Application of proteomics to understand the molecular mechanisms behind meat quality

The proteome is expressed from the genome, influenced by environmental and processing conditions, and can be seen as the molecular link between the genome and the functional quality characteristics of the meat. In contrast to traditional biochemical methods where one protein is studied at a time, several hundred proteins can be studied simultaneously. Proteomics is a promising and powerful tool in meat science and this is reflected by the increasing number of studies emerging in the literature using proteomics as the key tool to unleash the molecular mechanisms behind different genetic backgrounds or processing techniques of meat. Thus understanding the variations and different components of the proteome with regard to a certain meat quality or process parameter will lead to knowledge that can be used in optimising the conversion of muscles to meat. At present, there has been focus on development of techniques and mapping of proteomes according to genotypes and muscle types. In the future, focus should be more towards understanding and finding markers for meat quality traits. This review will focus on the methods used in the published proteome analyses of meat, with emphasis on the challenges related to statistical analysis of proteome data, and on the different topics of meat science that are investigated.     

Relationships between storage protein composition,protein content,growing season and flour quality of bread wheat

The storage protein composition from the Glu‐1, Glu‐3 and Gli‐1 loci encoding high and low molecular weight glutenin subunits (HMW‐GS and LMW‐GS) and gliadins, respectively, was determined on 30 wheat (T aestivum L) genotypes from three growing seasons. The gliadins and the LMW‐GS were identified as gliadin/LMW‐GS pairs. All samples were analysed by two one‐dimensional electrophoretic techniques, and selected samples were also subjected to two‐dimensional electrophoretic separation. Different statistical/data‐analytical techniques were evaluated in the study of how the presence or absence of the protein alleles, the protein content and the growing seasons are related to flour quality. The year of growth had a large impact on mixograph peak time. When predicting mixograph peak time from the presence or absence of significant proteins and the year of growth, 70% of the variability in mixograph peak time could be explained, whereas only 49% of the variability could be explained when the year of growth was deleted from the model. Protein had no effect on mixograph peak time as expected, and the well‐known positive effect of HMW‐GS 5 + 10, and the negative effects of 2 + 12 and 6 + 8 was observed. Furthermore, some of the gliadin/LMW‐GS combinations influenced mixograph peak time significantly. The gliadin/LMW‐GS at the combined Gli‐A1, Glu‐A3 loci b; ?? was positively related to mixograph peak time, whereas ??; ?? and a;a was negatively related. Although the LMW‐GS component ?? of the alleles b; ?? and ??; ?? alleles appear similar on one‐dimensional gels, two‐dimensional separation of selected samples may suggest that the ?? components in these alleles are different proteins. Cross‐validated partial least squares regression combined with empirical uncertainty estimates (jack‐knifing) of the parameters estimated in the model, gave similar results to ANOVA in identifying quality related protein alleles. The applicability of the multivariate approach in proteomics is, however, much wider. Copyright © 2004 Society of Chemical Industry     

Variation in the response to manipulation of post-mortem glycolysis in beef muscles by low-voltage electrical stimulation and conditioning temperature

The aim of this study was to investigate how manipulation of glycolytic rate by post-mortem processing conditions influences quality of aged beef of two bovine muscles of different physiological character, longissimus dorsi (LD) and adductor (AD). Post-mortem glycolysis was manipulated by low-voltage electrical stimulation (LV-ES) of half carcasses and by chilling rate of the muscles. Multivariate statistical analysis was used to visualise the data, while ANOVA was used to identify significant effects and interactions. As expected there was a significant effect of LV-ES on the pH decline in the first hours post-mortem in both muscles. Moreover, significant effects of LV-ES on WB shear force measured 2 and 8 days after slaughter were observed for LD at both chilling temperatures, while for AD no effect on WB shear force was observed. Furthermore, the results revealed a large individual variation in the response of LV-ES on both pH decline and WB shear force, and this variation did not always correlate for the two responses. Some animals showed no response of LV-ES on pH decline, but still had an improved WB shear force, and vice versa. The results from this study indicate that there probably are other mechanisms than accelerated pH decline and prevention of cold-shortening, by which LV-ES can affect meat tenderness.     

Dough and Hearth Bread Characteristics Influenced by Protein Composition, Protein Content, DATEM, and Their Interactions

ABSTRACT: The effects of protein composition, protein content, diacetyl tartaric acid ester of monoglycerides (DATEM), and their interaction on dough and bread characteristics were studied by small- and pilot-scale hearth bread baking, dough rheological testing using the Kieffer extensibility rig, and size distribution of proteins. Protein composition had the largest influence on dough and bread characteristics. There was interaction effect between protein quality and protein content on dough behavior. High percentage of sodium dodecyl sulfate-unextractable polymeric proteins in flours gave large area and good form ratio. Increased ratio between monomeric and polymeric proteins resulted in decreased form ratio. Addition of DATEM altered the rheological behavior of the dough and the bread-making performance. The effect was, however, small compared with the effect of the protein composition.     

Proteome changes in the insoluble protein fraction of bovine Longissimus dorsi muscle as a result of low-voltage electrical stimulation

Changes induced by low-voltage electrical stimulation (ES; 0-95 V for 8 s; 95 V for 32 s) in the insoluble protein fraction of bovine longissimus dorsi (LD) muscle at 1 and 24h post-ES were investigated by proteomics. Protein abundance patterns from ten Norwegian Red (NRF) young bulls were compared, and significant changes due to ES were found by rotation test and partial least square (PLS) regression analyses. Five protein spots showed lower abundance in ES samples at both sampling times, and in addition, 10 proteins at 1 h post-ES and 13 proteins at 24 h post-ES changed significantly in abundance due to ES. Reduced abundance of full-length structural proteins in ES samples indicates an accelerated proteolysis due to ES. Moreover, increased abundance of small heat shock proteins indicates earlier initiation of stress responses due to ES. These findings provide a better understanding of the biochemical processes taking place as a result of ES during post mortem storage of meat.     

Eicosapentaenoic acid-induced apoptosis depends on acyl CoA-synthetase

Marine n−3 FA are known to inhibit proliferation or induce cell death in several cancer cell lines. We have previously reported that EPA promotes apoptosis in the lymphoma cell line Ramos, whereas the U-698 cell line is insensitive to EPA. Furthermore, acyl-CoA synthetase (ACS) is expressed to a higher extent in Ramos cells compared to U-698 cells. To investigate the importance of ACS in EPA-induced apoptosis, we incubated Ramos cells with triacsin C, an inhibitor of ACS. This caused a 70% reduction in the amount of cell-associated EPA and diminished activation of EPA. In addition, triacsin C caused a 90% reduction in EPA-induced apoptosis. Several different approaches were tried to overexpress ACS4 in EPA-insensitive lymphoma cell lines, but we did not obtain viable cells with high expression of acyl-CoA activation. However, we show that overexpression of ACS4 in the more robust COS-1 cells caused up to a fivefold increase in activation of EPA and a 67% increase in the amount of cell-associated radiolabeled EPA. Furthermore, we observed 28% elevated cellular level of TAG in EPA-incubated COS-1 cells overexpressing ACS4. The present study provides new information about ACS as an important enzyme for EPA-induced apoptosis in Ramos cells. Our data offer a potential mechanism that may explain the effect of dietary marine n−3 PUFA on growth of certain malignant cells.     

Eicosapentaenoic acid promotes apoptosis in Ramos cells <Emphasis Type="Italic">via</Emphasis> activation of caspase-3 and -9

Eicosapentaenoic acid (EPA; 20∶5n−3) may reduce the cell number in cultured leukemia/lymphoma cells owing to reduced cell proliferation, induction of cell death, or a combination of these processes. EPA has been shown to promote apoptosis in Ramos cells, and our present study was focused on a possible cell cycle arrest and the pathways by which the apoptotic process is induced. Apoptosis may proceed along the intrinsic (mitochondrial) or the extrinsic (death receptor) pathway, which are mediated via different caspases. Caspases are a class of homologous cysteine proteases recognized as pivotal mediators of apoptosis. We investigated whether EPA affects progression of the cell cycle or promotes apoptosis directly. By incorporation of [³H]thymidine and [³H]valine, we showed that DNA, as well as protein synthesis, was reduced after incubation of Ramos cells with EPA for 6h. We monitored cell cycle distribution by 5-bromo-2′-deoxyuridine staining and observed no cell cycle arrest in the EPA-incubated cells. Incubation of cells with EPA caused PS-flipping, as demonstrated by annexin V-binding (flow cytometry), and cleavage of poly(ADP-ribose) polymerase measured by Western blot analysis. Furthermore, we observed increased activity of caspase-3 and-9, but not of caspase-8. Whereas inhibitors of caspase-3 and-9 reduced EPA-induced apoptosis, inhibition of caspase-8 did not. This suggests that EPA may promote apoptosis via the intrinsic pathway in Ramos cells. Thus, the reduction in cell number can be explained by a direct apoptotic effect of EPA rather than via cell cycle arrest.     

Lipid degradation and sensory characteristics of M. biceps femoris in dry‐cured hams from Duroc using three different processing methods

Hams from Norwegian Duroc pigs, reared and fed identically, were dry‐cured using three different processing methods: Spanish Serrano (SS), Norwegian Parma‐style (PS) and deboning before curing (ND). The fatty acid compositions of the green and dry‐cured hams were analysed in terms of their neutral lipid, phospholipid and free fatty acid contents and correlated with sensory attributes. Although the three dry‐curing processes were quite different, the hams′ lipid profiles, lipid degradation patterns and lipid‐associated sensorial characteristics differed only slightly. The phospholipids were the most extensively degraded lipid class (88, 89% and 84% degradation in PS, SS and ND hams, respectively) for all processing methods. The SS and PS hams had slightly riper sensory profiles due to their extensive conversion of fatty acids into aroma components. The free fatty acid contents of PS, SS and ND hams were 6.3, 6.2 and 7.5 times greater than those of green hams, respectively.     

Comparison of muscle proteome profiles in pure breeds of Norwegian Landrace and Duroc at three different ages

We have used proteomics as a tool to unravel the changes in protein composition between two pure pig breeds and three age groups. Forty two female pigs of Norwegian Landrace and Duroc breed slaughtered at 6, 9 and 12 months age were included in the study. Each of the breeds was raised in separate farms and was slaughtered at the same day in a commercial abattoir. A sample from the adductor muscle was collected approximately 45 min postmortem. Proteome analyses of the water soluble proteins using 2D electrophoresis showed that of the 1125 analyzed protein spots, 94 and 41 proteins are changed in abundance according to breed and age, respectively. A total of 63 changed proteins were identified by mass spectrometry. The identified proteins were classified as structural proteins, metabolic proteins, stress/defense proteins and other proteins. This demonstrates a difference in metabolism and muscle composition between breeds and age groups and shows that proteomics is a useful tool to uncover the molecular basis for physiological differences in muscles between pig breeds and age groups.     

Frequency multiplier measurements on heterostructure barriervaractors on a copper substrate

We have fabricated heterostructure barrier varactors (HBV) on a copper substrate, which offers reduced spreading resistance, and improved thermal conductivity compared to an InP substrate. The devices are fabricated without degrading the electrical characteristics. The three-barrier HBV material grown by MOVPE has a leakage current of only 0.1 μA/μm² at 19 V. The maximum capacitance is 0.54 fF/μm². In a frequency tripler experiment a maximum output power of 7.1 mW was generated at 221 GHz with a flange-to-flange efficiency of 7.9%