis of pathway impact by MetaboAnalyst (Figure 2), indicating a number of biologically meaningful di fferences in metabolic pathways between the two muscles. Such pathway analyses further a fforded new insights: pathways associated with thiamine, pentose phosphate, and NADH metabolism had a high impact on postmortem aging in both muscles. In other studies analyzing bovine LL, *psoas major* (PM), and/or *semimembranosus* muscles, intermuscular metabolomic di fferences in energy metabolism during 24 h aging [16], and di fferences in metabolites associated with meat color and lipid oxidation during 23 d aging [15] were also observed by LC–MS-based approaches. Thus, the skeletal muscle type is associated with the muscle metabolome as described above, in which postmortem aging makes the intermuscular metabolome di fferences inherent to live animals further complicated, as shown in the complexity of the di fferences in the final meat quality. Nevertheless, MEATabolomics approaches have shown its advantages to discriminate meat samples by muscle type and postmortem aging conditions.

**Figure 1.** Classification of pig LL and VI muscle samples of different postmortem aging period by PCA. Pig LL and VI muscles were aged during 0, 4, 24, 168 h postmortem.

**Figure 2.** Metabolic pathway impact analysis of pig LL and VI muscles during postmortem aging. Pathway impact values (horizontal axis) were calculated from the pathway topology analysis in MetaboAnalyst. A pathway with small *p* value and high impact is plotted as a red and large circle.

Regarding beef, metabolomic changes during postmortem aging was also analyzed by CE–MS [17] and NMR approaches [18,19] in *longissimus* muscle. In the CE–MS metabolomics targeting charged metabolites, changes in nutritionally important compounds such as choline, nicotinamide, and thiamine were observed during 14 d aging of JB cattle beef, some of which contributed to classification of the samples by the aging period [17]. On the other hand, NMR studies were conducted for metabolomic profiling during 10 weeks [18] and 21 days [19] postmortem. The former NMR study aimed to explore excellent biomarkers of postmortem JB beef aged up to 10 weeks, and revealed that leucine and creatine are available as biomarkers for long-term aging (>2 weeks) because of positive and negative correlation with aging duration, respectively [18]. The L-value, defined as the ratio of leucine to creatine in the study, was more appropriate to evaluate meat quality during long-term aging than the conventional K-value that is calculated by using the contents of ATP, ADP, AMP, and IMP. In addition, this study also showed that the NMR metabolomics was able to estimate unsaturation degree of triacylglycerol that was correlated to content ratio of unsaturated fatty acids (R<sup>2</sup> = 0.944) and melting point of intramuscular fat (R<sup>2</sup> = 0.871). This suggests that such an approach can evaluate not only postmortem meat aging and intramuscular fat content, but also palatability-associated quality traits such as juiciness and WHC that might be assessed through NMR-measured pH profile [18] in highly marbled JB beef. Besides, biomarker candidates for prolonged postmortem aging (up to 44 days) of Piedmontese LT beef was explored by the LC–MS approach [55]. The results indicated the possibility of glutamate, serine, and arginine being candidates of postmortem aging indicators due to their constant increase until day 44. The prolonged aging period likely enhanced the accumulation of amino acids and some other metabolites. In these studies, metabolomics approaches combined with statistical analyses have enabled screening of metabolic biomarker candidates of postmortem aging from the meat metabolome dataset. Thus, metabolomics have contributed not only to the understanding of postmortem muscle metabolisms but also to the finding of better biomarkers that the conventional studies have never assessed. These MEATabolomics of postmortem aging of pork and beef re-emphasize the possibility that the amino acid generation during cold storage is primarily due to postmortem protein degradation in the muscles, as shown in previous studies of aging [31,94–101]. Besides, the elucidation of flavor-associated compound generation from muscle metabolites has been accelerated with the accumulating knowledge of the uncovered meat metabolomes through postmortem aging studies on cooked meat [43].

Other optional postmortem storage conditions are also to be investigated due to the influences on a variety of metabolites associated with meat quality traits. A dry-aging condition of 3 ◦C with 0.2 m/s air-velocity for 3 weeks showed high improvement of beef palatability in a sensory panel evaluation compared to conditions of general wet-aging and other dry conditions, with higher contents of compounds including branched amino acids and IMP in a study by an NMR approach [56]. The air flow of this storage method might simply accelerate the up-concentration of the flavor-associated metabolites, but other undetermined factors are likely also involved. On the other hand, the chilling rate of lamb LT muscle during aging affected the muscle energy metabolism within 24 h postmortem, which was associated with meat tenderness in a study utilizing 1H–, 31P–NMR, and LC–MS [57]. MEATabolomics was also conducted to investigate roles of NADH in discoloration mechanism via mitochondria activity during postmortem wet-aging of bovine LL muscle [58]. The results suggested that discoloration during postmortem aging of beef was due to increased oxidative stress, mitochondrial damages, and decreasing metabolite sources to regenerate NADH.
