The potential of metabolomics in meat science: Current applications, trends, and challenges.

Metabolites are the final products of metabolism and provide insights into the biochemical balance of tissue systems. A cascade of reactions involving proteins, carbohydrates, and lipids affects meat color, tenderness, and flavor, specifically, metabolites that are key biomolecules in biochemical reactions associated with attainment of acceptable meat quality. Bioinformatics platforms, such as Kyoto Encyclopedia of Genes and Genomes (KEGG) databases and MetaboAnalyst, are utilized to help understanding the role of differentially abundant metabolites and characterizing their roles in cellular function/metabolism. However, the inability to identify all metabolites using a single platform and limited metabolite libraries specifically for meat/food remains a challenge. Therefore, the advances in metabolite separation, easy-to-use data processing, increased resolution of mass-spectrometry, and data analysis will help to make inferences or develop biomarkers related to meat quality. This review discusses how metabolomics can be exploited to characterize meat quality, the challenges, and current trends. SIGNIFICANCE: Metabolites play an important role in attaining consumer-preferred meat quality traits and nutritive value of foods. Visual appearance of fresh foods, such as muscle foods, are utilized by consumers to assess the quality at the retail market before making purchases. Similarly, tenderness and flavor of meats influence eating satisfaction and re-purchase decisions. Inconsistencies in meat quality lead to enormous economic losses to the food industry. For instance, consumers often associate a bright-cherry red color with freshness, and the US beef industry loses $3.74 billion annually due to discoloration during storage. Both pre-and post-harvest factors influence the extent of meat quality changes. Metabolomics offer robust tools to get a snapshot of small molecules such as acids, amino acids, glycolytic- and tricarboxylic acids, fatty acids, and sugars present in post-mortem muscle tissue and their role in meat quality. Further, using bioinformatics platforms enables characterizing the role of differentially present metabolites in meat quality as well as identifying biomarkers for desirable quality traits such as tender meat or color-stable carcasses. Innovative applications of metabolomics can be exploited to elucidate the underpinnings of meat quality and to develop novel strategies to enhance marketability of retail fresh meats.

Proteomic approaches to characterize biochemistry of fresh beef color.

Color of retail fresh beef is the most important quality influencing the consumers' purchase decisions at the point of sale. Discolored fresh beef cuts are either discarded or converted to low-value products, before the microbial quality is compromised, resulting in huge economic loss to meat industry. The interinfluential interactions between myoglobin, small biomolecules, proteome, and cellular components in postmortem skeletal muscles govern the color stability of fresh beef. This review examines the novel applications of high-throughput tools in mass spectrometry and proteomics to elucidate the fundamental basis of these interactions and to explain the underpinning mechanisms of fresh beef color. Advanced proteomic research indicates that a multitude of factors endogenous to skeletal muscles critically influence the biochemistry of myoglobin and color stability in fresh beef. Additionally, this review highlights the potential of muscle proteome components and myoglobin modifications as novel biomarkers for fresh beef color. SIGNIFICANCE: This review highlights the important role of muscle proteome in fresh beef color, which is the major trait impacting consumers' purchase decisions. In recent years, innovative approaches in proteomics have been exploited for an in-depth understanding of the biochemical mechanisms influencing color development and color stability in fresh beef. The review suggests that a wide range of factors, including endogenous skeletal muscle components, can affect myoglobin biochemistry and color stability in beef. Furthermore, the potential use of muscle proteome components and myoglobin post-translational modifications as biomarkers for fresh beef color is discussed. The currently available body of evidence presented in this review can have important implications in meat industry as it provides novel insights into the factors influencing fresh beef color and an up-to-date list of biomarkers that can be used to predict beef color quality.

Integrative proteomics and metabolomics profiling to understand the biochemical basis of beef muscle darkening at a slightly elevated pH.

Previous studies investigated the biochemical basis of dark-cutting conditions at elevated muscle pH (above 6), but the molecular basis at slightly above normal pH (between 5.6 and 5.8) is still unclear. The objective was to determine protein and metabolite profiles to elucidate postmortem muscle darkening at slightly elevated pH. Loins were selected based on the criteria established in our laboratory before sample collections, such as pH less than 5.8, L* values (muscle lightness) less than 38, and not discounted by the grader (high-pH beef with dark color are discounted and not sold in retail stores). Six bright red loins (longissimus lumborum) at normal-pH (average pH = 5.57) and six dark-colored strip loins at slightly elevated pH (average pH = 5.70) from A maturity carcasses were obtained within 72-h postmortem from a commercial beef purveyor. Surface color, oxygen consumption, metmyoglobin reducing activity, protein, and metabolite profiles were determined on normal-pH and dark-colored steaks at slightly elevated pH. Enzymes related to glycogen metabolism and glycolytic pathways were more differently abundant than metabolites associated with these pathways. The results indicated that oxygen consumption and metmyoglobin reducing activity were greater (P < 0.05) in darker steaks than normal-pH steaks. Enzymes involved with glycogen catabolic pathways and glycogen storage disease showed lower abundance in dark beef. The tricarboxylic acid metabolite, aconitic acid, was overabundant in darker-colored beef than normal-pH beef, but glucose derivative metabolites were less abundant. The majority of glycogenolytic proteins and metabolites reported as overabundant in the previous dark-cutting studies at high pH (>6.4) also did not show significant differences in the current study. Therefore, our data suggest enzymes involved in glycogen metabolism, in part, create a threshold for muscle darkening than metabolites.

A bright cherry-red color beef is ideal during meat retail and carcass grading. Any deviation from a bright red color, such as dark red color, at the interface of the 12th and 13th rib-eye area leads to carcass discounts. Various studies have determined protein, metabolite, and mitochondrial profiles to understand the biochemical basis of dark-cutting beef (muscle pH greater than 6); however, limited knowledge is currently available on muscle darkening at a slightly elevated pH. Bright red loins at normal muscle pH and darker color loins at slightly elevated pH (not discounted by a grader) were collected 72-h postmortem from a commercial beef purveyor. Surface color, oxygen consumption, metmyoglobin reducing activity, protein, and metabolite profiles were determined on normal-pH and dark-colored steaks at slightly elevated pH. The results indicated that oxygen consumption and metmyoglobin reducing activity were greater in darker steaks than normal-pH steaks. Furthermore, the protein abundance profiles of enzymes related to glycogen metabolism and glycolytic pathways were more differently abundant than metabolites associated with these pathways. Understanding the factors involved in the occurrence of dark color steaks help to minimize losses due to discount carcasses.

Recombinant Phytase Modulates Blood Amino Acids and Proteomics Profiles in Pigs Fed with Low-Protein, -Calcium, and -Phosphorous Diets.

A beneficial effect of corn-expressed phytase (CEP) on the growth performance of pigs fed with very low-protein (VLP) diets was previously shown. Little is known whether this improvement is related to alterations in the expression profiles of blood proteins and amino acids (AAs). The objective of this study was to investigate whether supplementation of VLP, low-calcium (Ca), and low-P diets with a CEP would alter the blood AAs and protein expression profiles in pigs. Forty-eight pigs were subjected to one of the following groups (n = 8/group) for 4 weeks: positive control (PC), negative control-reduced protein (NC), NC + low-dose CEP (LD), NC + high-dose CEP (HD), LD with reduced Ca/P (LDR), and HD with reduced Ca/P (HDR). Plasma leucine and phenylalanine concentrations were reduced in NC; however, the LD diet recovered the concentration of these AAs. Serum proteomics analysis revealed that proteins involved with growth regulation, such as selenoprotein P were upregulated while the IGF-binding proteins family proteins were differentially expressed in CEP-supplemented groups. Furthermore, a positive correlation was detected between growth and abundance of proteins involved in bone mineralization and muscle structure development. Taken together, CEP improved the blood profile of some essential AAs and affected the expression of proteins involved in the regulation of growth.

Dark-cutting beef mitochondrial proteomic signatures reveal increased biogenesis proteins and bioenergetics capabilities.

Mitochondria remain active in postmortem muscles and can influence meat color via oxygen consumption. Previous studies have shown that dark-cutting compared with normal-pH beef has greater mitochondrial protein and DNA content per gram of muscle tissue. However, the mechanism regulating mitochondrial content in dark-cutting vs. normal-pH beef is still unknown. Therefore, the objective was to compare mitochondrial proteomes of dark-cutting vs. normal-pH beef using LC-MS/MS-based proteomics and mitochondrial respiratory capacity using a Clark oxygen electrode. Dark-cutting compared with normal-pH beef has up-regulation of proteins involved in mitochondrial biogenesis, oxidative phosphorylation, intracellular protein transport, and cellular calcium ion homeostasis. Mitochondria isolated from dark-cutting phenotypes showed greater mitochondrial complex II respiration and uncoupled oxidative phosphorylation. However, mitochondrial membrane integrity and respiration at complexes I and IV were not different between normal-pH and dark-cutting beef. These results indicate that dark-cutting beef has greater mitochondrial biogenesis proteins than normal-pH beef, increasing mitochondrial content and contributing to dark-cutting beef. SIGNIFICANCE: Defective glycogen metabolism resulting from chronic stress before slaughter coupled with the greater mitochondrial protein and DNA content per gram of muscle tissue promotes muscle darkening in dark-cutting phenotypes in beef. However, the mechanistic basis for this occurrence in dark-cutting phenotypes is still unknown. In this work, we show that dark-cutting beef phenotype is caused, in part, as a consequence of over-proliferation of mitochondria. This is supported by the up-regulation of proteins involved in mitochondrial biogenesis, mitochondrial electron transport, calcium homeostasis, and fatty acid metabolism. Hence, the study of mitochondrial proteome changes provides a set of mitochondrial biogenesis proteins that could be used as potential candidate markers for detecting changes in pre-slaughter developmental events contributing to dark-cutting phenotypes in beef.

Changes in glycolytic and mitochondrial protein profiles regulates postmortem muscle acidification and oxygen consumption in dark-cutting beef.

Dark-cutting beef is a condition in which beef fails to have a characteristic bright-red color when the cut surface is exposed to oxygen. However, the mechanistic basis for this occurrence is not clear. Protein expression profiles were compared between dark-cutting and normal-pH beef using LC-MS/MS-based proteomics. Mass spectrometry analysis identified 1162 proteins in the proteomes of dark-cutting and normal-pH beef. Of these, 92 proteins had significant changes in protein abundance between dark-cutting versus normal-pH beef. In dark-cutting beef, 25 proteins were down-regulated, including enzymes related to glycogen metabolism, glucose homeostasis, denovo synthesis of adenosine monophosphate (AMP), and glycogen phosphorylase activity. In comparison, 27 proteins were up-regulated in dark-cutting beef related to oxidation-reduction processes, muscle contraction, and oxidative phosphorylation. Down-regulation of glycogenolytic proteins suggests decreased glycogen mobilization and utilization, while the up-regulation of mitochondrial transport chain proteins indicates a greater capacity to support mitochondrial respiration in dark-cutting beef. These results showed that changes in proteins involved in glycogenolysis and mitochondrial electron transport would promote the development of high-pH and greater oxygen consumption, respectively; thus limiting myoglobin oxygenation in dark-cutting beef. SIGNIFICANCE: The current understanding indicates that defective glycolysis causes less carbon flow, leading to less postmortem lactic acid formation and elevated muscle pH in dark-cutting beef. However, to the best of our knowledge, limited research has evaluated how changes in glycolytic and mitochondrial protein abundance regulate postmortem muscle acidification and oxygen consumption in dark-cutting beef. We utilized a shotgun proteomics approach to elucidate potential differences in protein profiles between dark-cutting versus normal-pH beef that may influence differences in postmortem metabolism and muscle surface color characteristics. Our study shows that down-regulation of glycolgenolytic and IMP/AMP biosynthetic proteins results in elevated postmortem muscle pH in dark-cutting beef. In addition, the up-regulation of mitochondrial protein content coupled with the higher muscle pH are conducive factors for enhanced oxygen consumption and less myoglobin oxygenation, contributing to a dark meat color typically associated with dark-cutting beef.

Impact of Up- and Downregulation of Metabolites and Mitochondrial Content on pH and Color of the Longissimus Muscle from Normal-pH and Dark-Cutting Beef.

Limited knowledge is currently available on the biochemical basis for the development of dark-cutting beef. The objective of this research was to determine the metabolite profile and mitochondrial content differences between normal-pH and dark-cutting beef. A gas chromatography-mass spectrometer-based nontargeted metabolomic approach indicated downregulation of glycolytic metabolites, including glucose-1- and 6-phosphate and upregulation of tricarboxylic substrates such as malic and fumaric acids occurred in dark-cutting beef when compared to normal-pH beef. Neurotransmitters such as 4-aminobutyric acid and succinate semialdehyde were upregulated in dark-cutting beef than normal-pH beef. Immunohistochemistry indicated a more oxidative fiber type in dark-cutting beef than normal-pH beef. In support, the mitochondrial protein and DNA content were greater in dark-cutting beef. This increased mitochondrial content, in part, could influence oxygen consumption and myoglobin oxygenation/appearance of dark-cutting beef. The current results demonstrate that the more tricarboxylic metabolites and mitochondrial content in dark-cutting beef impact muscle pH and color.