Ruth M. Hamill

Understanding meat quality through the application of genomic and proteomic approaches

During the past few decades, advances in molecular genetics have led to the identification of multiple genes or genetic markers associated with genes that affect traits of interest in livestock, including single genes of large effect and QTL (genomic regions that affect quantitative traits). Transcriptomics enables analysis of the complete set of RNA transcripts produced by the genome at a given time and provides a dynamic link between the genome, the proteome and the cellular phenotype. Through a functional genomics approach to understanding the molecular basis of meat quality, we can gain further insight into the complex interplay of gene expression events involved in the development of meat quality. Proteomics permits visualisation of the protein content of the cell under varying conditions, combining powerful separation techniques with highly sensitive analytical mass spectrometry. To date, both the human and bovine genome projects have advanced our understanding of gene expression and helped elucidate the function of large portions of the genome. Advantages from this research have permeated through to a broader spectrum of research including that of meat science. Meat quality is manifested through a complexity of events in the muscle and their interactions with many environmental stimuli in both the live animal and during the post-mortem period. A lot of progress has been made in our understanding of the biological processes that contribute to the delivery of consistent quality meat. Through the application of tools of genomics and proteomics we are gaining a deeper insight into these processes and their interaction with environmental factors. Knowledge gained from these approaches can be beneficial in defining and optimising management systems for quality, providing assurance of meat quality and in tailoring quality to suit market needs.

The impact of salt and fat level variation on the physiochemical properties and sensory quality of pork breakfast sausages.

The sensory and physiochemical properties of sausages with varying fat and salt levels were investigated. Twenty eight sausages were produced with varying concentrations of fat (22.5%, 27.5%, 32.5%, 37.5% w/w) and salt (0.8%, 1%, 1.2%, 1.4%, 1.6%, 2%, 2.4% w/w). Sausages were assessed instrumentally for colour, moisture, fat, cooking loss and texture profile analysis. Consumers (n = 25), evaluated each product in duplicate for colour, texture, tenderness, juiciness, salt taste, meat flavour, off-flavour and overall acceptability using a hedonic scale. Lowering fat produced products which consumers rated as less dark in colour, tougher, less juicy and taste less salty than higher fat products. However, no significant preferred sample was found amongst consumers. Salt reduction in products produced sausages which consumers rated as paler in colour, more tender and with greater meat flavour than higher salt containing products. The sausages containing 1.4% and 1.0% salt were significantly (P<0.01) found to be more acceptable to consumers than other salt levels.

Effect of varying salt and fat levels on the sensory and physiochemical quality of frankfurters

The sensory and physiochemical properties of frankfurters with varying fat and salt levels were investigated. Twenty frankfurter formulations were produced with varying concentrations of fat (10%, 15%, 20%, 25% w/w) and salt (1%, 1.5%, 2%, 2.5%, 3% w/w). Frankfurters were assessed instrumentally for colour, moisture, fat, cooking loss and texture profile analysis. Consumers (n=25) evaluated each product in duplicate for colour, coarseness, tenderness, juiciness, salt taste, meat flavour, off-flavour and overall acceptability using a hedonic scale. Salt levels below 1.5% were shown to have a negative effect on consumer acceptability, with 2.5% salt concentration being the most accepted (P<0.001) by consumers. However, frankfurters containing the lower fat levels 10% and 15% fat with higher salt levels (2.5-3%) were significantly the most acceptable variants to consumers. Samples containing less fat and salt were found to be tougher, less juicy and had greater cooking losses. Thus salt perception is very important for consumer acceptability, but fat levels can be potentially reduced without significantly affecting overall acceptability.

Centrifugal drip is an accessible source for protein indicators of pork ageing and water-holding capacity.

Achieving an improvement in water-holding capacity (WHC) of pork and a reduction in the incidence of pale, soft and exudative (PSE)- and dark, firm and dry (DFD)-like meat is a major challenge for the swine industry. Using proteomics, we sought to identify proteins associated with WHC and to monitor postmortem protein degradation. Twenty longissimus samples were categorised into WHC phenotypes. The centrifugal drip was subjected to SDS-PAGE and mass-spectrometry. Forty-four proteins were identified in the centrifugal drip proteome. Changes occurred in volume of five bands across the ageing period, with most significant changes representing increases between day 3 and day 7. Seven proteins were identified in these bands, most with functions in glycolysis. One band significantly differed in abundance across WHC phenotypes. Peptide signatures of the heat shock protein family were identified in this band.

Association analysis of single nucleotide polymorphisms in DGAT1, TG and FABP4 genes and intramuscular fat in crossbred Bos taurus cattle.

Previous studies have indicated that single nucleotide polymorphisms (SNPs) in the diacylglycerol-O-acyltransferase1 (DGAT1), thyroglobulin (TG) and adipose fatty acid binding protein (FABP4) genes are associated with intramuscular fat (IMF) levels or marbling scores in beef. The objectives were to estimate the frequency of SNPs in these candidate genes in purebred Irish cattle (n=459) and to determine if individual SNPs are associated with IMF values of longissimus thoracis et lumborum (LTL) and semimembranosus (SM) muscle of crossbred animals (n=138). Results indicated no significant association between the SNPs and IMF, despite the power of this study being sufficient to detect an association with SNPs in the DGAT1 and FABP4 genes. The results confirm the lack of an association found by many other studies and suggest that these SNPs are not influential on the divergent IMF levels in the crossbred population tested.

Effect of varying salt and fat levels on the sensory quality of beef patties.

The interactive effects of varying levels of salt and fat on the sensory and physiochemical properties of beef patties were investigated. Twenty beef patties with varying levels of fat (30% 40% 50% 60% w/w) and salt (0.5%, 0.75% 1.0% 1.25% 1.5% w/w) were manufactured. All samples were assessed instrumentally for colour, moisture, fat, cooking loss and texture profile analysis. Sensory consumer evaluation was conducted using 25 consumers. The consumers rated each coded product, in duplicate, in terms of colour, texture, tenderness, juiciness, salt, taste, meat flavour, off flavour and overall acceptability. The data indicate that the most consumer acceptable beef patty was that containing 40% fat with a salt level of 1%. This is a 20% decrease in fat and a 50% decrease in salt levels when compared to commercial patties available in Ireland and the UK.

Association of polymorphisms in candidate genes with colour, water-holding capacity, and composition traits in bovine M. longissimus and M. semimembranosus.

The objective of this study was to determine the association of single nucleotide polymorphisms (SNP) in selected candidate genes with sensory and technological meat quality traits in commercial cattle. SNP in seven candidate genes were genotyped in 130 crossbred Bos taurus cattle using PCR-RFLP. Reported associations between calpastatin (CAST) and Warner-Bratzler shear force and carboxypeptidase E (CPE) and intra-muscular fat were not confirmed. However, SNP in CAST, amp-activated protein kinase, gamma-3 subunit (PRKAG3), growth hormone receptor (GHR) and stearoyl coA desaturase (SCD) genes were significantly associated with colour traits (p<0.05). The PRKAG3 SNP was additionally associated with cook loss in M. longissimus thoracis et lumborum (p<0.05) and tended towards association in M. semimembranosus (p<0.1). An association with pH was identified for the SCD SNP (p<0.001). The GHR polymorphism was influential on moisture and intra-muscular fat in M. semimembranosus and protein content in both muscles (p<0.05). Only CPE was associated with sensory traits (flavour in M. longissimus, p<0.01).

Modelling the influence of inulin as a fat substitute in comminuted meat products on their physico-chemical characteristics and eating quality using a mixture design approach.

The effects of fat substitution using two commercial inulin products on the physico-chemical properties and eating quality of a comminuted meat product (breakfast sausage) were modelled using a specialised response surface experiment specially developed for mixtures. 17 treatments were assigned representing a different substitution level for fat with inulin. Sausages were formulated to contain pork shoulder, back fat/inulin, water, rusk and seasoning (44.3, 18.7, 27.5, 7 and 2.5% w/w). Composition, sensory, instrumental texture and colour characteristics were assessed. Fructan analysis showed that inulin was unaffected by heat or processing treatments. Models showed increasing inulin inclusions decreased cook loss (p<0.0017) and improved emulsion stability (p<0.0001) but also resulted in greater textural and eating quality modification of sausages. Hardness values increased (p<0.0001) with increasing inulin concentration, with panellists also scoring products containing inulin as less tender (p<0.0112). Optimisation predicted two acceptable sausage formulations with significantly lower fat levels than the control, which would contain sufficient inulin to deliver a prebiotic health effect.

2D DIGE proteomic analysis of early post mortem muscle exudate highlights the importance of the stress response for improved water-holding capacity of fresh pork meat

Variation in water‐holding capacity (WHC), which presents a major economic burden to the swine industry, is considered to be underpinned by variation at a molecular and biochemical level. High‐resolution 2D DIGE followed by MS analysis and Western blot were used to unravel the proteome of muscle exudate, collected following centrifugation, in the pH 4–7 range. A first 2DE‐based protein map of this substrate was produced where 89 spots were successfully characterised. Two phenotypes divergent for WHC plus one intermediate were compared with a view to deciphering the biochemical processes impacting on variation in WHC. Twenty spots were observed to be altered across the phenotypes. Of these, 14 represented sixteen proteins including metabolic enzymes, stress response proteins and structural proteins. Triosephosphate isomerase and transferrin showed a major difference between the two extreme phenotypes, and may have potential as biological markers for WHC prediction. Several members of the HSPs family were highlighted. This proteomic study makes an important contribution towards a more detailed molecular view of the processes behind WHC and will provide a valuable resource for future investigations.

Integrity of nuclear genomic deoxyribonucleic acid in cooked meat: Implications for food traceability

It is essential to isolate high-quality DNA from muscle tissue for PCR-based applications in traceability of animal origin. We wished to examine the impact of cooking meat to a range of core temperatures on the quality and quantity of subsequently isolated genomic (specifically, nuclear) DNA. Triplicate steak samples were cooked in a water bath (100 degrees C) until their final internal temperature was 75, 80, 85, 90, 95, or 100 degrees C, and DNA was extracted. Deoxyribonucleic acid quantity was significantly reduced in cooked meat samples compared with raw (6.5 vs. 56.6 ng/microL; P < 0.001), but there was no relationship with cooking temperature. Quality (A(260)/A(280), i.e., absorbance at 260 and 280 nm) was also affected by cooking (P < 0.001). For all 3 genes, large PCR amplicons (product size >800 bp) were observed only when using DNA from raw meat and steak cooked to lower core temperatures. Small amplicons (<200 bp) were present for all core temperatures. Cooking meat to high temperatures thus resulted in a reduced overall yield and probable fragmentation of DNA to sizes less than 800 bp. Although nuclear DNA is preferable to mitochondrial DNA for food authentication, it is less abundant, and results suggest that analyses should be designed to use small amplicon sizes for meat cooked to high core temperatures.