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Projeto de investigação
OptImization of novel value CHains for fish and seafood by developing an integraTed sustainable approacH for improved qualitY, safety and waSte reduction
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Proteomics of fish white muscle and Western blotting to detect putative allergens
Publication . Anjos, Liliana; Loukissas, A. Ζ.; Power, Deborah
A detailed workflow is provided for preparation from teleost fish white muscle of extracts for proteomics analysis. The protocol generates samples that can be analyzed by SWATH (Sequential Window data independent Acquisition of the Total High-resolution-Mass Spectra), a modern MS-based quantitative label free technology. The main steps for the extraction of three independent protein fractions, (1) soluble sarcoplasmic, (2) soluble myofibrillar, and (3) insoluble material, from fish white muscle are detailed. Coupled to the protein extraction protocol a Western blotting approach is outlined for detection of common fish allergens, in this case β-parvalbumin, in the white muscle sarcoplasmic protein fraction.
Proteome dataset of sea bass (Dicentrarchus labrax) skin-scales exposed to fluoxetine and estradiol
Publication . L, Anjos; PI Pinto, PPinto; Santos, Soraia; Estêvão, M. Dulce; Santa, Cátia; Manadas, Bruno; Canario, A.V.M.; Power, Deborah
Contamination of aquatic ecosystems with anthropogenic pollutants, including pharmaceutical drugs, is a major concern worldwide. Aquatic organisms such as fish are particularly at risk of exposure to pollutants. The surface of fish is the first point of contact with pollutants, but few studies have considered the impact of pollutants on the skin-scale barrier. The present proteome data are the basis of the findings discussed in the associated research article "Proteomics of sea bass skin-scales exposed to the emerging pollutant fluoxetine compared to estradiol" [1]. Juvenile sea bass were exposed by intraperitoneal injections to: a) the antidepressant fluoxetine (FLX), a widely prescribed psychotropic drug and an emerging pollutant; b) the natural estrogen 17 beta-estradiol (E2) and c) the vehicle, coconut oil (control). The scale proteome of fish exposed to these compounds for 5 days was analysed using quantitative label-free proteomics technology SWATH-MS (sequential windowed data-independent a cquisition of the total high-resolution-mass spectra). The proteome data generated was submitted to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD020983. LC-MS data from pooled protein extracts from the scales of all experimental groups was acquired using information-dependent acquisition (IDA) and 1,254 proteins were identified by searching against the sea bass genome database. 715 proteins were quantified by SWATH acquisition, and 213 proteins had modified levels (p < 0.05) between the E2- or FLX-exposed fish compared to the control. The main biological processes and KEGG pathways affected by E2 or FLX treatments were identified using Cytoscape/ClueGO enrichment analyses. (c) 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
Quantitative PCR assays as a monitoring tool for bacterial genera in fresh fish fillets
Publication . Pinto, Patricia IS; NAJAFPOUR, BABAK; Lima, Pedro; P. Machado; Aires, Tania; Engelen, Aschwin; Tsironi, T.; Anjos Guerreiro, Liliana Isabel Tomé; Power, Deborah Mary
Fresh fish fillets are a valuable but highly perishable food, and their rapid microbial deterioration is a drawback for food safety and sustainability of aquaculture, food and retail industries. Quantitative PCR (qPCR) assays based on 16S rRNA gene (16S) sequences were developed for the most abundant bacteria genera detected by metagenomics in fresh or processed fish fillets. The efficiency and specificity of six qPCR assays (for 16S of all bacteria or genera Shewanella, Pseudomonas, Carnobacterium, Janthinobacterium and Massilia) was verified using in silico predictions, cloning, sequencing and phylogenetic analyses of amplicons obtained from refrigerated control or high-pressure processed (HPP) European sea bass (Dicentrarchus labrax) fillets. In HPP sea bass fillets, significant decreases in total bacteria 16S and of Shewanella, Pseudomonas, Carnobacterium and Janthinobacterium 16S compared to control fillets were confirmed by qPCR, after 11 days of refrigerated storage. The qPCR assays were successfully applied to monitor microbial contamination during refrigerated storage of fresh fillets from commercial (retail) sea bass and gilthead sea bream (Sparus aurata). Significant increases in total bacterial and Shewanella, Pseudomonas, Carnobacterium and Janthinobacterium contamination were detected after 7-14 days. 16S copy number for total bacteria and the four target genera positively correlated with total viable counts using culture enumeration. 16S of Massilia, that is abundant in fresh fish fillets, did not significantly change during storage. The six validated qPCR assays developed are proposed as specific, sensitive, culture-independent methods for monitoring quality or processing outcomes for fish fillets during cold chain storage.
Valorisation of gilthead seabream by-products through recovery of antimicrobial proteins for active biopolymer formulations
Publication . Maurizzi, Enrico; Anjos Guerreiro, Liliana Isabel Tomé; Bigi, Francesco; Quartieri, Andrea; Mateus, Ana Patrícia; Volpelli, Luisa Antonella; Pulvirenti, Andrea; Power, Deborah Mary
In the seafish sector, industrial processing and by-catch currently lead to the waste of over 36 % of global fish production by weight. This is largely due to insufficient revalorization of by-products and the underdevelopment of sustainable practices to manage these discarded volumes, which are often disposed of or released into the environment, contributing to pollution. In this study, antimicrobial proteins were extracted from fish by-products for incorporation into biopolymer formulations. Specifically, the focus was on lysozyme, which was targeted using a molecular-proteomic approach. Protein extractions were conducted at various pH levels from Gilthead seabream (Sparus aurata) tissues (skin with scales and mucus, liver, and intestine) normally discarded during processing, to assess recovery of proteins from the selected tissues. The extracted proteins were separated using mild ion-exchange chromatography, followed by quantification and qualitative analysis via SDS-PAGE. The expression levels of lysozyme types-g and-c were quantified through Real-Time qPCR. The antimicrobial activity of the extracted proteins was assessed against Gram-positive (Listeria monocytogenes) and Gram-negative (Escherichia coli) bacteria using a Minimal Inhibitory Concentration (MIC) assay. The proteins were subsequently incorporated into biodegradable film-forming solutions based on hydroxypropyl methylcellulose/chitosan and hydroxypropyl methylcellulose/guar gum mixtures. These films were further tested against the same human pathogens. The results demonstrate the feasibility of extracting proteins from fish by-products using a non-targeted buffer pH extraction approach, which, even without further chromatographic purification, exhibited promising intrinsic antimicrobial activity for potential applications in the food industry.
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Entidade financiadora
European Commission
Programa de financiamento
H2020
Número da atribuição
872217