Actin as a candidate quality marker for food of animal origin

Most of the relevant meat and meat-product quality characteristics are the results of the chemistry of protein and peptides. Therefore, the inclusion of information on protein differences provided by protein markers can make a significant contribution to the improvement of meat and meat product quality. Actins are a family of highly conserved proteins, which play fundamental roles in nearly all aspects of eukaryotic cell biology. In the skeletal muscle, in addition to the highly specialized contractile apparatus, there are both the actin-associated costameric complexes and the functionally distinct cytoskeletal actin-based filaments. Ante mortem muscle biochemistry strongly influences post mortem biochemical processes which, in turn, are linked to final quality attributes, including texture which is one of the most important attributes of muscle foods. However, there is very little knowledge on the way in which ante mortem biochemistry changes the post mortem events and hence the resulting final quality. The understanding of post mortem mechanisms is essential for predicting the final quality in terms of texture. The histochemical and biochemical evidence indicates that much of the tenderization associated with post mortem aging is due to the action of the enzymes, which are known to be endogenous to the muscle. Interestingly, the most abundant proteins of the myofibril, actin and myosin, are not significantly degraded during post mortem aging. However, an extensive apoptosis could lead to a progressive degradation of cytoskeletal and thin filaments of actin, resulting to meat tenderization. The investigation on how actin degradation affects meat and meat product quality is still nascent, but the results reported in this PhD thesis seem promising. Indeed, the main activities have been directed to evaluate the potential use of actin as a biochemical marker for the prediction of muscle food quality in three Italian PDO dry-cured hams and in farmed sea bass. In the first part of this thesis, a brief general introduction on the muscle composition in mammals and fish and on the post mortem events occurring in muscle is reported, followed by a description of the characteristics of dry-cured hams and sea bass like food products of animal origin. Therefore, the two papers related to the above reported topics are attached, accompanied by some conclusive remark

The caspase and calpain proteolytic systems in Longissimus dorsi and Infraspinatus muscles of Italian Simmental young bulls

Calpains and capsases are two families of cytosolic proteases essential for a proper skeletal muscle function, which recognize specific target proteins for degradation. Moreover, \u3bc-calpain is believed to play a major role in post mortem proteolysis and meat tenderization, which in turn depends on fibre type composition. Recent data also support the involvement of caspases in the development of tender meat. In fact, it is plausible that the hypoxic conditions occurring in post mortem may activate these proteases, which function as vital executioners of apoptosis in hypoxia/ischemia and degrade structural components of the cytosckeleton. The goal of this work was to evaluate the activities of \u3bc-calpain and caspases in two different muscle types from bulls, immediately after slaughtering, by immunodetection of their specific proteolytic products and to compare such activities to their related mRNA expression level. In particular, caspases 9 and 3 have been analysed, which are an initiating and an executer caspase, respectively, to follow the entire cascade of events occurring during a hypoxic stress. Samples of Longissimus dorsi (white/type IIB muscle) and Infraspinatus (red/type I muscle) muscles were collected within 20 minutes from slaughtering of 16 Italian Simmental young bulls, and stored at -80\ub0 until the analysis. By SDS-PAGE the alpha II spectrin and its degradation products 145kDa (from \u3bc-calpain degradation) and 150kDda and 120kDda (from caspases degradation) were separated and then quantified in relative terms. Total RNA was extracted from the two muscles and cDNA was produced; the DNA was amplified by q-PCR technic. The genes analyzed were CAPN1 (coding for \u3bc-calpain), CASP3 (caspase 3), CASP9 (caspase 9). Genes coding for GAPDH, \u3b2-Actin, RPLP0 and Cyclophilin was used as reference genes. Both caspases and \u3bc-calpain were found active, having been recognized their target degradation products. In particular no difference in the level of alpha II spectrin fragment, derived from the caspase-3 activity, was found between the Longissimus dorsi and Infraspinatus muscles, in agreement with gene expression results. On the other hand, the \u3bc-calpain activity was influenced by the type of muscles

Proteolytic resistance of actin but not of myosin heavy chain during processing of Italian PDO (protected designation of origin) dry-cured hams

Proteomics is the approach of choice to study the fate of specific proteins, and it is very useful in identifying quality molecular markers. A combination of immunochemical and mass spectrometry analysis was used to assess the occurrence of proteolytic changes of actin and myosin heavy chain (MHC) proteins in pig biceps femoris skeletal muscle during processing of three Italian PDO drycured hams. Early post-mortem muscle displayed low levels of actin and myosin fragments. In spite of a high proteolysis index and the presence of active cathepsin D until the final ripening phase, during dry-cured ham processing, very low actin proteolysis and no generation of fragments from \u3b1-skeletal muscle isoform were found, while the identified fragments derived mainly from the cardiac actin isoform. On the other hand, MHC showed a remarkable degradation of its catalytic head, generating a C-terminal 135-kDa fragment. Based on its ability to interact with actin in vitro, this MHC fragment might have a role in stabilisation of actin. In conclusion, these results suggest that maintenance of skeletal muscle \u3b1-actin could reflect limited dismantling of the sarcomeric structure and be a useful marker to monitor the events that result in the typical texture of dry-cured ham

Effects of ozone processing on chemical, structural and functional properties of whey protein isolate

High concentration of gaseous ozone was used to treat whey protein isolate (WPI) powder for different times ranging from 30 to 480. min. The aim of the present work was to study the changes in protein structure and evaluate their consequences on selected functional properties. Surface hydrophobicity, free sulphhydryl groups, turbidity, FTIR, SDS-PAGE, HPLC analysis are performed to evaluate the chemical and structural effects of ozone. The effect of ozonation on the solubility and the foaming properties of proteins are also determined. Results show a reduction of free sulphhydryl groups and an increase of surface hydrophobicity, indicating a self-rearrangement in the protein structure following ozonation. Thus, ozonation allows creation of a more flexible structure without formation of a strong network of disulphide bond or aggregations, which is corroborated by the turbidity analysis and SDS-PAGE. Ozone processing induces modifications that improve the foaming capacity and foam stability, however, a slight reduction in the solubility is encountere

Effect of confinement and starvation on stress parameters in the (Homarus americanus)

The American lobster (Homarus americanus) is one of the most important crustacean resources in North America. In Italy and Europe, this fishery product is available throughout the year and it has a high and increasing commercial demand. American lobsters are traditionally marketed live and stocked, without feed, in temperature controlled recirculating systems for several weeks before being sold in the market places. The current Italian legislation does not fix a maximum length of time for the crustacean confinement and specific welfare requirements. In the present research, a 4-week experiment was carried out using 42 adult H. americanus reared in 4 recirculating aquaculture tanks. After one month of confinement, mean glucose, protein and total haemocyte count levels in the hemolymph of H. americanus were stable and similar (P>0.05) to the values observed at the beginning of the experiment. Results of the proximate analysis of the abdominal muscles of H. americanus showed no significant differences in concentrations of crude protein, lipid and ash during the trial. At the end of the experiment, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis revealed a marked degradation of the muscle myofibrillar proteins. A number of fragments, possibly from myosin, were evident in the range between 50 and 220 kDa between time t0 and t28. Results of this study show that the main hemolymphatic variables and degradation analysis of the muscle myofibrillar proteins can be used as sensitive indicators of the crustacean stress response to confinement and starvation

The effect of slaughtering methods on actin degradation and on muscle quality attributes of farmed European sea bass (Dicentrarchus labrax).

In the current study, two different slaughtering procedures, spiking vs immersion in water/ice slurry, were applied on electrically stunned European sea bass (Dicentrarchus labrax) and the effects on actin degradation and fillet quality attributes were investigated. Rigor mortis index was similar for the two slaughtering techniques, whereas the shear force measurement indicated that rigor mortis occurred more quickly and intensely in the water/ice handled fish than in those slaughtered by spiking. The water/ice immersion procedure also resulted in higher amount of actin fragments than spiking. Muscle tissue apparent viscosity and water holding capacity were lower in sea bass treated in water/ice slurry compared with fish handled by spiking, whereas the acidification patterns of the two groups were not easily distinguishable. In conclusion, spiking appeared to preserve actin integrity better that water/ice and it seemed to ensure less muscle damage as indicated by the higher viscosity and water holding capacity values. Finally, a role of actin as a biochemical marker for the prediction of fish quality was suggested

Development of a risk-tailored approach and dashboard for efficient management and monitoring of investigator-initiated trials

Abstract Background Most randomized controlled trials (RCTs) in the academic setting have limited resources for clinical trial management and monitoring. Inefficient conduct of trials was identified as an important source of waste even in well-designed studies. Thoroughly identifying trial-specific risks to enable focussing of monitoring and management efforts on these critical areas during trial conduct may allow for the timely initiation of corrective action and to improve the efficiency of trial conduct. We developed a risk-tailored approach with an initial risk assessment of an individual trial that informs the compilation of monitoring and management procedures in a trial dashboard. Methods We performed a literature review to identify risk indicators and trial monitoring approaches followed by a contextual analysis involving local, national and international stakeholders. Based on this work we developed a risk-tailored management approach with integrated monitoring for RCTs and including a visualizing trial dashboard. We piloted the approach and refined it in an iterative process based on feedback from stakeholders and performed formal user testing with investigators and staff of two clinical trials. Results The developed risk assessment comprises four domains (patient safety and rights, overall trial management, intervention management, trial data). An accompanying manual provides rationales and detailed instructions for the risk assessment. We programmed two trial dashboards tailored to one medical and one surgical RCT to manage identified trial risks based on daily exports of accumulating trial data. We made the code for a generic dashboard available on GitHub that can be adapted to individual trials. Conclusions The presented trial management approach with integrated monitoring enables user-friendly, continuous checking of critical elements of trial conduct to support trial teams in the academic setting. Further work is needed in order to show effectiveness of the dashboard in terms of safe trial conduct and successful completion of clinical trials

The effect of the pre-slaughter feeding/fasting schedule on post mortem quality traits of rainbow trout

Few studies have investigated the influence of pre-slaughter feeding-fasting history of cultured fish on quality traits of raw fillets. Short pre-slaughter fasting periods (3-9 days) led to slight changes in Salmonid fillet quality, whereas longer starvation (35 days) resulted in improved pre-slaughter resistance to stress and delayed rigor (L\uf2pez-Luna et al., 2014; Bermejo-Poza et al., 2015; M\uf8rk\uf8re et al., 2008). The aim of this study was to evaluate the effect of a 3-week pre-slaughter fasting or feed restriction period on the evolution of post mortem quality traits of fillet of large rainbow trout (O. mykiss). Fish groups, each consisting of 40 specimens (695g) kept at 13.2\ub10.1\ub0C in 2.5 m3 circular tanks, were fed a full ration (FR, 0.66% biomass day-1) or a restricted one (RR, 30% of FR) or were kept starved (R0) over three weeks before slaughtering. Fish were rapidly sacrificed by percussion and five fish per treatment were stored on ice for recording evolution of rigor mortis. Other fish were subjected to biometry and dressing out yield measurements. Fillets were analyzed for proximate analysis and physicochemical traits over 72 h of cold storage (+4\ub0C). Dorsal muscle samples were quickly excised and stored in liquid nitrogen until actin degradation analysis. At slaughter, fish previously fed a full ration gained some weight (+14%) while a weight loss occurred in starved fish (-4.4%) and in those given a restricted ration (-1.6%). Fillet proximate composition was not affected (P>0.05) by the pre-slaughter treatment. A pre-slaughter restricted ration induced higher variability in fish size and certain quality traits such as WHC and texture. It also resulted in faster muscle actin degradation during the first post mortem hours (P<0.05). Despite the weight loss and decreased muscle redness (P<0.05), pre-slaughter starved fish had higher slaughter yield, better muscle firmness after 24 h in cold, extended pre-rigor phase, higher pH and improved WHC values after 7 h, than both full or restricted fed fish (P<0.05). The present study suggest that an extended pre slaughter fasting period could result in improved technological quality traits in rainbow trout