Identification of the metabolites responsible for colour stability from different lamb sires and elucidation of the underlying biochemical pathways : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Biochemistry at Lincoln University

Abstract

The browning of lamb meat colour during retail display is generally undesirable for consumers. This is especially important for the New Zealand meat industry that relies heavily on high-value, export chilled lamb products which are worth $400 – 630 million annually. The main objective of my study was to elucidate the biochemical pathways that are responsible for lamb meat colour stability. To achieve this, the study was designed through complementary work of objective colour measurements and the application of “Omics technologies”. The combination of both approaches would be able to characterise and quantify the relative abundance of extracellular and cellular molecules which correlate with colour stability. Progeny from rams that were identified based on genomic breeding values for meat colour characteristics were used in this study. These animals were Terminal Sire composites and Texels mated to a variety of maternal breeds. The hypothesis was tested by collecting and analysing the objective colour measurements of a single muscle, the longissimus lumborum (n=196), of the selected sires in 2017, 2018 and 2019. The outcome showed a significant, consistent difference in colour measurement values between the colour stable and labile sire groups. Colour stable sire group exhibited significantly higher a* (redness), higher R630/R580 (redness: browness), lower Δa* and lower Hue angle throughout the retail display period. The data analysis confirmed the sire differences over 3 years of colour data and led to the need to understand the mechanism which underpins the differences in colour stability between the colour stable and labile sire group. This objective was accomplish through utilising the “omics technologies” tools, which include proteomic, metabolomic and lipidomic. In proteomics, the combination of gel-based 2D-DIGE and mass spectrometry identified pyruvate kinase isozymes M1/M2 isoform 4, aldehyde dehydrogenase (mitochondrial) and ATP synthase subunit d; whereas the label-free proteomics discovered dihydrolipoamide S-succinyltransferase, fructose-bisphosphatase 2, Heat Shock Protein 10, superoxide dismutase, thioredoxin domaincontaining protein, tubulin beta chain, annexin 1, Beta 1- metal binding globulin, Tripartite motif family protein, galectin, Apolipoprotein A1, LIM domain binding 3 and myoglobin in differential relative abundance between the colour stable and labile groups. In metabolomics, using a normal phase hydrophilic interaction chromatography-mass spectrometry (HILIC-MS) approach, taurine, glutamic acid, aspartic acid and cytidine were identified. As a complementary measure to HILIC by using the reverse-phase C18 column in lipidomics, the approach has discovered Phosphatidylcholine(PC), phosphatidylethanolamine(PE) and Phosphatidylserine(PS) in differential relative abundance between the colour stable and labile sire groups. Those previously mentioned compounds were mapped together to provide an overview of how different omics approaches intertwined. The functionality of each compound could be interconnecting and the trend could be used to understand the meat colour stability. This approach identified three trends: the first trend included compounds of higher relative abundance in colour labile group at 0h and 72h retail display; the second trend was compounds of higher relative abundance in colour stable group at 0h and 72h and the final trend was compounds that showed high relative abundance in colour stable group at 0h, but at 72hr, the reverse was observed, a higher relative abundance was observed in colour labile group. Based on the functionality of the compound, the work concluded that compounds that promote and control the movement of molecules and ions in the intermembrane space and within the inner membrane of mitochondria (such as PE, PC, ATP synthase, pyruvate kinase, aldehyde dehydrogenase), govern the molecules motion activity and energy metabolism that takes place between the cytoplasm and mitochondria outer membrane and within the cytoplasm (such as PC, annexin, beta 1-metal binding globulin, galectin, Dihydrolipoamide S-succinyltransferase, fructose-bisphosphatase 2); compounds that retain cell structure integrity (such as tubulin, TRIM72, thioredoxin domaincontaining protein family, Heat Shock Protein 10, apolipoprotein A1); compounds that promote the generation of NADH (aspartic acid and glutamic acid) and poses antioxidant properties such as taurine, are associated with meat colour stability.