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

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

ANALISI COMPARATIVA DEL MITOGENOMA COMPLETO DI ALCUNE SPECIE DI SPARIDI E VALUTAZIONE DI UN NUOVO MARKER MOLECOLARE PER PAGELLUS ERYTHRINUS

Il Pagello fragolino (Pagellus erythrinus) è una delle specie di Sparidi più pescate e commercializzate nel Mar Mediterraneo e nell’Oceano Atlantico. Tale specie è molto apprezzata nei mercati Europei ed è spesso soggetta a frodi per sostituzione di specie. Le sostituzioni avvengono soprattutto mediante utilizzo di altri Sparidi con differente pregio commerciale. Le sequenze di DNA mitocondriale (mt) maggiormente utilizzate per l'identificazione di specie ittiche sono il citocromo b-CYTB, la citocromo c ossidasi I-COI, i geni che codificano per le subunità ribosomiali 16S e 12S. Ricerche recenti hanno dimostrato che lo studio dell'intero mtDNA consente di identificare nuovi marcatori molecolari specie-specifici e quindi più efficaci. In particolare, il gene NAD5 ha un'elevata capacità discriminatoria per le specie appartenenti alla famiglia Sparidae. Tuttavia, l'utilizzo di tutti questi markers molecolari richiede diversi steps analitici, come l'amplificazione del frammento target ed il sequenziamento. Quest’ultima fase rappresenta un momento critico dell’identificazione di specie, poiché richiede tempi lunghi. Inoltre, tale analisi deve essere effettuata in laboratori altamente specializzati, poiché la lettura errata di pochi nucleotidi può falsare la corretta identificazione della specie. Scopo di questo studio è stato quello di analizzare e confrontare l'intera sequenza di mtDNA di alcune specie di Sparidi al fine di identificare un marcatore molecolare utile per riconoscere la specie P. erythrinus, evitando la fase di sequenziamento. In questo studio sono stati confrontati i mitogenomi di n°13 Sparidi. L’allineamento degli mtDNA è stato condotto mediante l’utilizzo del software UGENE. L'algoritmo Hamming Distance è stato utilizzato per valutare la percentuale di diseguaglianza genetica tra le specie ed i geni. La p-genetic distance è stata valutata utilizzando il Maximum Composite Likelihood model. La variabilità della sequenza nucleotidica è stata determinata allineando le sequenze gene-by-gene utilizzando MEGA 6.0. I primers sono stati disegnati a mano in seguito ad allineamento multiplo delle sequenze complete di mtDNA mediante il software BioEdit Sequence Alignment Editor. L'efficienza dei primers per l'identificazione di P. erythrinus è stata testata mediante PCR end-point. Sono stati testati n°10 campioni di P. erythrinus con differente provenienza geografica e n°30 campioni di filetti appartenenti ad altre specie ittiche. I risultati della Hamming Distance analysis, della valutazione della p-genetic distance e dell’analisi di variabilità della sequenza nucleotidica hanno consentito di identificare il gene NAD2 come potenziale marcatore molecolare per gli Sparidi. La reazione PCR ha confermato la capacità di discriminazione del gene NAD2. In particolare, l'amplificazione di un frammento selezionato del gene NAD2 è stata possibile solo per la specie P. erythrinus. Tale risultato può consentire di verificare la specie senza necessità di sequenziamento, riducendo tempi e costi delle analisi ed aumentando l’efficienza dei risultati. La valutazione di presenza/assenza della specie P. erythrinus può essere ottenuta in poche ore di lavoro di laboratorio. Ulteriori indagini sono in corso al fine di identificare primers specie-specifici anche per altre specie appartenenti alla famiglia degli Sparidi. In accordo con il Regolamento (UE) 1379/2013, questo studio contribuisce alla tracciabilità molecolare dei prodotti della pesca

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

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

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

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

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)