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The discarding of agri-food by-products is a stringent problem due to their high environmental impact. Recovery strategies can lead to a reduction of waste and result in new applications. Agri-food waste represents a source of bioactive molecules, which could promote health benefits. The primary goal of this research has been the assessment of the antioxidant activity of milk permeate, a dairy farm by-product, and the isolation and identification of peptide fractions endowed with antioxidant activity. The chromatographic extraction of the peptide fractions was carried out, and the peptides were identified by mass spectrometry. The fractions showed radical scavenging activity in vitro. Moreover, the results in the Caco-2 cell model demonstrated that the peptide fractions were able to protect from oxidative stress by stimulating the Keap1/Nrf2 antioxidant signaling pathway, increasing the transcription of antioxidant enzymes. In addition, the bioactive peptides can affect cellular metabolism, increasing mitochondrial respiration. The action of the peptide fractions was also assessed in vivo on a zebrafish model and resulted in the protection of the whole organism from the adverse effects of acute cold stress, highlighting their strong capability to protect from an oxidative insult. Altogether, the results unveil novel recovery strategies for food by-products as sources of antioxidant bioactive peptides that might be utilized for the development of functional foods.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the loss of motor neurons in the brain and spinal cord. While the exact causes of ALS are still unclear, the discovery that familial cases of ALS are related to mutations in the Cu/Zn superoxide dismutase (SOD1), a key antioxidant enzyme protecting cells from the deleterious effects of superoxide radicals, suggested that alterations in SOD1 functionality and/or aberrant SOD1 aggregation strongly contribute to ALS pathogenesis. A new scenario was opened in which, thanks to the generation of SOD1 related models, different mechanisms crucial for ALS progression were identified. These include excitotoxicity, oxidative stress, mitochondrial dysfunctions, and non-cell autonomous toxicity, also implicating altered Ca 2+ metabolism. While most of the literature considers motor neurons as primary target of SOD1-mediated effects, here we mainly discuss the effects of SOD1 mutations in non-neuronal cells, such as glial and skeletal muscle cells, in ALS. Attention is given to the altered redox balance and Ca 2+ homeostasis, two processes that are strictly related with each other. We also provide original data obtained in primary myocytes derived from hSOD1(G93A) transgenic mice, showing perturbed expression of Ca 2+ transporters that may be responsible for altered mitochondrial Ca 2+ fluxes. ALS-related SOD1 mutants are also responsible for early alterations of fundamental biological processes in skeletal myocytes that may impinge on skeletal muscle functions and the cross-talk between muscle cells and motor neurons during disease progression.

Bioactive peptides are a group of molecules with health beneficial properties, deriving from food matrices. They are protein fragments consisting of 2-20 amino acids that can be released by microbial fermentation, food processing and gastrointestinal digestion. Once hydrolyzed from their native proteins, they can have different functions including antioxidant activity, which is important for cell protection by oxidant agents. In this work, fermented soy products were digested in vitro in order to improve the release of bioactive peptides. These were extracted, purified and analyzed in vitro and in a cellular model to assess their antioxidant activity. Peptide sequences were identified by LC-MS/MS analysis and a molecular docking approach was used to predict their ability to interact with Keap1, one of the key proteins of the Keap1/Nrf2 pathway, the major system involved in redox regulation. Peptides showing a high score of interaction were selected and tested for their antioxidant properties in a cellular environment using the Caco-2 cell line and examined for their capability to defend cells against oxidative stress. Our results indicate that several of the selected peptides were indeed able to activate the Keap1/Nrf2 pathway with the consequent overexpression of antioxidant and phase II enzymes.

Due to their beneficial properties, fermented foods are considered important constituents of the human diet. They also contain bioactive peptides, health-promoting compounds studied for a wide range of effects. In this work, several antioxidant peptides extracted from fermented milk proteins were investigated. First, enriched peptide fractions were purified and analysed for their antioxidant capacity in vitro and in a cellular model. Subsequently, from the most active fractions, 23 peptides were identified by mass spectrometry MS/MS), synthesized and tested. Peptides N-15-M , E-11-F , Q-14-R and A-17-E were selected for their antioxidant effects on Caco-2 cells both in the protection against oxidative stress and inhibition of ROS production. To define their action mechanism, the activation of the Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2(Keap1/Nrf2) pathway was studied evaluating the translocation of Nrf2 from cytosol to nucleus. In cells treated with N-15-M , Q-14-R and A-17-E, a higher amount of Nrf2 was found in the nucleus with respect to the control. In addition, the three active peptides, through the activation of Keap1/Nrf2 pathway, led to overexpression and increased activity of antioxidant enzymes. Molecular docking analysis confirmed the potential ability of N-15-M , Q-14-R and A-17-E to bind Keap1, showing their destabilizing effect on Keap1/Nrf2 interaction., Competing Interests: The authors declare no conflict of interest.