Mitochondrial Analysis of Sparidae Species to Detect a New DNA Barcoding Marker for Dentex gibbosus to Utilize against Fraud.

Dentex gibbosus (Pink dentex) is a fish species of increasing economic interest in the Mediterranean Sea that is consumed both whole and processed. The growing value of this sparid in European markets is responsible for its substitution with fraudulent species. The distinctive morphologic feature of D. gibbosus is the conspicuous hump on the forehead in the older and larger specimens. However, the head is regularly convex in young individuals, requiring high skills and competencies for correct identification. Authentication becomes even more challenging in the case of prepared and processed products. Therefore, the molecular characterization of Pink dentex plays a crucial role in preventing commercial fraud with species substitution. This paper proposes a comparative mitogenome analysis between 19 sparid species of commercial interest as a tool to accurately design species-specific primers targeting a fragment of the NAD2 gene for the identification of D. gibbosus. We successfully detected Pink dentex DNA both using endpoint and real-time PCR. The findings showed the high specificity of the designed primers, demonstrating this a suitable, fast, and cost-effective method that could be used for the unambiguous identification of Pink dentex. This innovative approach for sparid authentication is expected to contribute to seafood traceability, public health assurance, integrity, and the credibility of the seafood industry.

Antimicrobial activity evaluation of pure compounds obtained from Pseudoalteromonas haloplanktis against Listeria monocytogenes: Preliminary results.

L. monocytogenes is a foodborne pathogen responsible for a serious disease with a high mortality rate, particularly in vulnerable consumers. Recently, the scientific community has shown increasing attention to the search for new natural molecules with antimicrobial activity, aimed at preventing the spread of foodborne diseases. Extremophilic microorganisms, typical of extreme temperature environments, are a valuable source of these molecules. The present work aimed to study the antibacterial activity of four pure compounds derived from a molecule, the pentadecanal, produced by the Antarctic bacterium Pseudoalteromonas haloplanktis, against two different pathotypes of L. monocytogenes. Growth assays were performed in 96-well polystyrene plates with serial dilutions of the tested compounds at different concentrations (0.6, 0.3, 0.15, 0.07 mg/mL). The plates were incubated at 37°C for 24 h, with a spectrophotometric reading at OD 600 nm. Preliminary results of this study showed that pentadecanal inhibits the growth of L. monocytogenes, with a MIC (Minimum Inhibitory Concentration) of 0.6 mg/mL. Acetal, carboxylic acid, and ester did not demonstrate antibacterial activity at the concentrations tested. These findings suggest the possibility of using pentadecanal as a natural antibacterial to improve safety standards along the food supply chain.

The complete mitochondrial genome of the zebra seabream Diplodus cervinus (Perciformes, Sparidae) from the Mediterranean Sea.

The complete nucleotide sequence of the mitochondrial (mt) genome of the demersal zebra seabream Diplodus cervinus (Lowe, 1838) was determined for the first time. The double stranded circular molecule is 16,559 base pairs (bp) in length and encodes for the typical 37 metazoan mitochondrial genes, and 2 non-coding regions (D-loop and L-origin). The gene arrangement of the D. cervinus mt genome follows the usual one for fishes. The nucleotide sequences of the mt protein coding and ribosomal genes of D. cervinus mt genome were aligned with orthologous sequences from representatives of the Sparidae family and phylogenetic relationships were inferred. Maximum likelihood analyses placed D. cervinus as a sister species of Diplodus sargus (Linnaeus, 1758).

DNA barcoding and nutritional analysis as a tool for promoting the market of inland fish species.

The increasing world market demand for seafood requires an expansion of product categories available to consumers. Inland fish are usually considered having unmarked taste and are less appreciated by consumers; thus, they have low commercial value. Therefore, the marketing of the lake's fresh and processed fish is limited to the local market and consumers are currently uninformed and mistrustful about these species. In this study, six different fish species were caught in the Fondi lake (Lazio, central Italy): Anguilla anguilla, Tinca tinca, Carassius gibelio, Cyprinus carpio, Micropterus salmoides, Chelon ramada. All the samples were subjected to nutritional and DNA barcoding analysis. Moisture, protein, fat, carbohydrates, ash, and sodium content were measured. As regards the fatty acids profile, the most abundant were MUFAs with the highest value in Anguilla anguilla (45.97%). Oleic acid (C18: 1 n9 cis) was particularly high in Cyprinus carpio (55.46%). The fraction of polyunsaturated fatty acids (PUFA) revealed a higher DHA content (C22: 6 n3) in Anguilla anguilla than the other species (>12 %) while Chelon ramada presented both higher EPA content (C 20: 5 n3) and total fraction of omega 3 PUFAs. Concerning molecular analysis, a 655 bp fragment of cytochrome C oxidase subunit I (COI) gene was successfully used for the identification at the species level using both BOLD and BLAST public databases. The present study gives the basis for improving the knowledge and promoting inland fish' market and traceability along the supply chain.

Effect of beetroot (Beta vulgaris) extract on Black Angus burgers shelf life.

Beef burgers are meat preparations with easy perishability. To ensure a longer shelflife, the Regulation EU 1129/11 allows the use of some additives. However, healthconscious consumers prefer products which do not contain synthetic substances. Aim of the present study was to evaluate the effect of Red Beetroot (Beta vulgaris) integration on Black Angus made burgers shelf life. Red beet was prepared as powder and added to meat mixture as the same or in water solution. The study was split into 2 trials to assess the extract activity also in burgers vacuum-packaged stored. Burgers were analysed (up to 9 days at 4°C) in terms of sensory properties, microbiological profile, pH, aw and lipid oxidation (TBARS). At the end of storage, treated samples showed the highest values of redness and the lowest content of malondialdehyde, probably due to antioxidant properties of red beet towards myoglobin and lipid oxidation processes. Moreover, results highlighted that Red Beetroot activities were dose-dependent and intensified if dissolved in water. The aw values did not appear to be conditioned by extract integrations, unlike the pH that was lower in treated samples than control ones. Microbiological analyses identified beetroot as a potential antimicrobial substance, especially in high concentration. In conclusion, Beta vulgaris extract could be proposed as natural compound exploitable in beef burgers to preserve qualities and extend their shelf-life.

Dentex dentex Frauds: Establishment of a New DNA Barcoding Marker.

The common dentex (Dentex dentex (Linnaeus, 1758)) is an iconic fish in the Mediterranean diet. Due to its commercial and organoleptic importance, this sparid is highly appreciated in European markets and is often subjected to species substitution frauds. Comparative mitogenomics is a suitable approach for identifying new and effective barcode markers. This study aimed to find a molecular tag useful for unequivocally discriminating the sparid species D. dentex. The comparison of the complete mitochondrial DNA (mtDNA) sequences of 16 sparid species allowed us to highlight the potential of the NAD2 gene for direct identification purposes. Common dentex-specific primers were created and successfully evaluated by end-point and real-rime PCR (Polymerase Chain Reaction) for several fish species, achieving amplification only in the D. dentex. The method proposed in this study appears fast, simple, and inexpensive and requires affordable instrumentation. This approach provides unambiguous results for the common dentex authentication without the sequencing step. The presence/absence assay for D. dentex can be executed in a few hours of lab work. Therefore, national authorities responsible for food safety and traceability could apply and make full use of DNA-testing methods for deterring operators from false seafood declarations.

Effects of Different Levels of Inclusion of Apulo-Calabrese Pig Meat on Microbiological, Physicochemical and Rheological Parameters of Salami during Ripening.

This study focused on the characterization of salami produced with meat from different pig breeds. The aim consisted in evaluating the added value of the inclusion of Apulo-Calabrese meat in the production of salami, which was characterized by production until the end of maturation (1, 30, 60, and 120 days). The experimental design involved three types of salami, two of which were produced by partial inclusion of 50 and 75% of the Italian breed pork meat (S50 and S75, respectively). Physicochemical (pH, aw, fatty acid analysis, and malondialdehyde concentration), rheological parameters (texture analyses and color measurement), and bacterial biodiversity were evaluated. Results showed that the partial inclusion of Apulo-Calabrese meat influences the fatty acid profile of final products, which were characterized by a higher percentage of monounsaturated fatty acids compared to traditional salami; however, due to the high content of unsaturated fatty acids, S50 and S75 showed higher values of secondary lipid oxidation up to the 120th day. The linoleic and palmitic acid content significantly affected hardness and brightness. Overall, the ripening process was able to control the microbiological profile and the S50 formulation appeared as a suitable choice that could satisfy consumers for nutritional expectations and sensory profiles.

The complete mitochondrial genome of the white seabream Diplodus sargus (Perciformes: Sparidae) from the Tyrrhenian sea.

The white seabream Diplodus sargus (Linnaeus, 1758) is a species of interest for commercial fisheries throughout its range of distribution and it is also reared using aquaculture techniques. Herein, we present the first complete sequence and annotation of the mitochondrial genome of this species. The D. sargus mitogenome is 16,515 base pairs in length and contains 13 protein-coding genes, 2 rRNA, 22 tRNA, and 2 non-coding regions (D-loop and L-origin). The overall nucleotide composition is: 27.3% A, 28.9% C, 26.8% T, and 17.0% G. Maximum likelihood analyses placed D. sargus as a sister species of Diplodus puntazzo. This study provides valuable information for further studying identification methods and evolutionary relationships of Sparidae species.

Anisakis spp. Larvae in Deboned, in-Oil Fillets Made of Anchovies (Engraulis encrasicolus) and Sardines (Sardina pilchardus) Sold in EU Retailers.

Sardina pilchardus and Engraulis encrasicolus are considered the principal target species for commercial fishing in Europe and are widely consumed as semipreserved products. Although they are considered shelf-stable products, if treatment is not correctly applied, their consumption may represent a public health risk in regard to anisakiasis and allergic reactions. Little is known about the prevalence of Anisakis spp. in ripened products. This study aimed to evaluate the presence of Anisakis spp. larvae in deboned, in-oil anchovy and sardine fillets marketed in the EU to assess the influence of processing techniques on the prevalence of larvae. Ninety semipreserved anchovy and sardine products deriving from the Mediterranean Sea or Atlantic Ocean were collected from different EU retailers and examined using chloropeptic digestion to evaluate the presence of larvae and identify them. Thirty nonviable Anisakid larvae-A. pegreffii (30%) and A. simplex (70%)-were found. The frequency of larvae was higher in anchovies (28.8%). The low frequency of parasites found proved that processing technologies can influence the presence of larvae in final products, but it is important that visual inspection is performed only by trained people. The sources of raw materials should be considered in the production flow chart.

A Rapid Method for the Identification of Fresh and Processed Pagellus erythrinus Species against Frauds.

The commercialization of porgies or seabreams of the family Sparidae has greatly increased in the last decade, and some valuable species have become subject to seafood substitution. DNA regions currently used for fish species identification in fresh and processed products belong to the mitochondrial (mt) genes cytochrome b (Cytb), cytochrome c oxidase I (COI), 16S and 12S. However, these markers amplify for fragments with lower divergence within and between some species, failing to provide informative barcodes. We adopted comparative mitogenomics, through the analysis of complete mtDNA sequences, as a compatible approach toward studying new barcoding markers. The intent is to develop a specific and rapid assay for the identification of the common pandora Pagellus erythrinus, a sparid species frequently subject to fraudulent replacement. The genetic diversity analysis (Hamming distance, p-genetic distance, gene-by-gene sequence variability) between 16 sparid mtDNA genomes highlighted the discriminating potential of a 291 bp NAD2 gene fragment. A pair of species-specific primers were successfully designed and tested by end-point and real-time PCR, achieving amplification only in P. erythrinus among several fish species. The use of the NAD2 barcoding marker provides a rapid presence/absence method for the identification of P. erythrinus.

The complete mitochondrial genome of the sharpsnout seabream Diplodus puntazzo (Perciformes: Sparidae).

The sharpsnout seabream Diplodus puntazzo Walbaum, 1792 is a target species of small-scale fishery activities and is cage-cultured for human consumption. Nonetheless, genetic information on this species is limited. We here first sequence its complete mitochondrial genome. The sequence is composed of 16,638 base pairs, accounting for 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 2 non-coding regions (D-loop and L-origin). The overall nucleotide composition is: 27.4% A, 28.9% C, 26.9% T, and 16.8% G. Maximum likelihood analyses placed D. puntazzo close to Acanthopagrus and some Pagellus species.

The Inhibitory Effect of Plant Extracts on Growth of the Foodborne Pathogen, Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen responsible for about 1600 illnesses each year in the United States (US) and about 2500 confirmed invasive human cases in European Union (EU) countries. Several technologies and antimicrobials are applied to control the presence of L. monocytogenes in food. Among these, the use of natural antimicrobials is preferred by consumers. This is due to their ability to inhibit the growth of foodborne pathogens but not prompt negative safety concerns. Among natural antimicrobials, plant extracts are used to inactivate L. monocytogenes. However, there is a large amount of these types of extracts, and their active compounds remain unexplored. The aim of this study was to evaluate the antibacterial activity against L. monocytogenes of about 800 plant extracts derived from plants native to different countries worldwide. The minimal inhibitory concentrations (MICs) were determined, and scanning electron microscopy (SEM) was used to verify how the plant extracts affected L. monocytogenes at the microscopic level. Results showed that 12 of the plant extracts had inhibitory activity against L. monocytogenes. Future applications of this study could include the use of these plant extracts as new preservatives to reduce the risk of growth of pathogens and contamination in the food industry from L. monocytogenes.

Draft Genomic Sequences of Three Escherichia coli Sequence Type 131 Isolates (H45, H43ii, and H43iii) from Patients in Lagos, Nigeria.

Escherichia coli sequence type 131 (ST131) has recently emerged as a leading multidrug-resistant pathogen that causes urinary tract and bloodstream infections in humans. Here, we report the draft genomic sequences of three E. coli ST131 isolates, H45, H43ii, and H43iii, from urine samples of patients in Lagos, Nigeria.

Comparison of mitochondrial DNA enrichment and sequencing methods from fish tissue.

Sparid fish species have different commercial values related to their organoleptic features. Mitochondrial (mt) DNA provides a potential tool to distinguish species, but the enrichment of high-quality mtDNA from total genomic DNA is critical to obtain entire mtDNA sequences. Conventional mtDNA isolation is relatively low-cost and proficient. However, high numbers of PCR cycles can lead to artefacts (10-6 mutations/bp). We describe a rapid protocol for mtDNA extraction and enrichment from fish tissues, based on conventional miniprep columns and paramagnetic bead-based purification, without the need to employ PCR amplification. This newly described method generates a substrate for next-generation sequencing (NGS) analysis and is likely to have wider applications for mitochondrial studies in other fish families to help ensure traceability and differentiation of fish with high commercial values.

The complete mitochondrial genome of the common dentex, Dentex dentex (perciformes: Sparidae).

The common Dentex (Dentex dentex, Linnaeus 1758) has a significant economic importance and is a highly valued table fish in the Mediterranean region. The paucity of genetic information relating to sparids, despite their growing economic value, provides the impetus for exploring the mitogenomics of this fish group. Here, we sequenced D. dentex complete mitochondrial genome. The sequence is comprised of 16,652 bp and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a 2 non-coding regions (D-loop and L-origin). The overall nucleotide composition is: 27.5% of A, 28.7% of C, 26.9% of T, and 16.9% of G.

The complete mitochondrial genome of the axillary seabream, Pagellus acarne (Perciformes: Sparidae).

The axillary seabream (Pagellus acarne, Risso 1827) belongs to the Sparidae family, order Perciformes. This high-valued commercial fish species is distributed along the northern and eastern Atlantic coasts from Norway to Senegal, and throughout the Mediterranean Sea. Its complete mitochondrial genome is 16,486 bp in length, consisting of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions (D-loop, 808 bp and L-origin, 29 bp). Its overall base composition is A: 26,8%, C: 29,0%, G: 17.6%, and T: 26.6%.

The complete mitochondrial genome of the Pink dentex Dentex gibbosus (Perciformes: Sparidae).

The Pink dentex (Dentex gibbosus, Rafinesque 1810) is one of the most commercially important Sparidae species and it is often subjected to fraud. Here, we report the complete mitochondrial genome of D. gibbosus. The mitogenome is 16,771 bp in length and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and 2 non-coding regions. The overall base composition of D. gibbosus mtDNA is: 27.8% for A, 28.60% for C, 16.5% for G, 27.05% for T.

The complete mitochondrial genome of the common pandora Pagellus erythrinus (Perciformes: Sparidae).

The common pandora (Pagellus erythrinus, Linnaeus 1758), one of the most popular sea bream species in the Mediterranean Sea, has high potential for aquaculture development. In this investigation, we analyzed the complete mitochondrial genome of P. erythrinus. The sequence has 16,828 bp in length and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a two non-coding regions (D-loop and L-origin). The overall nucleotide composition is: 27.5% of A, 28.2% of C, 27.5% of T, and 16.8% of G.

Development of a Method to Extract and Amplify the Complete Mitogenome of Some Sparidae Species.

Previous studies showed that fish mitochondrial DNA (mtDNA) is set up by a closed circular molecule of 16-17 kilobases (kb), comprising 2 ribosomal RNA genes (rRNA), 22 transfer RNA genes (tRNA), 13 protein-coding genes and 2 non-coding regions. The analysis of single mtDNA genes, such as Cytb, COI, 16S and 12S, or short segment of them, has been widely used against species substitution in both fresh and processed fish products. The analysis of the complete mitochondrial genome of fishery products allows to better study and characterise fish species. The aim of this research was to extract and amplify the complete mtDNA of some fish species of commercial interest belonging to the Sparidae family. The studied species were Dentex dentex, Dentex gibbosus, Dentex nufar, Pagellus acarne and Pagellus erythrinus. The entire mitogenome was obtained by gene amplification using long polymerase chain reactions. The analysis of the complete mitochondrial sequences will allow to gain further insights on these species and to find polymorphic sites that assess the degree of genetic variability of the species belonging to the family Sparidae.

Evaluation and Comparison of Four Protein Extraction Protocols for Mono- and Two-Dimensional Electrophoresis in Mytilus Galloprovincialis.

In this study, four protein extraction protocols from Mytilus galloprovincialis were evaluated with the aim to identify the most practical, efficient and reproducible method. Four extraction protocols frequently used for mussels and organic matrices were selected and compared. The methods were based on the use of: i) TRIzol reagent; ii) Lysis buffer; iii) phenylmethanesulfonyl fluoride; iv) trichloroacetic acid-acetone. Protein concentration was measured by the Bradford method. Three specimens of mussels were studied and the analysis was conducted in triplicate for each of the four protocols. Results indicated that the four methods could extract significantly different protein profiles. The highest number of protein spots resolved in 2DE gels and the best reproducibility was obtained using trichloroacetic acid-acetone protocol. Results afforded the selection of a suitable extraction protocol to be used for ecotoxicoproteomics studies from mussels and for other proteomic studies conducted by particularly complex tissues such as Mytilus galloprovincialis.