Valutazione Progetti SIR

Tenderness is one of the more appreciated characteristics of beef by consumers, which are willing to pay more for more tender beef. Among the factors that influence beef tenderness, proteolysis seems to be the most important one during aging. After animal bleeding, the state of hypoxia and ischemia lead to cell death. The onset of cell death, that corresponds to the first step of the conversion of muscle into beef, seems to have an apoptotic nature and to be associated to the caspase proteolytic system activation. Mitochondria are responsible of the aerobic production of cellular energy and they have been proposed as main regulators of apoptosis. Therefore their energetic dysfunction seems decisive in the subsequent muscle cell responses influencing the proteolysis and thus, the final beef tenderness. Aims of this project are to study the mitochondria post mortem fate and their role in triggering the apoptotic process, and to verify whether specific proteins, whose expression can be influenced or associated with mitochondria dysfunction during meat aging, can be considered as proteomic predictors of beef tenderness. Given the transdisciplinary nature of this project, methods ranging from human to animal science will be applied. Food scientists, biologist, biochemistry researcher and animal physiologist will be involved, allowing to cover all the multidisciplinary aspects of the project. Two experiments will be performed. In the first one, samples of Longissimus lumborum m (LL) and Sternomandibularis m (SM), which differ for fiber-type composition and tenderness, from 6 Italian Simmental (IS) young bulls will be excised immediately after exsanguination and at different times of aging within 48 h of slaughter. Mitochondria bioenergetic function will be assessed by measurements of oxygen consumption, membrane potential, activities of the oxidative phosphorylation complexes and cellular ATP content. To evaluate their involvement in apoptosis, the release of pro-apoptotic factors from mitochondria will be quantified and related to the caspase 3 and 9 activation and the level of DNA fragmentation. The occurrence of mitochondrial fission and autophagy will be assessed by quantification of specific markers and related to the degree of oxidative stress, evaluated by the mitochondrial protein carbonyl content. The expression levels of the mitochondrial pro-apoptotic factors and of those proteins influenced by mitochondria dysfunction during aging will be assessed by RT-PCR, RNA-seq and immunoblotting and related to the shear force (WBSF) measured after 48 h and 7 days of ageing, considering also the collagen content of beef. In the second experiment, the 6 young bulls showing extreme WBSF values among 20 animals (IS) will be considered. On these animals, the mRNA-seq will be carried out. The expression levels of the proteins considered in Experiment 1 and/or those identified by RNA-seq as differently expressed in LL and SM will be quantified and related to beef tenderness taking into account also the collagen content. Considering that the mechanisms of proteolysis during aging are not completely clear, and studies regarding the post mortem fate of mitochondria and their role in apoptosis and in tenderization process of beef are still lacking, the results of this project will provide a new approach for beef tenderness research, and will advance our knowledge of the tenderization mechanisms triggered by mitochondrial dysfunction associated to post mortem hypoxic/ischemic conditions. These new knowledge could allow to improve current industrial techniques focused on beef tenderness, such as electric stimulation of carcasses or addition of Calcium chloride to muscle that may act on mitochondria, or to propose new industrial technologies. Furthermore, the expression level of the proteins studied could provide new markers for the evaluation and prediction of beef tenderness.