Sviluppo di metodiche basate sulla caratterizzazione molecolare per l’identificazione di frodi nel comparto ittico

L’aumento dell’attività di pesca a livello mondiale e il depauperamento delle risorse nazionali in risposta all’incremento della domanda di mercato dei prodotti ittici hanno determinato il progressivo aumento delle importazioni, sia da Paesi Comunitari che da Paesi Terzi, in particolar modo dalla Cina. Nel settore ittico, la Cina produce, allo stato attuale, oltre il 38% del fatturato mondiale, grazie soprattutto alla promozione di strategie commerciali estremamente concorrenziali. Tali livelli produttivi non sono supportati, però, dalla messa in atto di procedure efficienti, ai fini di una corretta tracciabilità dei prodotti alimentari lungo la filiera produttiva, con notevoli ripercussioni a livello commerciale e sanitario. Inoltre, in risposta alla crescente domanda da parte del consumatore di alimenti pronti al consumo e di facile utilizzo, negli ultimi anni si è assistito ad un aumento della commercializzazione di prodotti ittici trasformati e precotti. Il riconoscimento di specie assume un’importanza fondamentale per la prevenzione di fenomeni di falsificazione e contraffazione a tutela e protezione del consumatore. Considerando che la tecnica di identificazione di specie su base morfologica, unica riconosciuta in Italia in sede legale, risulta inapplicabile in tali tipologie di prodotto, l’attenzione è stata rivolta all’individuazione di strumenti analitici alternativi. Le metodiche analitiche basate sull’analisi del DNA finalizzate all’identificazione di specie sono quelle maggiormente utilizzate in virtù dell’elevato grado di resistenza dell’acido nucleico a trattamenti fisico-chimici e a fenomeni di degradazione, offrendo un valido strumento per il controllo della tracciabilità nelle diverse tipologie di prodotto (fresco, trasformato, conservato). In quest’ottica, i progetti di studio sviluppati sono stati rivolti sia al miglioramento delle tecniche per l’estrazione del DNA in matrici fresche o conservate, in grado di garantire soddisfacenti caratteristiche quali-quantitative dell’acido nucleico ottenuto ed un minore impegno dell’operatore, sia allo sviluppo di strumenti analitici basati sull’utilizzo della PCR per la discriminazione di specie diretta o post sequenziamento del target selezionato. Sono state sviluppate con successo metodiche per la discriminazione diretta tra specie di provenienza cinese e specie mediterranee o atlantiche di maggior valore commerciale e realizzati studi di caratterizzazione di prodotti ittici reperibili in esercizi commerciali etnici e di mangimi per animali domestici, ai fini del controllo di conformità su quanto dichiarato in etichetta. Infine, le metodiche sviluppate sono state utilizzate per effettuare controlli su prodotti commerciali acquistati presso la piccola e grande distribuzione, evidenziando come il fenomeno di sostituzione di specie, volontaria o involontaria, rappresenti una problematica reale, la cui entità suggerisce la necessità di potenziare le attività di controllo da parte degli organi ufficiali e ulteriori studi. In questo contesto, un adeguato livello di tutela del consumatore non può prescindere dallo sviluppo di metodiche analitiche sempre più rapide e performanti. The global increase of fishing activities, together with the depletion of national marine resources, in response to the rising demand for seafood, led to a progressive growth in imports, both from European Union countries and Third countries, particularly from China. In the fishery sector, China produces, at present, over 38% of the world output, thanks to the promotion of very competitive commercial strategies. These production rates are not supported, however, by the implementation of efficient systems of traceability of food products along the supply chain, with significant repercussions on trade and health issues. In addition, in recent years the commercialization of processed and precooked fish products has increased, in response to the growing demand for ready to cook and ready to eat food products. Thus, species identification acquires fundamental importance in the prevention of phenomena of counterfeiting and adulteration for consumer protection. Considering that the morphological identification of seafood species, the only official method approved in Italy, is inapplicable in these types of products, alternative analytical tools are needed. DNA-based analytical methods are the most applied for species identification due to the high resistance of nucleic acid to physical-chemical treatments and degradation, representing a valuable tool for traceability in different types of products (fresh, processed, canned). In this perspective, the research activities were aimed: I) to the improvement of DNA extraction techniques in fresh or processed matrices , in order to ensure satisfactory qualitative and quantitative characteristics of the obtained nucleic acid and simplify the laboratory workflow, II) to the development of analytical tools based on PCR for direct or post sequencing species discrimination using selected targets. Methods for direct discrimination between Chinese fish species and Mediterranean or Atlantic species of higher commercial value have been successfully developed, as well as studies on the identification of fish products sold in ethnic shops and of pet food, with the aim of evaluating the label correctness. Finally, the developed methods have been used to carry out analysis on commercial products directly purchased in small and large retailers, highlighting that the phenomenon of species substitution, whether deliberate or accidental, represents a real problem, suggesting the need for the implementation of further Official control activities and research studies. In this context, the development of rapid and performing analytical tools is essential to ensure an adequate level of consumer protectio

Fish species identification in canned pet food by BLAST and Forensically Informative Nucleotide Sequencing (FINS) analysis of short fragments of the mitochondrial 16s ribosomal RNA gene (16S rRNA)

Nowadays, pet food claiming high-valued fish among ingredients is largely available on the market. Unfortunately, the modifications induced by processing make species identification by visual inspection difficult and hinder the enforcement of the legislation on traceability. In this work, after aligning 819 sequences of Clupeidae, Engraulidae, Salangidae and Scombridae families, we developed new universal primers for the amplification and sequencing of 2 short fragments (±118 and ±213) of the mitochondrial 16s ribosomal RNA (16S rRNA) gene. Once tested on 130 DNA reference samples, these primers were used in the analysis of highly degraded DNA extracted from 43 canned cat food containing whole minnows (whitebait) (M) and tuna, or bonito or mackerel fillets (F). Three M and 2 F samples were analyzed for each can. A BLAST and a FINS analysis, the latter performed only on the 118 bp fragment, were performed separately on the sequences obtained from M and F samples. All the M samples were identified at the species or genus level by both BLAST and FINS analysis. This allowed to highlight an impressive rate of mislabeling (100%). F samples, for which FINS was less performing in species identification, resulted mislabeled in 40% of the products

Multiple DNA BARCODING for fish species identification in sushi products

The aim of this work was to perform a molecular survey based on DNA barcoding to identify the seafood species used in the preparation of ethnic products (sushi). Twenty-one raw products (each composed of 3 to 8 pieces, for a total of 88 samples) were purchased in ethnic restaurants in the provinces of Pisa (11), Lucca (2), Livorno (3) and Florence (5). The total DNA extracted (1) was evaluated by gel electrophoresis and amplified using universal primers for mitochondrial (COI, 16SrRNA) or nuclear genes (PEPCK) depending on the species (fish, mollusk or crustacean) and the level of DNA degradation. Different primers (2,3,4,5,6,7) for the amplification of a long (~700 bp) or a short (~139-200 bp) fragment were used. Ninety-five PCR products were obtained (for some products two genes were analyzed). Of these, 30 have already been sequenced (Experimental Zooprophylactic Institute of Latium and Tuscany (Rome)). The sequences were elaborated with Clustal W in Bioedit 7.0.9.0, and analyzed by a BLAST analysis on GenBank and by using the Identification System on BOLD. A top match with a sequence similarity of at least 98% was used to designate potential species identification (8). DNA was degraded in almost one third of the samples. This was probably due to rice acidification, to repeated cycles of freezing/thawing or to prolonged storage. The degradation was confirmed by PCR amplification. In fact, we obtained long amplicons in 72.6% of the cases (n=69) and short amplicons for 27.3% of the samples (n=26). The average length of the long sequences was 595 bp for the COI FDB and 490 bp for the PEPCK gene, while the length of the short sequences was ~210bp for the 16S rRNA and 139bp for the COI MDB. All the samples were identified at least at the genus level, with identity values ranging from 99 to 100%. Although for some samples it was impossible to achieve a specific identification, the results were informative enough to verify the information given by the producers. No samples were found mislabeled. Even though the COI gene represents the most exploited target for seafood species identification, issues were found during amplification and comparison with the databases. Thus, in order to increase the PCR output, new universal primers, able to amplify a wide range of taxa, would be desirable. Finally, in case of degraded DNA samples, where the number of diagnostic mutation is limited, a multiple gene analysis is advisable

Implementation of a pilot checklist for control standardization on fishery goods consignments at the Livorno border control post

To standardize control activities, it is necessary to introduce checklists to support the control of consignments entering the European Union through border control posts (BCPs). This study aimed to develop a pilot checklist for the control of fishery consignments, preliminarily identified as the predominant group of goods entering the Livorno (Italy) BCP. The design of the pilot checklist was preceded by i) a revision of the current European and national legislation on the general and specific objectives of border control activities on fishery products and ii) a comparative analysis of two checklists (one of the Ministry of Health and one of the former Livorno border inspection post) developed on the basis of the repealed legislation. This comparison aimed to define the pilot checklist structure, verification objectives, and selection of assessment scores to be included in defining consignment compliance and acceptability. Once developed, the clarity and ease of use of the first draft of the pilot checklist were verified through its use in a field test during the control of 64 fishery product consignments. 22 regulatory sources (18 European and 4 national) were selected as reference legislation. The pilot checklist was structured as a dynamic “read-do” document based on the workflow of control activities described in the current legislation. The field test was useful in improving the clarity of the verification objectives within the documentary, identity, and physical control sections and in facilitating the use of the checklist and the collection of evidence during the control activity. This study, which focused on fishery products, can provide a practical approach for the development of checklists for all the other categories of goods under the responsibility of BCPs

Pentaplex PCR as screening assay for jellyfish species identification in food product

Salted jellyfish, a traditional food in Asian Countries, is nowadays spreading on the Western markets. In this work, we developed a Pentaplex PCR for the identification of five edible species (Nemopilema nomurai, Rhopilema esculentum, Rhizostoma pulmo, Pelagia noctiluca, and Cotylorhiza tuberculata), which cannot be identified by a mere visual inspection in jellyfish products sold as food. A common degenerated forward primer and five specie-specific reverse primers were designed to amplify COI gene regions of different lengths. Another primer pair targeted the 28SrRNA gene and was intended as common positive reaction control. Considering the high level of degradation in the DNA extracted from acidified and salted products, the maximum length of the amplicons was set at 200 bp. The PCR was developed using 66 reference DNA samples. It gave successful amplifications in 85.4% of 48 ready to eat products (REs) and in 60% of 30 classical salted products (CPs) collected on the market

DNA barcoding reveals substitution of Sablefish (Anoplopoma fimbria) with Patagonian and Antarctic Toothfish (Dissostichus eleginoides and D. mawsoni) in online market in China: How mislabeling opens door to IUU fishing

China's rapid economic development has determined profound changes in seafood consumption patterns, and nowadays besides the traditional luxury seafood, high-quality marine fish are consumed. Among these is Anoplopoma fimbria (Sablefish), a highly priced species on the Chinese market. A recent molecular survey on products sold online in China found that all the analyzed products sold as Yin Xue, used to indicate A. fimbria, were instead Dissostichus spp., a genus of fish extremely vulnerable to overfishing (Xiong et al., 2016). Considering this and the lack of a standardized naming system for seafood species in China, an initial search was conducted to identify all the possible Chinese names indicating A. fimbria. The aim of the present study was to assess the challenges of the online market with regards to frauds for fish species substitution. DNA barcoding was employed to verify the identity of 42 products sold on e-commerce platforms as Sablefish. Moreover, the information reported on the webpage and on the label was analyzed according to the Chinese regulation in force. All the PCR products gave readable sequences. By using the IDs analysis on BOLD and the BLAST analysis on GenBank all the samples were unambiguously identified at the species level. Of the 42 products sold as Sablefish, only 6 (14.3%) were molecularly identified as this species, while 32 (76.2%) were identified as Dissostichus eleginoides (Patagonian Toothfish) and 4 (9.5%) as D. mawsoni (Antarctic Toothfish), highlighting an alarming overall misrepresentation rate of 85.7% and implications for the management of these species' fisheries. The combined analysis of all the information of the webpages and the labels allowed us to hypothesize unintentional and intentional mislabeling. Our findings suggest the possible existence of a trade pattern enabling IUU fishing operators to launder illegal catches of Toothfish through mislabeling

Assessment of a Sampling Plan Based on Visual Inspection for the Detection of Anisakid Larvae in Fresh Anchovies (Engraulis encrasicolus). A First Step Towards Official Validation?

The presence of anisakid larvae in fish is a public health issue, and effective risk management procedures are needed to avoid that heavily infected products reach the market. Currently, an official sampling plan for fresh fish defining sample size, inspection methods, and criteria to accept or reject the merchandise is lacking at the European and Italian level. In this study, we compared the visual inspection proposed by the sampling plan of the Lombardy Region (Italy) to the UV press method and to an optimized digestion procedure with the aim to assess its ability in detecting visible parasites. Thirty-one batches of Engraulis encrasicolus, each composed of ∼30 specimens, were collected and subsequently analyzed with the three techniques. The mean abundance (MA) was calculated after each procedure and compared on the basis of a threshold value. The results showed that the visual inspection performed similarly to the digestion method, with a sensitivity of 93 %, a specificity of 100 %, and an accuracy of 97 %. Overall, the comparison showed that, in the proposed sampling plan, the visual inspection is effective in rejecting unmarketable anchovies and in preventing the commercialization of unsafe products. This method is simple, less demanding than digestion in terms of time and equipment, and thus suitable as a standardized procedure to be routinely applied by food business operators. The hazard characterization, performed by sequencing the mtDNA cox2 gene, has identified the visible larvae as Anisakis pegreffii in 98 % of the cases, highlighting the zoonotic potential of the parasites found and the need for preventive measure

Is raw better? A multiple DNA barcoding approach (full and mini) based on mitochondrial and nuclear markers reveals low rates of misdescription in sushi products sold on the Italian market

New dietary habits have favored an ever growing popularity of Eastern country cooking style and in particular of sushi. Even though the Reg. (EU) 1379/2013 does not apply to restaurants and caterers, the Reg. (EU) 1169/2011 establishes that all the information they provided to the final consumer have to meet the transparency requirements as regards the description of the ingredients used for the preparation of food. The present study aimed at performing a molecular based survey to identify the seafood species used in the sushi preparations at the retail level. A total of 185 samples were collected from sushi venues and supermarkets and DNA barcoding, followed by a pairwise divergence and Neighbor Joining clustering analysis, was applied in order to verify the information declared at purchase. Rather than to a proper training of Food Business Operators working at the catering level, the low mislabeling rate found in this study (3.4%) could be ascribed to the standardization of the products sold in ethnic restaurants. In fact, the common practice of proposing standardized menus always relying on the same species of fish could limit the risk of mislabeling occurrence