Rigor development and meat quality of Murrah buffalo from different production systems

The purpose of this study was to evaluate the rigor mortis development and its effects on striploins quality of Murrah buffalos from meat (males) and dairy (females) productions. The rigor was monitored during carcass chilling (up to 22 h postmortem) and the meat physicochemical traits was accessed after 72 h postmortem. Glycolytic rate, measured by decreasing pH and glycogen content, was higher (p < 0.05) in female than in male buffalo, leading to a different onset rigor time (5-6 h and 9-10 h, respectively). Male meat had (p < 0.05) lower fat (1.41 vs 3.58%) and insoluble collagen (1.18 vs 1.58 mg g-1) contents, but higher soluble collagen content (5.16 vs 20.38%), sarcomere length (1.65 vs 1.84 µm), myofibrillar fragmentation (334 vs 295) and shear force (39.38 vs 25.92 N) than females. These results suggest that dairy buffalo meat is more tender and can be used on the market as high-quality meat such as from buffalo raised to meat production.

Collagen Cross‐Linking Is Associated With Cardiac Remodeling in Hypertrophic Obstructive Cardiomyopathy

Background Collagen cross‐linking is covalent bonds among collagen fibers from catalysis of lysyl oxidase (LOX) and advanced glycation end products (AGEs). We aimed to evaluate the formation of enzymatic and nonenzymatic collagen cross‐linking and its clinical significance in patients with hypertrophic obstructive cardiomyopathy. Methods and Results Forty‐four patients with hypertrophic obstructive cardiomyopathy who underwent surgical myectomy were consecutively enrolled. Cardiovascular magnetic resonance parameters of left atrial/left ventricular function were measured, including peak filling rate (PFR) and early peak emptying rate (PER‐E). Total collagen was the sum of soluble and insoluble collagen, which were assessed by collagen assay. The myocardial LOX and AGEs expression were measured by molecular and biochemical methods. Compared with patients without atrial fibrillation, insoluble collagen ( P =0.018), insoluble collagen fraction ( P =0.017), and AGEs ( P =0.039) were higher in patients with atrial fibrillation, whereas LOX expression was similar ( P =0.494). The insoluble collagen fraction was correlated with PFR index (PFR normalized by left ventricular filling volume) (r=−0.44, P =0.005), left atrial diameters (r=0.36, P =0.021) and PER‐E index (PER‐E normalized by left ventricular filling volume) (r=−0.49, P =0.001).Myocardial LOX was positively correlated with total collagen (r=0.37, P =0.025) and insoluble collagen fraction (r=0.53, P < 0.001), but inversely correlated with PFR index (r=−0.43, P =0.006) and PER‐E index (r=−0.35, P =0.027). In multiple regression analysis, myocardial LOX was independently associated with PFR, while insoluble collagen fraction showed independent correlation with PER‐E after adjustment for clinical confounders. Conclusions Collagen cross‐linking plays an important role on heart remodeling in hypertrophic obstructive cardiomyopathy. Myocardial LOX expression is independently correlated with left ventricular stiffness, while accumulation of AGEs cross‐links might be associated with the occurrence of atrial fibrillation in patients with hypertrophic obstructive cardiomyopathy.

Studying the Effects of Collagenase (Type 1) on the Collagen in Woody Breast Meat

Abnormal collagen infiltration in the Pectoralis major, breast muscle, of fast-growing big broilers has led to woody breast (WB) myopathy resulting in meat quality issues. Mechanisms to degrade the collagen were investigated to potentially improve WB texture. Freshly deboned WB fillets (n = 5 per trial; 3 trials) were ground and divided in to 25 g portions. Aqueous collagenase Type I solution (1 mL) from concentrations of 2.5, 5, and 10 mg/mL were incorporated in ground WB samples (n = 3 samples/treatment × 3 trials). Ground WB with 1 mL water acted as a control. All the samples were placed at 4 °C for 24 h and frozen at −80 °C. Control samples without any treatment or water addition (n = 3/trial) were frozen immediately upon grinding. Data collected on total (TC), soluble (SC), and insoluble collagen (IC) content was analyzed using one-way ANOVA with Tukey’s honestly significant difference (HSD) (p ≤ 0.05). Fresh WB fillets had TC, SC, and IC content of 19.5, 4.9, and 14.6 mg/g, respectively. The addition of collagenase decreased (p ≤ 0.05) the IC to 5.8 mg/g in the 10 mg/mL treatment after 24 h. Converting IC to SC using collagenase can potentially help the poultry industry to reduce WB toughness.

Influence of collagen changes on the tenderness of yak rumen smooth muscle during storage

The aim of the present study was to investigate the influence of collagen changes on the tenderness of yak rumen smooth muscle during storage. Yak rumen smooth muscle was stored at 3 ± 1 °C for 7 d and the Warner–Bratzler shear force (WBSF); total, soluble, and insoluble collagen content (TCC, SCC); collagen solubility (CS); and histological structure were monitored. Label-free mass spectrometry was used to validate the changes in collagen. Collagen changes significantly influenced yak rumen smooth muscle tenderness. A significant positive relationship was found between WBSF and TCC, SCC, as well as CS. The label-free mass spectrometry results validated that the collagen of smooth muscle was degraded during storage. Histological analysis revealed that yak rumen smooth muscle structure was destroyed during storage. Smooth muscle underwent a similar change in tenderness as skeletal muscle, and the weakening of connective tissue was the main reason for smooth muscle tenderness development.

Relationship between heifer carcass maturity and beef quality characteristics

Our objective was to determine the relationship between heifer carcass maturity and beef palatability of the longissimus lumborum (LM) and biceps femoris (BF). Left sides of A (n = 30), B (n = 30), and C (n = 30) maturity heifer carcasses under 30 mo of age by dentition were used. Carcasses were selected to ensure similar marbling scores across maturity groups (Small to Modest). Beef strip loins (LM) and outside rounds (BF) were obtained from these carcasses. Steaks were used to measure color stability, lipid oxidation (thiobarbituric acid reactive substances; TBARS), Warner-Bratzler shear force (WBSF), soluble and insoluble collagen, and consumer sensory perceptions. Heifer carcass maturity did not affect pH, fluid loss, WBSF, or collagen content of LM or BF steaks (P &gt; 0.29). In LM and BF steaks, a maturity × day of retail display interaction occurred for TBARS, in which B maturity steaks had lower levels of lipid oxidation compared with A and C maturity steaks from the fourth day to the end of the retail display (P &lt; 0.01). Nevertheless, LM steaks from B maturity carcasses tended to have lower overall acceptability (P = 0.08) and juiciness (P = 0.09) than steaks from C maturity carcasses, but steaks from B and C maturity carcasses did not differ from LM steaks obtained from A maturity carcasses. No differences in tenderness or flavor were observed due to maturity (P &gt; 0.24). Similarly, maturity had no effect on sensory characteristics of BF steaks (P &gt; 0.30). In conclusion, our results indicate that advanced physiological maturity does not decrease palatability of strip loin or outside round steaks from carcasses of heifers under 30 mo of age.

The Impacts of Feeding Natursafe (an Immune Support Product) on Beef Quality

ObjectivesNaturSafe® (Diamond V, USA) is a Saccharomyces cerevisiae fermentation product developed as an animal feed supplement for the further manufacture of nutritionally balanced feeds for beef cattle. This immune support product (Association of American Feed Control Officials number 96.8, 73.046 and International Feed Name number 7–05–520, 8–08–034) has been specifically formulated to optimize beef cattle health and performance, antibiotic stewardship, and food safety. Research has shown that NaturSafe supports optimal rumen and liver health, overall health and immune function, consistency of feed intake, daily gain, feed conversion, and antibiotic effectiveness. As no research previously has assessed the impacts of this feed ingredient on the quality of meat, the objective of this research was to characterize the effects of feeding NaturSafe on meat quality characteristics in beef.Materials and MethodsCrossbred steers (N = 60, n = 12 per treatment; mean hot carcass weight = 421 kg), through an antibiotic free production system, were individually fed diets containing 12, 15, or 18 g/d of NaturSafe or a control diet without (–AB) antibiotics or a control with antibiotics (+AB; 330 mg monensin + 110 mg tylosin·steer–1·d–1) for 112 d. Strip loins were collected and aged for 13 or 29 d postmortem prior to fabrication. Steaks (m. Longissimus) were then evaluated for Warner-Bratzler shear force, pH, sarcoplasmic calcium concentration, troponin-T degradation, fatty acid profile, proximate composition, sarcomere length, total collagen and insoluble collagen. After each aging period, steaks were evaluated for lipid oxidation, and color characteristics (L*, a*, b*, discoloration percentage, and percentage surface oxymyoglobin, metmyoglobin and deoxymyoglobin), during and/or after a 7 d simulated retail display period. A subset of samples at various aging and retail display periods were analyzed for lactic acid bacteria (LAB), psychotrophic plate counts (PPC), and aerobic plate counts (APC). Animal was considered the experimental unit and hot carcass weight and marbling score were used as covariates in the analysis.ResultsTreatment had no effect on pH, sarcomere length, troponin-T degradation, fatty acid profile, proximate composition, total collagen, insoluble collagen, LAB, PPC, APC, lipid oxidation, oxymyoglobin percentage, or metmyoglobin percentage. Meat from cattle fed 18 g/d of NaturSafe was (1) equal to–AB controls and had higher shear force values compared to all other treatments (P < 0.01), (2) had higher (P < 0.05) sarcoplasmic calcium levels than +AB controls and cattle fed 12 g of NaturSafe/d, (3) was redder (higher a* values, P < 0.05) than all other treatments, and (4) was yellower (higher b* values, P < 0.01) than the 12 or 15 g dose and the–AB control. There were no differences among treatments fed NaturSafe for lightness (L*) at either aging time. There were no differences for meat from animals fed 12 or 15 g NatureSafe/d, except deoxymyoglobin percent and discoloration, which were both minimal. Discoloration values were low for all treatments (< 10%).ConclusionThese data indicate that feeding NaturSafe had few discernible effects on meat quality characteristics.

PSVIII-32 Estimates of genetic parameters for sub-primal and meat quality traits in Canadian commercial crossbred swine populations

The hypothesis that genetic relationships exist between loin muscle collagen characteristics and sub-primal and meat quality traits was tested. Data from 500 pigs from crosses between Duroc sires and hybrid Large White ✕ Landrace sows with pedigree back to about eight generations were used. Significant fixed effects (slaughter group and company) and a random additive effect were fitted in bivariate animal models to estimate phenotypic and genetic correlations using ASReml 4.1. Moderate heritabilities were obtained for sub-primal traits ranging from 0.21 for bone weight to 0.44 for loin muscle weight with a low estimate of 0.10 being obtained for loin weight. Meat quality traits were low to moderately heritable with the highest estimate being found for intramuscular fat (0.42). The heritability estimates for percentages of heat soluble and insoluble collagen were 0.12 and 0.15, respectively, while 0.33 was found for total collagen. Moderate to relatively high heritabilities imply the possibility of improving these traits through selective breeding. In general, moderate to high phenotypic and genetic correlations were obtained for sub-primal traits, whilst meat quality traits had moderate phenotypic and moderate to high genetic correlations. Strong negative genetic correlations between moisture traits and fat traits and a further negative correlation between fat and muscling traits were estimated confirming that selecting for improved muscling over time can negatively affect fat traits and indirectly decrease meat eating quality. The strong genetic correlation between pH and L* (-0.95) suggested possible pleiotropic gene effects on these traits. Warner-Braztler shear force (WBSF) had moderate genetic correlations with insoluble collagen (0.42) and soluble collagen (-0.38) suggesting a potential relationship between some of the genes impacting these traits. Genetic correlations between WBSF and collagen characteristics indicate that despite the relative youthfulness of pigs at slaughter, genetic selection for collagen solubility may decrease pork toughness.

Allosteric communications between domains modulate the activity of matrix metalloprotease-1

ABSTRACTAn understanding of the structure-dynamics relationship is essential for understanding how a protein works. Prior research has shown that the activity of a protein correlates with intra-domain dynamics occurring at picosecond to millisecond timescales. However, the correlation between inter-domain dynamics and the function of a protein is poorly understood. Here we show that communications between the catalytic and hemopexin domains of matrix metalloprotease-1 (MMP1) on type-1 collagen fibrils correlate with its activity. Using single-molecule FRET (smFRET), we identified functionally relevant open conformations where the two MMP1 domains are well-separated, which were significantly absent for catalytically inactive point mutant (E219Q) of MMP1 and could be modulated by an inhibitor or an enhancer of activity. The observed relevance of open conformations resolves the debate about the roles of open and closed MMP1 structures in function. A sum of two Gaussians fitted histograms, whereas an exponential fitted autocorrelations of smFRET values. We used a two-state Poisson process to describe the dynamics and used histograms and autocorrelations of conformations to calculate the kinetic rates between the two states. All-atom and coarse-grained simulations reproduced some of the experimental features and revealed substrate-dependent MMP1 dynamics. Our results suggest that an inter-domain separation facilitates opening up the catalytic pocket so that the collagen chains come closer to the MMP1 active site. Coordination of functional conformations at different parts of MMP1 occurs via allosteric communications that can take place via interactions mediated by collagen even if the linker between the domains is absent. Modeling dynamics as a Poisson process enables connecting the picosecond timescales of molecular dynamics simulations with the millisecond timescales of single molecule measurements. Water-soluble MMP1 interacting with water-insoluble collagen fibrils poses challenges for biochemical studies that the single molecule tracking can overcome for other insoluble substrates. Inter-domain communications are likely important for multidomain proteins.Statement of SignificanceIt is often challenging to distinguish functionally important dynamics because proteins are inherently flexible. MMP1 is a model enzyme because both the catalytic and hemopexin domains are necessary to degrade triple-helical type-1 collagen, the highly proteolysis-resistant structural component of the extracellular matrix. We report, for the first time, measurements of MMP1 inter-domain dynamics on type-1 collagen fibrils. We have identified functionally relevant MMP1 conformations where the two domains are far apart. Mutations and ligands can allosterically modulate the dynamics that correlate with activity. The dynamics follow a two-state Poisson process that connects the picosecond timescales of MD simulations with the millisecond timescales of experiments. The two domains can functionally communicate via collagen even when the physical linker is absent.

Dynamic Stretching of Fibrillar Collagen Enhances Cross-linking by Transglutaminas

Category: Basic Sciences/Biologics Introduction/Purpose: New approaches to improve tendon repair after injury are an active area of research. Critical properties of tendons are governed by the production and assembly of fibrillar collagens. Cross-linking of fibrillar collagen is a primary factor in determining the function and mechanical properties of the collagen fibers comprising Enzymatic cross-linking by lysyl oxidase in the telopeptide domain of collagen I and III is one determinant of collagen fibril assembly and is the best characterized biochemical cross-link. Transglutaminase catalyzes the modification of lysine residues that in turn form an n-e-glutamyl lysine bond between proteins in the extracellular space. We hypothesize that transglutaminase-dependent modification of collagen in tendons is also a principal determinant of tendon strength and function and is dependent upon tension. Methods: 3-D collagen gels were generated from acid solubilized type I collagen with telopeptides (Advanced BioMatrix). Collagen gels were plated and loaded into a MechanoCulture FX apparatus (CellScale). Gels were subjected to a 10% stretch for 24 hrs at 37°C at 2hz (dynamic) or no stretch, static controls. Gels exposed to enzymatic cross-linking were incubated with either 2.4 ng of recombinant Transglutaminase 2 (Axxora) in a 10 mM Ca2+ solution. Inhibition and labeling of transglutaminase substrates was performed by incubation of collagen gels with 0.2 mM aminopentyl biotinamide in DMSO. Soluble collagen was separated from insoluble collagen by centrifugation at 10,000G. Insoluble fractions were boiled in SDS-Laemmli buffer prior to separation by SDS-PAGE. Collagen in soluble and insoluble fractions was evaluated by Coomassie stain whereas transglutaminase modification was detected via western blot using streptavidin conjugated horse radish peroxidase to detect biotinylated proteins. Results: Evaluation of collagen gels subjected to dynamic versus static stretch revealed minor differences in insoluble collagen incorporation in the two conditions. Notably, higher molecular weight cross-linked forms of collagen appeared to be higher in dynamic versus static gels. In the presence of transglutaminase, differences in higher molecular weight cross-linked forms of collagen, beta-bands, were also detected. Finally, incorporation of biotinylated transglutaminase substrate into collagen alpha bands was enriched in dynamic versus static cultures. Hence, preliminary results support a differential role for transglutaminase modification in collagen under cyclic tension versus static conditions. Conclusion: A better understanding of the role of dynamic stretching and differential tension in the regulation of collagen cross- link formation is predicted to contribute to improved strategies to treat injured tendons.

Analysis of “old” proteins unmasks dynamic gradient of cartilage turnover in human limbs

Unlike highly regenerative animals, such as axolotls, humans are believed to be unable to counteract cumulative damage, such as repetitive joint use and injury that lead to the breakdown of cartilage and the development of osteoarthritis. Turnover of insoluble collagen has been suggested to be very limited in human adult cartilage. The goal of this study was to explore protein turnover in articular cartilage from human lower limb joints. Analyzing molecular clocks in the form of nonenzymatically deamidated proteins, we unmasked a position-dependent gradient (distal high, proximal low) of protein turnover, indicative of a gradient of tissue anabolism reflecting innate tissue repair capacity in human lower limb cartilages that is associated with expression of limb-regenerative microRNAs. This association shows a potential link to a capacity, albeit limited, for regeneration that might be exploited to enhance joint repair and establish a basis for human limb regeneration.