Myofibrillar Fraction Research Articles

Skeletal Muscle Proteome Modifications following Antibiotic-Induced Microbial Disturbances in Cancer Cachexia.

Cancer cachexia is an involuntary loss of body weight, mostly of skeletal muscle. Previous research favors the existence of a microbiota-muscle crosstalk, so the aim of the study was to evaluate the impact of microbiota alterations induced by antibiotics on skeletal muscle proteins expression. Skeletal muscle proteome changes were investigated in control (CT) or C26 cachectic mice (C26) with or without antibiotic treatment (CT-ATB or C26-ATB, n = 8 per group). Muscle protein extracts were divided into a sarcoplasmic and myofibrillar fraction and then underwent label-free liquid chromatography separation, mass spectrometry analysis, Mascot protein identification, and METASCAPE platform data analysis. In C26 mice, the atrogen mafbx expression was 353% higher than CT mice and 42.3% higher than C26-ATB mice. No effect on the muscle protein synthesis was observed. Proteomic analyses revealed a strong effect of antibiotics on skeletal muscle proteome outside of cachexia, with adaptative processes involved in protein folding, growth, energy metabolism, and muscle contraction. In C26-ATB mice, proteome adaptations observed in CT-ATB mice were blunted. Differentially expressed proteins were involved in other processes like glucose metabolism, oxidative stress response, and proteolysis. This study confirms the existence of a microbiota-muscle axis, with a muscle response after antibiotics that varies depending on whether cachexia is present.

Effect of reduced crude protein diets supplemented with free limiting amino acids on body weight, carcass yield, and breast meat quality in broiler chickens

This study investigated the effect of reducing dietary crude protein (CP) content in the grower and finisher diets of broiler chickens on breast meat quality, muscle protein functionality, growth, carcass yield, and meat yield. To achieve this, a total of 1,269 one-day-old male Ross 308 chicks were fed 1 of 3 diets replicated 9 times each in a randomized complete block design with 9 blocks. The diets included a control (20.4% and 19.5% CP in the grower and finisher phase, respectively), a diet with a 1.5% reduction (CP-1.5%) and a diet with a 3.0% reduction (CP-3.0%) in CP content in both the grower and finisher phases. At the end of the experiment, the reduced-CP diets had no impact on body weight, feed intake, or feed conversion ratio. However, reduced-CP diet resulted in reduced (P < 0.001) total nitrogen intake (-7.46 and -11.94% in CP-1.5% and CP-3.0%, respectively). Breast meat quality was assessed (n=36 birds/group), and the experimental diets were associated with a slightly increased (P=0.07) ultimate pH (5.75, 5.79, and 5.81 for the control, CP-1.5%, and CP-3.0%, respectively). Breast fillets from the CP-1.5% and CP-3.0% groups had lower yellowness (b*, P < 0.001) and lower cooking loss (CL, P < 0.001) values than the control. Moreover, the solubility, emulsion activity, and stability indices of the sarcoplasmic and myofibrillar fractions of muscle proteins were not influenced by the diets. CP-1.5% and CP-3.0% diets were associated with an increased (P < 0.001) breast yield (18.39, 19.21, and 19.61% for the control, CP-1.5%, and CP-3.0%, respectively) while leg yield remained unchanged. Additionally, breast meat nutritional properties including protein and lipid contents were not impacted by the experimental diets. In conclusion, the CP content in the grower and finisher diets of broiler chickens can be reduced by as much as 3.0% without detrimental effects on performance or on meat quality as long as birds' amino acid requirements are adequately met.

Relationship between pre-rigor temperature of pork longissimus muscle, myofibril-bound calpain activity and protein degradation

The effect of pre-rigor temperature incubation on the activity and distribution in sarcoplasmic and myofibrillar fractions of calpains, and meat quality attributes was investigated. Porcine longissimus thoracis muscles were incubated pre-rigor at 14, 22, 30 and 38 °C to 6 h postmortem, followed by another 2 h incubation at 14 °C. Thereafter, muscles were stored at 2 °C for 1 or 4 days. With higher pre-rigor temperature, sarcoplasmic Ca2+ concentration, purge loss and myofibril-bound calpain-1 content increased, while shear force declined. Water-holding capacity of isolated myofibrils was lower after pre-rigor incubation at 38 °C. Desmin and troponin T degradation, and myofibril fragmentation was greater upon incubation of isolated myofibrils with added Ca2+ in the order 800 μM Ca2+ > 40 μM Ca2+ > no Ca2+, suggesting that calpain-1 and calpain-2 were associated to myofibrils and proteolytically active with sufficient Ca2+. Activity of myofibril-bound calpain-1 in muscle incubated pre-rigor at 22 and 30 °C were higher than when incubated at 14 and 38 °C. These results indicate that calpains translocate from the sarcoplasm onto myofibrils with higher pre-rigor temperature to 30 °C and the proteolytic potential of myofibril-associated calpains is thereby increased.

Cytokine Pattern of Peripheral Blood Mononuclear Cells Isolated from Children Affected by Generalized Epilepsy Treated with Different Protein Fractions of Meat Sources.

The objective of the present study was the evaluation of cytokine patterns in terms of TNF-α, IL-10, IL-6, and IL-1β secretion in peripheral blood mononuclear cell (PBMC) supernatants isolated from blood of children affected by generalized epilepsy and treated in vitro with myofibrillar, sarcoplasmic, and total protein fractions of meat and fish sources. Children with generalized epilepsy (EC group, n = 16) and children without any clinical signs of disease, representing a control group (CC group n = 16), were recruited at the Complex Structure of Neuropsychiatry Childhood-Adolescence of Policlinico Riuniti (Foggia, Italy). Myofibrillar (MYO), sarcoplasmic (SA), and total (TOT) protein fractions were obtained from longissimus thoracis muscle of beef (BF) and lamb (LA); from pectoralis muscle of chicken (CH); and from dorsal white muscle of sole (Solea solea, SO), European hake (Merluccius merluccius, EH), and sea bass fish (Dicentrarchus labrax, SB), respectively. PBMCs were isolated from peripheral blood of EC and CC groups, and an in vitro stimulation in the presence of 100 μg/mL for each protein fraction from different meat sources was performed. Data were classified according to three different levels of cytokines produced from the EC group relative to the CC group. TNF-α, IL-10, and IL-6 levels were not affected by different meat fractions and meat sources; on the contrary, IL-1β levels were found to be significantly affected by the tested proteins fractions, as well as different meat sources, in high-level cytokine group. On average, the protein fractions obtained from LB, BF, and CH meat sources showed a higher level of IL-1β than the protein fractions obtained from EH and SB fish samples. When all cytokine classes were analyzed, on average, a significant effect was observed for IL-10, IL-1β, and TNF-α. Data obtained in the present study evidence that the nutritional strategy based on protein from fish and meat sources may modulate the immunological cytokine pattern of infants with generalized epilepsy.

Overexpression of Multiple E3 Ubiquitin Ligases in Gastrocnemius Muscles from Mice

E3 ubiquitin ligases are commonly studied in skeletal muscle under atrophy‐inducing conditions. MuRF1 is a recognized marker of muscle atrophy and we have recently identified Fbxl22 to be involved in the process of denervation‐induced muscle atrophy. Thus, we sought to investigate the role of MuRF1 and Fbxl22 together in the process of skeletal muscle atrophy. Male C57BL/6 mice (16 to 20‐week old) were subjected to in vivo electroporation with either a mouse specific MuRF1 or Fbxl22 plasmid (20ug) in the gastrocnemius (GA) muscles with the contralateral leg serving as a vector control (EV). An additional cohort of mice was electroporated with MuRF1 and Fbxl22 plasmids simultaneously. After 7 days, lateral (LGA) and medial (MGA) gastrocnemius muscles were collected, weighed and sectioned for cross‐sectional area (CSA) and GFP visualization. Western blotting was performed on sarcoplasmic and myofibrillar fractions to determine levels of ubiquitin and sarcomeric proteins in the transfected GA muscles.MuRF1 overexpression (OE) resulted in significant loss of total GA muscle mass (‐8.8%) after 7 days alone. In comparison, Fbxl22 OE led to no loss in total GA muscle mass, although significant reductions in LGA mass (‐3.4%) were observed. Combined OE of MuRF1 and Fbxl22 together resulted in significant loss of GA muscle mass (‐5.5%) but the level of atrophy was not as great as compared to MuRF1 OE alone. In transfected LGA muscles, total ubiquitin levels were significantly elevated in the Fbxl22 OE and combined E3 ligase group for the sarcoplasmic fraction. In the myofibrillar fraction of LGA transfected muscles, only the combined E3 ligase group displayed increased total ubiquitin levels after 7 days. In transfected Fbxl22 LGA muscles, we observed significant increases in vimentin, alpha‐actinin and Troponin T in the sarcoplasmic fractions. Gene manipulation of the E3 ubiquitin ligases, MuRF1 and Fbxl22 in basal skeletal muscle resulted in skeletal muscle atrophy and divergent changes in ubiquitination and sarcomeric proteins were observed following E3 overexpression. Future studies will seek to identify potential unique and shared substrates targeted by Fbxl22 and MuRF1 for ubiquitination.

Proximate composition and changes in muscle proteins of dried salted abo (Otolithes ruber)

Dried salted abo is a popular fishery product in San Miguel Bay (SMB), Philippines. However, the development of this food product has not been fully explored. In this study, the proximate composition and muscle protein profile of dried salted abo was investigated after confirming the molecular identification of abo found in SMB. Sequence analysis of 16S ribosomal RNA and cytochrome c oxidase subunit I genes suggested that abo in SMB could be Otolithes ruber. Abo fillets were brined in 4%, 8%, and 12% salt concentrations (w/w) and sun-dried for 6 and 12 hrs. In both 6 hrs and 12 hrs drying time, moisture content decreased in samples brined with 12% salt, suggesting that the salt concentration could be lowered between 8–12% with 6 hrs drying time. Additionally, the proteolytic profiling by SDS-PAGE analysis detected 17 bands in the myofibrillar protein fraction. Subsequent mass spectrometry identified nine proteins, of which the myosin heavy chain, actin, and keratin were degraded while the other proteins remained stable. Moreover, at 12% salt concentration, changes in staining intensities in several proteins were observed at both 6 hrs and 12 hrs drying time. These findings revealed that the applied salting and drying conditions led to the degradation of myosin heavy chain and polymerization of actin, in addition to an overall alteration of protein profile, which could affect the texture of dried abo product. Overall, this study demonstrated molecular evidence for species identification of abo for the first time. The information on proximate composition and changes in the protein profile of dried salted abo obtained from this study could help the local dried fish industry develop high-quality dried abo products with low salt content.

New Insights on the Impact of Cattle Handling on Post-Mortem Myofibrillar Muscle Proteome and Meat Tenderization.

This study investigated the effect of different cattle management strategies at farm (Intensive vs. Extensive) and during transport and lairage (mixing vs. non-mixing with unfamiliar animals) on the myofibrillar subproteome of Longissimus thoracis et lumborum (LTL) muscle of “Asturiana de los Valles” yearling bulls. It further aimed to study the relationships with beef quality traits including pH, color, and tenderness evaluated by Warner–Bratzler shear force (WBSF). Thus, comparative proteomics of the myofibrillar fraction along meat maturation (from 2 h to 14 days post-mortem) and different quality traits were analyzed. A total of 23 protein fragments corresponding to 21 unique proteins showed significant differences among the treatments (p < 0.05) due to any of the factors considered (Farm, Transport and Lairage, and post-mortem time ageing). The proteins belong to several biological pathways including three structural proteins (MYBPC2, TNNT3, and MYL1) and one metabolic enzyme (ALDOA) that were affected by both Farm and Transport/Lairage factors. ACTA1, LDB3, and FHL2 were affected by Farm factors, while TNNI2 and MYLPF (structural proteins), PKM (metabolic enzyme), and HSPB1 (small Heat shock protein) were affected by Transport/Lairage factors. Several correlations were found between the changing proteins (PKM, ALDOA, TNNI2, TNNT3, ACTA1, MYL1, and CRYAB) and color and tenderness beef quality traits, indicating their importance in the determination of meat quality and their possible use as putative biomarkers.

Identification of meat species in processed meat products by using protein based laser induced breakdown spectroscopy assay

The detection of meat fraud and mislabeling in processed meat products is a raising concern for consumers. The aim of this study was to develop and demonstrate the potential of protein-based laser induced breakdown spectroscopy (LIBS) method to be used for the identification of beef, chicken, and pork in fermented sausage and salami products. In this respect, bulk protein and protein fractions rich in sarcoplasmic and myofibrillar protein of sausage and salami products were obtained and subjected to LIBS analysis. LIBS spectrum was evaluated with chemometric methods to classify meat species and determine adulteration ratio by using principal component analysis and partial least square analysis, respectively. Limit of detection values for chicken and pork adulteration in beef sausage were found as 3.68 and 3.83% for myofibrillar fraction, while those values in beef salami were found as 3.80 and 3.47% for sarcoplasmic fraction, respectively.

Impact of Extraction Method on the Detection of Quality Biomarkers in Normal vs. DFD Meat.

The objective of this work was to demonstrate how the extraction method affects the reliability of biomarker detection and how this detection depends on the biomarker location within the cell compartment. Different extraction methods were used to study the sarcoplasmic and myofibrillar fractions of the Longissimus thoracis et lumborum muscle of young bulls of the Asturiana de los Valles breed in two quality grades, standard (Control) or dark, firm, and dry (DFD) meat. Protein extractability and the expression of some of the main meat quality biomarkers—oxidative status (lipoperoxidation (LPO) and catalase activity (CAT)), proteome (SDS-PAGE electrophoretic pattern), and cell stress protein (Hsp70)—were analyzed. In the sarcoplasmic fraction, buffers containing Triton X-100 showed significantly higher protein extractability, LPO, and higher intensity of high-molecular-weight protein bands, whereas the TES buffer was more sensitive to distinguishing differences in the protein pattern between the Control and DFD meat. In the myofibrillar fraction, samples extracted with the lysis buffer showed significantly higher protein extractability, whereas samples extracted with the non-denaturing buffer showed higher results for LPO, CAT, and Hsp70, and higher-intensity bands in the electrophoretic pattern. These findings highlight the need for the careful selection of the extraction method used to analyze the different biomarkers considering their cellular location to adapt the extractive process.

Heterochronic Plasma Transfer Alters Proteostatic Maintenance in Skeletal Muscle

Studies using heterochronic blood transfer (HBT) and heterochronic plasma transfer (HPT) show that circulating factors in young blood and plasma can rejuvenate the ability of aged skeletal muscle to respond to damage. Additionally, the transfer of old blood and plasma to young mice inhibits skeletal muscle regeneration. These changes in the ability of skeletal muscle to regenerate in response to modifications in the systemic environment are largely mediated by satellite cells. However, the role of satellite cells in maintaining normal muscle function is unclear. Therefore, how the systemic environment influences skeletal muscle in the absence of extreme muscle damage is still unknown. Proteostatic maintenance, which includes protein synthesis and breakdown, is necessary for cellular stability in skeletal muscle. One measure of proteostatic maintenance is protein:DNA ratio (i.e. how much protein is synthesized for cellular maintenance verses cellular proliferation). Here we determined if altering the systemic environment of old mice with young plasma and the systemic environment of young mice with old plasma would change proteostatic maintenance of skeletal muscle. We hypothesized that compared to old mice that receive old plasma (OOp), old mice that underwent HPT from young mice (OYp) would improve proteostatic maintenance. Additionally, we hypothesized that compared to young mice that receive young plasma (YYp), young mice that underwent HPT from old mice (YOp) would decrease proteostatic maintenance. To test this, we implanted young (5-month, n=3 YYp and 5 YOp) and old (24-months, n=5 OOp, and 5 OYp) C57BL/6J mice with a jugular catheter. All mice underwent heterochronic plasma transfer (HPT), receiving 100μl plasma injections, every three days for 24 days. The day before HPT, mice began stable isotope labeling with a bolus injection of 99% deuterium oxide (D2O) followed by 8% D2O-enriched drinking water. After 4 weeks, the quadriceps, gastrocnemius, and tibialis anterior muscles were harvested and analyzed for DNA and protein synthesis to calculate the protein:DNA ratios. The protein:DNA ratio for the mitochondrial fraction trended toward a higher ratio in the tibialis anterior of the OYp versus OOp (11.93 ± 1.161 and 8.928 ± 1.128, respectively: p=0.10). The protein:DNA ratio for the myofibrillar fraction trended toward being lower in the gastrocnemius of the YOp compared to YYp (8.338 ± 0.429 and 6.281 ± 0.737, respectively: p=0.06). These results suggest that manipulating the systemic environment with young or old plasma can change proteostatic maintenance of skeletal muscle so that the muscle reflects the systemic environment. Therefore, strategies to improve the systemic environment, like with exercise, may be a feasible strategy to improve proteostatic maintenance in aged skeletal muscle. Further research is needed to determine what circulating factors alter protein and DNA turnover in skeletal muscle.

Comparative Study on Pale, Soft and Exudative (PSE) and Red, Firm and Non-Exudative (RFN) Pork: Protein Changes during Aging and the Differential Protein Expression of the Myofibrillar Fraction at 1 h Postmortem.

In this paper, the protein changes during aging and the differences in the myofibrillar protein fraction at 1 h postmortem of pale, soft and exudative (PSE), and red, firm and non-exudative (RFN) pork longissimus thoracis (LT) were comparatively studied. The PSE and RFN groups were screened out based on the differences in their pH and lightness (L*) at 1 h, and their purge loss at 24 h postmortem. Based on the measured MFI, desmin degradation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, PSE meat presented more significant changes in the myofibrillar protein fraction compared to RFN meat during postmortem aging. Through liquid chromatograph-mass spectrometer/mass spectrometer (LC-MS/MS) analysis, a total of 172 differential proteins were identified, among which 151 were up-regulated and 21 were down-regulated in the PSE group. The differential proteins were muscle contraction, motor proteins, microfilaments, microtubules, glycolysis, glycogen metabolism, energy metabolism, molecular chaperones, transport, and enzyme proteins. The AMP activated protein kinase (AMPK) signaling pathway, HIF-1 signaling pathway, calcium signaling pathway, and PI3K-Akt signaling pathway were identified as the significant pathways related to meat quality. This study suggested that the different changes of the myofibrillar protein fraction were involved in the biochemical metabolism in postmortem muscle, which may contribute to the molecular understanding of PSE meat formation.

Identifying Regulators of O‐GlcNAcylation in the Broken Heart

The post‐translational modification of intracellular proteins by monosaccharides of O‐linked b‐N‐acetylglucosamine (O‐GlcNAc) has emerged as a critical regulator of cellular function. Previous research has demonstrated that enhanced O‐GlcNAcylation activates cytoprotective pathways in cardiac models of ischemia reperfusion (I/R) injury. The cycling of O‐GlcNAc is regulated by the collective efforts of two enzymes: O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA), which catalyze the addition and hydrolysis of O‐GlcNAc respectively. The goal of these studies is to understand the mechanisms by which the heart regulates O‐GlcNAcylation in response to injury. Sex and gender have been linked to the pathophysiology of cardiovascular disease; however, the impact of sex on O‐GlcNAc cycling has not been studied. Thus, to address our goal, we harvested male and female hearts from mice and extracted the total heart proteome and assessed the levels of O‐GlcNAc, OGT, and OGA as well as key enzymes within the hexosamine biosynthetic pathway (HBP). We observed a modest elevation in O‐GlcNAc levels in female hearts. Counter‐intuitively, OGA levels appeared to be elevated in the female hearts. To assess the impact of sex on O‐GlcNAc cycling in response to injury, we used the Langendorff perfused mouse heart model to collect male and female hearts exposed to either control, ischemic or I/R conditions. Hearts were partitioned into a soluble fraction enriched in cytosolic and mitochondrial proteins, as well as a myofibrillar fraction enriched in contractile proteins. Our data demonstrate that the majority of the O‐GlcNAc‐modified proteins are detected in the soluble fraction. Ischemia results in a suppression of O‐GlcNAcylation; however, O‐GlcNAc levels were modestly elevated in hearts subjected to I/R injury. Future work will assess the impact of sex on the activity of OGT and OGA, as well as the nucleotide sugar UDP‐GlcNAc. Collectively, these findings will provide a foundation for understanding whether sex is an important factor to consider when assessing molecular mechanisms regulating O‐GlcNAc cycling in the heart.Support or Funding InformationK12 HL141952, U01 CA2300978, R01 HL129640, P01 HL107153

Phosphoproteome analysis of sarcoplasmic and myofibrillar proteins in stress-induced dysfunctional broiler pectoralis major muscle

Postmortem changes of sarcoplasmic and myofibrillar protein phosphorylation in pectoralis major (PM) muscle of broilers under pre-slaughter stress were investigated. Broiler chickens were randomly distributed to unstressed control and transport under high environmental temperature groups. PM muscle samples of transport-stressed broilers were classified into normal or pale, soft and exudative (PSE)-like. Sarcoplasmic fraction in PSE-like meat had a higher global phosphorylation level than that in normal meat at the early postmortem stage. The myofibrillar proteins showed diverse phosphorylation patterns at different postmortem times. The stress-induced highly phosphorylated sarcoplasmic proteins were glycometabolic enzymes, which partially contributed to accelerated glycolysis rate. The phosphorylation levels of most sarcomeric proteins identified in the myofibrillar fraction were affected by postmortem time, implying their roles in regulating muscle rigor mortis development. This work contributes to a deeper understanding of the biochemical processes that may lead to stress-induced changes in meat quality.

Effect of Feeding a Low Vitamin a Diet to Beef Steers on Calpain 1 Activation during Meat Aging

ObjectivesThe objective of the study was to determine if a vitamin A deficient diet during beef finishing influences calpain 1 activation during meat aging.Materials and MethodsSixty-four steers of approximately 7 mo of age were subjected to a 14-d acclimation period followed by a 95-d growing period on a low vitamin A diet (1017 IU vitamin A/kg DM) designed to deplete liver vitamin A stores. Steers were assigned to a randomized complete blocked design with a 2 × 2 arrangement of treatments (breed: commercial Angus, n = 32, and purebred Simmental, n = 32; and a Low Vitamin A diet or a control diet). The low Vitamin A (LVA) treatment was a finishing diet with no supplemental vitamin A (723 IU vitamin A/kg DM). The control (CON) treatment was the LVA diet plus supplementation with 2200 IU vitamin A/kg DM for a total of 2923 IU vitamin A/kg DM. Serum retinol concentrations were monitored at the beginning and end of treatment. Upon completion of finishing, steers were slaughtered in two groups at a commercial plant. After fabrication, boneless strip loins (IMPS 180) were collected and transported to NDSU. Samples (approximately 40 g) were collected from the anterior portion of the strip loin on d-2 and d-7 of aging and immediately frozen. Protein was extracted from meat samples in fractionation buffers to yield sarcoplasmic and myofibrillar portions, separated by SDS-PAGE, and transferred to PVDF membranes. Immunoblot analysis was done using anti-desmin (d-2 and d-7) and anti-calpain 1 (d-2) antibodies, and results were visualized and documented. A pooled control was run on all membranes and set to a value of one for normalizing results. All experimental data were analyzed using the Proc Mixed procedure of SAS with breed of steers, dietary treatments, their interaction and slaughter date used as a fixed effect.ResultsCalpain 1 autolysis in the sarcoplasmic protein fraction of the d-2 aged loin samples were not affected by treatment or breed. The myofibrillar protein fraction from Angus loins had greater (P = 0.02) accumulation of the 76 kDa calpain 1 autolysis product than that from the Simmental loins; the myofibrillar fraction of the loins from the LVA treatment tended (P = 0.07) to have more 76 kDa calpain 1 autolysis product than that from the CON. There were not any differences (P &gt; 0.19) in the 80 kDa calpain 1 band or the 78 kDa calpain 1 intermediate autolysis product in the myofibrillar fraction. There was a treatment by breed interaction (P = 0.01) for desmin in the d-7 aged loins where Angus loins from the CON treatment had less accumulation of the 46 kDa band than Angus loins on the LVA treatment and Simmental loins from either treatment.ConclusionVitamin A restriction increased protein proteolysis in Angus but not in Simmental steers. The increased calpain 1 autolysis in Angus vs. Simmental, regardless of Vitamin A treatment, indicates a genetic difference that may be the driver for the increased protein degradation in steers a restricted vitamin A diet.

Effect of hydroxyl-radical on the biochemical properties and structure of myofibrillar protein from Alaska pollock (Theragra chalcogramma)

The aim of the study was to investigate the effect of hydroxyl-radical on myofibrillar protein properties. Myofibrillar protein isolated from Alaska pollock muscles were incubated with oxidant agents containing different H2O2 concentrations of 0, 0.1, 1, 5, 10 and 20 mmoL/L for 1, 3 and 5 h respectively. Incubation at higher concentrations of H2O2 induced a significant decrease of protein total sulfhydryl group and an increase in protein carbonyl content in the myofibrillar fraction. Moderate H2O2 concentration (1 mmoL/L) enhanced water-holding capacity and textural properties of a gel, while lower (0.1 mmoL/L) and higher (5, 10 & 20 mmoL/L) H2O2 concentrations significantly reduced the water-holding capacity. At higher H2O2 concentration, oxidized gel revealed some resistance toward the change in gel properties. Additionally, the results from SDS-PAGE and microstructure confirmed the structural changes through aggregation and denaturation of myofibrillar components that induced changes on gelation of myofibrillar protein. The whiteness of the myofibrillar protein gel decreased significantly (p < 0.05) with increasing the concentration of H2O2. Oxidation of myofibrillar protein revealed the slight improvement of textural properties of the heat set gel at a moderate concentration of oxidant agent.

Effect of Feeding a Low Vitamin a Diet to Beef Steers on Calpain 1 Activation during Meat Aging

ObjectivesThe objective of the study was to determine if a vitamin A deficient diet during beef finishing influences calpain 1 activation during meat aging.Materials and MethodsSixty-four steers of approximately 7 mo of age were subjected to a 14-d acclimation period followed by a 95-d growing period on a low vitamin A diet (1017 IU vitamin A/kg DM) designed to deplete liver vitamin A stores. Steers were assigned to a randomized complete blocked design with a 2 × 2 arrangement of treatments (breed: commercial Angus, n = 32, and purebred Simmental, n = 32; and a Low Vitamin A diet or a control diet). The low Vitamin A (LVA) treatment was a finishing diet with no supplemental vitamin A (723 IU vitamin A/kg DM). The control (CON) treatment was the LVA diet plus supplementation with 2200 IU vitamin A/kg DM for a total of 2923 IU vitamin A/kg DM. Serum retinol concentrations were monitored at the beginning and end of treatment. Upon completion of finishing, steers were slaughtered in two groups at a commercial plant. After fabrication, boneless strip loins (IMPS 180) were collected and transported to NDSU. Samples (approximately 40 g) were collected from the anterior portion of the strip loin on d-2 and d-7 of aging and immediately frozen. Protein was extracted from meat samples in fractionation buffers to yield sarcoplasmic and myofibrillar portions, separated by SDS-PAGE, and transferred to PVDF membranes. Immunoblot analysis was done using anti-desmin (d-2 and d-7) and anti-calpain 1 (d-2) antibodies, and results were visualized and documented. A pooled control was run on all membranes and set to a value of one for normalizing results. All experimental data were analyzed using the Proc Mixed procedure of SAS with breed of steers, dietary treatments, their interaction and slaughter date used as a fixed effect.ResultsCalpain 1 autolysis in the sarcoplasmic protein fraction of the d-2 aged loin samples were not affected by treatment or breed. The myofibrillar protein fraction from Angus loins had greater (P = 0.02) accumulation of the 76 kDa calpain 1 autolysis product than that from the Simmental loins; the myofibrillar fraction of the loins from the LVA treatment tended (P = 0.07) to have more 76 kDa calpain 1 autolysis product than that from the CON. There were not any differences (P > 0.19) in the 80 kDa calpain 1 band or the 78 kDa calpain 1 intermediate autolysis product in the myofibrillar fraction. There was a treatment by breed interaction (P = 0.01) for desmin in the d-7 aged loins where Angus loins from the CON treatment had less accumulation of the 46 kDa band than Angus loins on the LVA treatment and Simmental loins from either treatment.ConclusionVitamin A restriction increased protein proteolysis in Angus but not in Simmental steers. The increased calpain 1 autolysis in Angus vs. Simmental, regardless of Vitamin A treatment, indicates a genetic difference that may be the driver for the increased protein degradation in steers a restricted vitamin A diet.

The effect of ultrasound treatments on the tenderizing pathway of goose meat during conditioning.

In order to figure out the effect of ultrasound treatments (UT) on the tenderizing pathway of goose meat, breast muscles of 32 Eastern Zhejiang White Geese were treated with different ultrasound powers (the control, 300 and 600W) at 40kHz for 30min. Shear force, cooking loss, myofibrillar fraction index (MFI), particle size, morphological analysis of actin filaments and the levels of F-actin and G-actin were investigated during 168h storage. Results showed that 600W group had the lowest shear force and cooking loss at 24, 48, 96 and 168h among treatments, while 300W UT decreased shear force and cooking loss compared to the control. UT increased MFI and induced myofibrillar small particles (D3,2) by disrupting actin filaments for myofibril and transforming of F-actin to G-actin compared to the control. We concluded that UT tenderized goose meat by fragmenting actin filaments and myofibrillar fraction.

Use of liquid isoelectric focusing (OFFGEL) on the discovery of meat tenderness biomarkers

Protein biomarkers of meat tenderness are known to be of primary importance for the prediction of meat quality, and hence, industry profitability. Proteome analysis was performed on meat from 8 Main Anjou beef cattle, previously classified as tender or tough meats by Warner Bratzler shear force measurements. Myofibrillar fraction of Longissimus thoracis muscle was separated by a novel fractionation approach based on liquid isoelectric focusing (OFFGEL) and further analyzed by SDS-PAGE and liquid chromatography coupled to tandem mass spectrometry.Obtained OFFGEL fraction profiles were reproducible allowing the comparison of both meat qualities and revealing 7 protein bands capable to discriminate between tender and tough samples. The proteins present in these bands were troponin T, Heat Shock protein beta-1, creatine kinase, actin, troponin C, myosins 1 and 2 and myozenin-1. The latter protein has not been previously reported as a marker of meat tenderness. SignificanceThis study introduces an innovative proteomic approach for the study of muscle proteome. The fact of obtaining fractions in liquid state after OFFGEL fractionation allows for a faster analysis of proteins by mass spectrometry, being an interesting alternative to more classical proteomic approaches based on two dimensional gel electrophoresis (2-DE).

The physical and biochemical effects of pre-rigor high pressure processing of beef

High pressure processing (HPP) of pre-rigor longissimus thoracis (strip loin) from prime and bull animals substantially decreased the shear force and improved consumer eating attributes of the final meat product. The improved tenderness in both prime and bull meat was associated with a lower myofibrillar fragmentation index and reduced calpain 1 activity which indicated the mechanism of tenderisation was different from that which occurred in chill aged meat. Light microscopy showed disruption to the fibre packing within the muscle and electron microscopy confirmed significant disruption of the Z discs and M lines and disappearance of the A lines. Thus, HPP is associated with a reduction in the structural integrity and strength of the sarcomeres. These effects were consistent in strip loins sourced from prime and bull stock. HPP also led to the movement of glycogen phosphorylase from the sarcoplasmic fraction to the insoluble myofibrillar fraction in all animals and this was associated with a higher pH at 24 h.

The effect of ATP marination on the depolymerization of actin filament in goose muscles during postmortem conditioning

In order to study the tenderization mechanism of ATP treatments by depolymerizing actin filaments, breast muscles of Eastern Zhejiang White Geese were randomly divided into 3 groups: control, 10 and 20mM groups. Shear force (SF), sarcomere length (SL) and myofibrillar fraction index (MFI), the content of F-actin and G-actin, the expression of actin associated proteins (cofilins and tropomodulins) were investigated during conditioning. In 20mM group, cofilins content increased from 48 to 168h, while tropomodulins decreased; the content of F-actin decreased from 24 to 168h, while the increased G-actin was observed upto 48h. In the control, the degraded tropomodulins were observed at 168h, and the increased cofilins and G-actin were detected at the same time; the increase of MFI and decrease of F-actin content were shown at 96 and 168h. Compared to control group, 20mM group accelerated the transformation of F-actin into G-actin; it showed higher SL and MFI, and lower SF at 48, 96 and 168h, respectively. We concluded that depolymerization of actin filaments, which was regulated by cofilins and tropomodulins, contributed to myofibrillar fraction and low SF during conditioning.