FOODCONT-D-19-01294. (num. Manuscript)
Labelling compliance and species identification of herring products sold at large scale retail level withinon the Italian market
Tinacci L.a*, Guardone L.a, Castro-Palomino Rubio J.b, Riina M. V.c, Stratev, D. d, Guidi A.a, Armani A.a
aFishLab, Department of Veterinary Sciences, University of Pisa, Viale delle Piagge 2, 56124, Pisa (Italy).
b.Faculty of Veterinary Medicine, Córdoba University, Carretera Madrid-Cádiz, Km 396,
14071, Córdoba (Spain).
cIstituto Zooprofilattico sperimentale di Piemonte Liguria e Valle D’Aosta, Torino, via
Bologna 148, 10154 Turin, Italy;
d Department of Food Hygiene and Control, Veterinary Legislation and Management,
Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria
*Corresponding author:
Postal address: Department of Veterinary Sciences, University of Pisa, Viale delle Piagge 2, 56124, Pisa (Italy) Email: lara.tinacci@unipi.it Abstract 1 2 3 4 5 6 8 9 10 11 12 13 14 15 18 19 20 21 22 23 24 25 26 27
The present study aimed at verifying labelling compliance to the current EU legislation, as well as the products’ identity against substitution incidents, of processed herring currently available on the Italian market. Totally, one hundred and twenty-two herring samples including whole smoked exemplars, smoked fillets, pickled and canned products were collected. The labels were analyzed against the requirements of Regulation No.1169/2011 and No. 1379/2013 and the product identity was verified by the use of a full and a mini-DNA barcoding protocol. Shortcomings and labelling issue included the absence of a clear allergens highlight in 9.8% of the products analyzed, the incomplete mention to the catching area and the lack of fishing gear declaration in 40% and 33% of the products falling within the scope of Regulation No. 1379/2013. The COI barcodes selected in the study allowed the univocal species identification of all the products which were confirmed belonging to the species Clupea harengus. The substantial labelling compliance and the absence of mislabelling incidents confirmed a high level of food business operators training and control along the herring supply chain and a strong will to protect and enhance the consumers’ informed choice.
Keywords
Seafood labelling, herring, processed products, COI, full length barcode, mini-barcode
1. Introduction 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
Mislabelling and species substitution are the most often reported fraudulent incidents among the illegal activities affecting the seafood sector (Reilly, 2018). They usually imply a deliberate substitution of high value with low value species with the aim of deceiving the consumer regarding the nature of fishery products, generally for economical gain (Lewis & Boyle, 2017; Oceana, 2014; Oceana, 2018; Pardo, Jiménez, & Pérez-Villarreal, 2016). Ready to eat (RTE) products are, among the variety of seafood products available on the market, those particularly exposed to Economically motivated adulteration (EMA) due to their generally high commercial value and the impossibility to recognize the species contained in the products using visual inspection (morphological analysis) (Everstine, Spink & Kennedy 2013). In this respect, seafood traceability is a key aspect to protect consumers’ interests and health and guarantee the sustainability of the stock exploitation.
According to the EU requirements, information on fisheries and aquaculture products should therefore be available throughout the supply chain. In particular, specific mandatory information are requested by the Regulation (EU) No 1379/2013 for prepacked and non-prepacked seafood products, including: the commercial designation, the species scientific name, the production method and the catching or farming area according to the FAO fishing areas classification, the product durability and whether the product has been defrosted (with limited exceptions). This information complements the mandatory provisions specified in the Articles 9 and 10 of the Regulation (EU) No. 1169/2011 on the provision of food information to consumers.
The Regulation (EU) n. 1379/2013 should be applied to seafood products falling within the CN0302, CN0303, CN0304, CN0305 customs commodity codes described in the Commission Implementing Regulation (EU) No. 1925/2017 and specifically represented by fresh and frozen unprocessed or filleted products and salted, dried and smoked seafood. On the contrary, it doesn’t refer to processed product such as canned and prepared, preserved and 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76
semi-preserved products (CN1604 and CN1605 codes), which therefore exclusively fall within the provisions of the Regulation (EU) No. 1169/2011. This lack may represent a shortcoming for the market transparency. Factually, even though the enforcement of the Regulation (EU) No 1379/2013 seems to have produced positive feedbacks in terms of reduction of seafood mislabelling rate within the EU market (Armani et al., 2017; Bénard-Capelle et al., 2015; Mariani et al., 2015), several studies highlighted that unrecognizable processed products (skinned, chopped, filleted, sliced, prepared or cooked) are more prone to be involved in fraudulent replacement than whole fish (Galal-Khallaf, Ardura, Mohammed-Geba, Borrell, & Garcia-Vazquez, 2014; Helyar et al., 2014; Khaksar et al., 2015). Therefore, the enforcement of specific survey aimed at authenticating the species declared on certain kind of seafood products marketed in EU are needed.
Currently, fish species belonging to the Scombridae family (Tuna and tuna like and mackerel species), Salmonidae (salmon and trout species) and small pelagic species belonging to Engraulidae (anchovy) and Clupeidae (Sardine and herring) family are the most relevant seafood on the European market (EUMOFA, 2018). These fish groups are commonly available on the internal market in different products categories, either falling in or out of the scope of the Regulation EU No. 1379/2013.
The term herring usually refers to species belonging to the genus Clupea, which currently includes 3 accepted species: C. harengus, C. manulensis and C. pallasii (subspecies C. pallasii pallasii, C. pallasii marisalbi, C. pallasii suworowi) (World Register of Marine Species, WORMS; FishBase database). Among the abovementioned species, Atlantic herring (C. harengus) and Pacific herring (C. pallasii sub pallasii and marisaldi) are the most exploited for human consumption. They present a distinct geographical distribution, North Atlantic and Pacific respectively, although a partial overlapping catching area on White sea 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100
and the south-eastern Barents seas has been confirmed (Laakkonen, Lajus, Strelkov, & Väinölä, 2013).
Within the intra-EU commerce, herring species represent the 18% of the traded small pelagic fish and one of the main commercial species contributing to the rise of the average per capita consumption of fishery products with a registered increase of consumer demand, in 2016, from 0.93 kg to 1.23 kg (EUMOFA, 2018). In the 2017, the 44% and 33% of herring volumes was sold as frozen and fresh while the remaining 27% was represented by processed products responsible in fact for the highest value share (55%) (EUMOFA, 2018).
Processed herring products sold at retail level generally consist of whole, beheaded or filleted smoked exemplars, ready-to-eat, marinated or pickled, and canned delicacies, all of them also available on the Italian market (Guardone et al., 2019). Unlike tuna, sardine and anchovy products, for which the mislabelling rate on the European market has already been extensively investigated (Cutarelli, Galiero, Capuano, & Corrado,2018; Giusti et al., 2019; Jérôme et al., 2008; Lago, Herrero, Vieites, & Espiñeira, 2011; Sotelo et al., 2018; Velasco, Aldrey, Pérez-Martín, & Sotelo, 2016) data on identity and labeling compliance of the different herring products at retail level are barely available (Günther, Raupach, & Knebelsberger, 2017).
DNA Barcoding technique represents an effective tool for seafood authentication by means of suitable analytical workflow (Tinacci et al., 2018). The 650 bp fragment of the mitochondrial gene coding for cytochrome c oxidase 1 (COI), designated as DNA barcode for animal species identification (Hebert, Cywinska, Ball, & Dewaard, 2003), has been successfully applied to the authentication of commercial seafood, including herring products (Günther et al., 2017; Hu, Huang, Hanner, Levin, & Lu, 2018). However, DNA fragmentation, induced by the exposure to heat and other physical stressors during the processing methods, represents the main limiting factor to the full-length target amplification 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125
(Günther et al., 2017; Nicolé et al., 2012; Shokralla, Hellberg, Handy, King, & Hajibabaei, 2015). Nevertheless, in the presence of highly degraded DNA samples, the use of highly informative COI mini-barcodes (100-400 bp), has been confirmed effective for species identification and the mini-barcoding approach has been applied in several studies of variously processed seafood products authentication (Fields, Abercrombie, Eng, Feldheim, & Chapman, 2015; Günther et al., 2017; Shokralla et al., 2015).
This considered, in the present study, both full and mini-length DNA barcoding analytical approach were used to molecularly identify smoked, canned and marinated herring products collected at large scale retail level at retail withinon the Italian market. The compliance of the information labelled on the seafood products to the requirement of the current European legislation was also verified in order to provide data on labelling accuracy of this highly appreciated products. This study, by providing data on the mislabelling rate and species substitution in highly-appreciated products sold on the Italian market, furnishes an up-dated picture of the current impact of these issue on the seafood compart.
2. Materials and Methods
2.1 Sampling
A total of 122 herring products, 79 prepacked and 43 non-prepacked wrapped at sale on consumer’s demand were collected. Among these, 7 “smoked whole” products (SW) were collected at the Border Inspection Post (BIP) of Livorno-Pisa while the remaining 115 products were directly sampled at different points of both Italian and European sale of large-scale retail retailers and discount companie chains .distribution in Tuscany (Northern Italy)
between from October 2017 to October 2018 . The sampling was conducted from October
2017 to October 2018 and the products were collected from both Italian and European large
retailers and discount companies chains.The sampling has been structured to include a
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proportional number of products per type, according to the market supply and the brands variety.
The productsy included: 35 SW, 25 “smoked filleted” (SF), 28 “marinated fillets” (MF) and 27 “canned fillets” (CF) (Table 1).
In addition, two reference DNA samples belonging to C. harengus (C. har-A; C. har-B), obtained by fresh morphologically identified specimens collected in this study, and 2 reference DNA samples of C. pallasii pallasii (UW 048005; UW 049684), obtained by ethanol preserved tissue samples, which were kindly provided the Washington University, were included as positive controls for both the full length barcode (FLB) and the mini-barcode (MB) amplification.
2.2 Label analysis
Labels correctness was evaluated in the light of the mandatory information required by Regulation (EU) No 1169/2011. In detail, for each prepacked product, the presence of the commercial denomination, the ingredient list, allergens highlight, the net and drained weight, the conservation instructions, the best before date, the company name or code and the nutrition declaration were assessed by the analysis of the labels reported on the package. In case of SW and SF products, the presence of mandatory information (the commercial designation of the species and its scientific name, the production method, the area where the product was caught, and the category of used fishing gear and the date of minimum durability) required by Regulation (EU) No 1379/2013 was also verified. For CF other voluntary claims were considered when available. In the case of seafood offered for sale as non-prepacked products, the presence of mandatory information set out in Italian legislation (Dl.gs 109/92 Article 16: sale of products packed on consumer’s demand) according to the requirements of the Regulation No. 1169/2011 (Article 44) and the mandatory information imposed by the Regulation No. 1379/2013 were verified by the analysis of both products catalogues available 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174
for consumers consultation at the fishery desk and labels printed on the pack wrapped on the sales premises. Finally, as regards the 7 products collected at the BIP of Livorno the labelling information on the commercial document accompanying the product was analyzed. The information to be reported on the document must comply those requested by the Commission Implementing Regulation EU No. 1204/2011.
2.3 Total DNA extraction and evaluation. Total DNA extraction and evaluation was
performed starting from ~150 mg of tissue following Armani et al. (2014). The DNA integrity for each sample was evaluated by gel electrophoresis as described by Armani et al. (2017) and, according to the UV visible pattern the DNA samples were classified as 1) low or no fragmented (>1000 bp or complete); 2) medium fragmented (500-1000 bp) and 3) highly fragmented (<500bp).
2.4 Primers selection and PCR amplification
The primer pair to be used for the amplification of the DNA samples were selected according to the results of their degradation level assessed by gel electrophoresis (section 2.3). 2.4.1 Full length DNA Barcoding. For samples not showing degradation the primer pair
designed by Handy et al., 2011 (FISHCOILBC_tsa
5’-CACGACGTTGTAAAACGACTCAACYAATCAYAAAGATATYGGCAC -3’ and
FISHCOIHBC_tsa
5’-GGATAACAATTTCACACAGGACTTCYGGGTGRCCRAARAATCA -3’) was selected for obtaining a COI fragment of 705bp (655 bp without primers) (FLB) according to the protocol described in Tinacci et al., (2018).
2.3.2 Mini DNA barcoding. A mini DNA barcoding approach was instead considered in the presence of medium-highly fragmented total DNA (fragment <500 bp) as eventually highlighted by electrophoresis (section 2.3.1). To this end, mitochondrial COI gene DNA reference sequences belonging to valid species within the genus Clupea sp., in accordance 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199
with the Global Information System on fishes (fishbase.org), were aligned with Clustal W in BioEdit version 7.0.9 (Hall, 1999) to highlight the region with the highest interspecies variability within the full-length barcode. Overall 55 sequences were collected and analyzed. In particular, 24 reference sequences belonging to C. harengus, 6 belonging to C. pallasii pallasii and 25 deposited as Clupea pallasii were aligned. No reference sequences for the species C. manulensis and the sub species C. pallasii suworowi and C. pallasii marisaldi were available. The interspecies variability was estimated as within group and between group mean distance by the use of kimura 2-parameter model (Kimura, 1980) with 1000 nonparametric bootstrap replicates and an unrooted Neighbor Joining (NJ) phylogram (Saitou & Nei, 1987) was produced to visualize the clustering pattern of the selected sequence dataset in accordance with distance-based identification technique provided by the Identification System (ID’s) in the Barcode of Life Data Systems (BOLD) identification engine. The analysis was computed on MEGA 6.0 software package (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013).
Thus, two different software, Tool Primer BLAST
(https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and Sequence Manipulation Suite: PCR primer state (http://www.bioinformatics.org/sms2/pcr_primer_stats.html), were applied for the primer design and quality check (melting temperature, GC content, molecular weight, extinction coefficient, self-annealing and hairpin formation). Two distinct primer pairs CLUP-FOR5 (25 bp, CCCTCGAATAAACAATATGAGCTTC-3’)/ CLUP-REV4 (21 bp, TCCTCTATTCTAGGGGCCATT-3’) and CLUP-FOR9 (21 bp,
5’-ATTCCTCTTATGATCGGAGCG-3’) / CLUP-REV9 (25 bp,
5’-TCTAGGGGCCATTAATTTCATTACC-3’) were finally designed for the amplification of a 205bp and 252 bp amplicons (157 bp and a 206 bp without primers). The interspecies variability of each of the mini-barcodes selected was checked by calculating the pairwise 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223
distance matrix and performing the NJ analysis previously applied on full length barcodes dataset.
The mini-barcode (MB) PCR reaction was prepared with the same protocol described for the FLB while the amplification protocol was set as follow: denaturation at 95 °C for 3 min; 40 cycles at 95 °C for 30 s, 54°C for 30 s, and 72°C for 20 s; final extension at 72°C for 5 min.
2.5 Sequencing.
The successful amplification of PCR products with the expected length (655 bp or 206/159 bp) was verified by gel electrophoresis on a 2.0% or 2.5% agarose gel stained with GelRed™ Nucleid Acid Gel Stain (Biotium, Hayward, CA, USA), comparing the amplicons with the standard molecular marker SharpMass™50-DNA (Euroclone Spa, Milano). All the PCR products were purified with EuroSAP PCR Enzymatic Clean-up kit (EuroClone Spa, Milano) and sequenced by the Istituto Zooprofilattico di Piemonte Liguria e Valle D’Aosta (Turin, Italy) as described in Guardone et al., (2018).
2.6 BLAST analysis: species identification and mislabelling assessment
The obtained sequences were visualized, aligned and edited with Clustal W in Bio Edit version 7.0.9 (Hall, 1999) and the final sequences were queried for species identification against the reference sequences available in GenBank (http://www.ncbi.nlm.nih.gov) and BOLD (http://www.boldsystems.org/) databases, as described in Tinacci et al., (2018). The species identification was allocated when the identity rate showed less than 2% difference with reference sequences associated to 100% query coverage. Outcomes from this phase were compared to data obtained from the labelling analysis. The mislabelling rate in relation to the scientific denomination was calculated according to the product category: on (a) SW and SF products declaring the species scientific name (as mandatory information), (b) MF and CF voluntary reporting the species scientific name.
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3. Results and discussion
3.1 Samples collection: product categories and commercial value of the products
The sampling, conducted at retail, specifically included products belonging to both Italian and European large-scale retail chains and European discount chains well represented and spread also outside the regional territory. The small supplies of gastronomic specialties, prepared exclusively for the regional market, were purposely excluded.
The samples collection was conducted focusing the attention on the principal herring products available at national retail level.purchase. At the points of sale visited during the sampling, all the products types offered for sale were collected without any preliminary selection based on the product price or the presentationmethod.
Whole and filleted exemplars individually prepacked or packed on demand at purchase, which constituted together 55% of the sampled products, represent the more widespread products on the Italian market as already observed by Bernardi, Tirloni, & Cattaneo (2014) and Guardone et al., (2018). The remaining 45% was equally distributed between marinated delicacies and canned products which only marginally belonged to national production and were instead generally imported from Lithuania, Sweden and Norway, the countries detaining
well-established herring processing plants
(http://www.eumofa.eu/documents/20178/96604/Monthly+Highlights+-+No.+2-2017.pdf). The price of the products within each category ranged around 10-12 €/kg for silver or golden smoked whole herrings and fillets; 12-16 €/kg for marinated products and 10-25 €/kg for canned products. Comparing the average price of each category to the price of the fresh herring at retail (10-12 €/kg) a relevant increase of the marinated or canned products was highlighted in accordance with the trend registered at European level (EUMOFA, 2018). According to the assumptions of Everstine et al., (2013) and on the basis of the results 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272
obtained by the price analysis, the RTEs products were thus confirmed as the best candidates for EMA perpetration.
3.2 Labels analysis
3.2.1 Labeling information compliance to the requirements of Regulation EU No. 1169/2011. Each product was presented for sale with the commercial designation “herring” eventually accompanied, in the case of SW and SF products, by the adjective “silver” (26/67) or “golden” (16/67) according to the product’s curing. According to the Norway traditional processing, herring products are indeed identified on the basis of the duration of the smoking process and classified as silver type with a smoking process lasting in 12 hours, golden type if smoked up to three days and hard cured when the smoking process is prolonged to 12 days (http://slowfood.com/slowfish/pagine/eng/pagina.lasso?-id_pg=122). In this light, according to article 17 of the Regulation EU No. 1169/2011, the commercial name acquires a descriptive connotation since the adjective offer an immediate information of the organoleptic properties of the product to the final consumer.
All the products were generally found compliant to the requirements of the aforesaid Regulation with the exception of 12 prepacked marinated products (9.8%, 12/122) didn’t including a clear highlight of the allergens among the ingredients list (Table 1).
3.2.2 Compliance to and voluntary application of the Regulation EU No. 1379/2013 for the products labelling. As abovementioned, with the exception of smoked fish and fillets which fully fall within the scope of the Regulation (EU) n. 1379/2013, the declaration of the other mandatory information on the other processed products labels is exclusively subject to the will of the Food Business Operators (FBOs) although strongly advocated by the European Parliament to allow an informed consumers’ choice (European Parliament Resolution No 2016/2532). 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296
The label analysis performed on smoked products (n=67) confirmed a general fulfilment of the Regulation requirements. All the products’ commercial designation was associated to the species scientific name C. harengus in accordance with the current Italian list of official commercial designations (MIPAAF Decree No. 19105 of September 22nd, 2017). Production method and catching area was provided in the 100% of the prepacked or non-prepacked products although in 40% (27/67) of the products a full catching area description and the FAO sub-area or subdivision wasn’t reported.
In this respect, the Italian Ministry of Agricultural and Forestry Politics, in the 2014, in order to clarify the operational obligations of each food operator in the fish supply chain with provisions set by current European legislation has issued a clarification guideline regarding specific requirements for the application of EU Regulation No. 1379/2013 including the list of the accepted descriptive denominations for each FAO catching areas, sub-areas and divisions (FAO 27 and 37) (MIPAAF December 12th, 2014)
Moreover, the mention to the specific fishing gear lacked in 33% (22/67) of the products. The relatively high percentage observed may be plausibly recollected to the fact that this aspect constituted a totally new element with respect to the legislation prior to EU Reg. No. 1379/2013 and probably requires a longer time for adjustment by all seafood sector operators.
Finally, as regards the other processed specialties not included under the scope of Reg EU No. 1379/2013, the analysis highlighted that all the Regulation requirements (Art. 35) were voluntarily and correctly reported in all the CF. As regard MF the only information voluntarily reported in 43% (12/28) of them was the scientific name. Interestingly, when present it was always associated to a correct commercial designation according to the official list. These outcomes confirm the FBOs’ will to follow the European Parliament recommendation for the protection of consumers’ informed choice (European Parliament Resolution No. 2016/2532). 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321
3.3 Molecular analysis
3.3.1 Assessment of DNA fragmentation and primer selection. Different combination of physical-chemical treatments (dehydration, pH lowering, high temperature and pressure, smoking) commonly applied during food manufacturing processes are known to induce random breaks and partial oxidation in the DNA strands significantly reducing the number of DNA fragments with suitable size for the following PCR analysis ( Armani et al., 2015a ; Lo & Shaw, 2018).
In this respect, as previously highlighted in a study aimed at authenticating species in processed seafood (Shokralla et al., 2015), the tested samples showed a variable DNA fragmentation degree in relation to the different product categories. In the present study, total DNA samples belonging to SF, generally characterized by medium to low fragmentation pattern (60% 15/25), appeared as the less affected by the processing degradative effect followed by SW products DNA samples presenting medium (31% 13/42) to high fragmentation degree (69% 29/42). The remaining DNA samples extracted from MF and CF were characterized by a high degree of fragmentation, with a hardly visible electrophoretic pattern above 500 bp (Table 3 and Figure 1). The higher fragmentation pattern highlighted in the two latter categories is plausibly to be recollected to the sterilization temperature (121,1°C) applied in accordance with standard canning principles (http://www.fao.org/docrep/003/T0007E/T0007E02.HTM) and to the pH value (pH ~ 4.5) reached inside the final products (http://www.fao.org/wairdocs/tan/x5932e/x5932e01.htm) which are known as major physical stressors contributing to DNA damage (Nicolé et al., 2012). In particular, the results obtained are analogous to those obtained by Piskata et al., (2017) in a comparative study on DNA extraction techniques and DNA quality evaluation from yellowfin tuna samples subdued to different preserving (boiling, smoking and canning) techniques. 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346
Although the reliability of short DNA MBs within the standard COI barcoding region to seafood authentication has been thoroughly demonstrated, the final species allocation can be limited by the lack of diagnostic sites in the MB fragment Armani et al., (2017). In this respect the primer REVSH-1 set in a previous study within the FLB for the analysis of phylogenetically distant taxa (Armani et al., 2015b) was preliminarily verified on alignment of Clupea harengus and C. pallasii reference sequences. Despite the high degree of primer match on the annealing region, the analysis highlighted the unsuitability of the MB selected due to the absence of diagnostic sites and substantial overlapping of the DNA sequences belonging to the two species. Thus, the alignment of Clupea harengus and C. pallasii reference sequences was set again in order to identify higher variability sites within FLB and two alternative primer pairs for the amplification of two short DNA targets shorter than 500bp were designed. The kimura 2-parameter analysis confirmed interspecies variability of both the MB fragments selected and showed a decrease of the divergence estimated within taxa groups towards the divergence rate calculated on the FLB (Table 2). The inferred NJ tree obtained by the analysis of the reference FLB showed two distinct clusters, the first including all the sequences belonging to C. harengus specimens, the second collecting both the sequences deposited as C. pallasii and C. pallasii subsp. pallasi (Figure 2). The NJ analysis conducted on MB, although confirming a significant decrease of divergence, showed the maintenance of clear separation between the two species cluster (Figure 3-4).
3.3.2 Amplification and sequencing. All the DNA samples were successfully amplified. In detail, 14 FLB belonging to SW (n=8) and SF (n=6), 95 MB of 250 bp and 13 MB of 205 bp were obtained as summarized in Table 3. All PCR products were then purified, successfully sequenced and subdued to the further BLAST analysis. Nevertheless, for none of the FLB a full length sequence was obtained, and the length of the sequences ranged from 69% to 89% of the expected final amplicon (Table 2).
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The selection of the proper barcode length generally allowed the final recovery of one PCR product with a minimum final concentration of 5ng/µl by the setting of a unique PCR reaction according to the preliminary assessment of the DNA fragmentation. Relevant exceptions were represented by 14 DNA samples, belonging to 5 SW and 9 SF, ascertained as medium fragmented and initially amplified using the FLB protocol. In this case the final recovery of a suitable PCR product for the following sequencing was only obtained following the re-amplification of the samples with the primer pair CLUP-FOR9/REV9 (252 bp/206 bp without primers). The remaining 14 FLB PCR products although presenting the minimum final concentration required for the sequencing reaction, highlighted a significantly lower amplification efficiency compared to that shown on the two reference DNA samples (Figure 1). Failures in the amplification of long PCR products from smoked DNA samples has also been recently highlighted by Piskata et al., (2017) and by Günther et al. (2017) in a DNA barcoding survey for the identification of differently processed seafood. Well-known pivotal chemical constituents (aldehydes and phenolic compounds) produced during the smoking process are responsible for PCR inhibiting effects associated with cross-linking of histones on DNA which act as major obstacles to the progression of Taq polymerase imposing constraints on the size of the PCR products (Zagga et al., 2013).
The post sequencing control of the FLB forward and reverse sequences, manually checked and edited with Bioedit 7.0 software, confirmed the hypothesis of a plausibly marked degradation of the DNA extracted from the smoked products. Particularly, the chromatogram analysis highlighted the presence of small peaks underneath all peaks in the sequence read tails, mixed peaks, impossibility in the univocal assignment of nucleotides which were recollected to poor quality, low concentrated PCR products and a multiple nucleotide misincorporation during PCR amplification (Shokralla et al., 2015).
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3.3.3 BLAST analysis and assessment of mislabelling rate
By the combination of BLAST and BOLD ID's system results verified by Neighbor-Joining clustering analysis on Kimura 2-parameter distance model (Kimura, 1980; Saitou & Nei, 1987) directly provided on BOLD ID's system, all samples were identified at species level as belonging to C. harengus regardless to the final length of the barcode amplified and sequenced (FLB or MB) (Table 1). Thus, no mislabelling in SW and SF products or discrepancies between the scientific name voluntary declared on MF and CF products and the species molecularly identified were highlighted. Analogous results were found in Hu et al., (2018) study, in which herring products, although marginally included in the sampling, were all confirmed compliant with the information declared by the food operators. In contrast, in the same study, Pacific herring (Clupea pallasii) was found as substitute species of Ethmidium maculatum in a product labelled with the commercial designation “menhaden” commonly referring to the species although not appearing among the official commercial designations admitted in Canada, the country in which the study was carried out. The authors recollected the incident to an involuntary substitution plausibly related to the similar morphology of the species, both belonging to Clupeidae family. Previous studies that marginally analyzed herring products within a heterogeneous sampling basket also include Günther et al., 2017 and Nicolé et al., 2012, surveys in which RTE herring products (chilled pickled herring) and smoked herring products were considered respectively, without highlighting any mislabelling on this kind of products.
4. Conclusions
The present study is the first survey specifically conducted to verify the accuracy and compliance of labeling of herring products. The molecular analysis reaffirmed the smoking procedure as a major factor affecting DNA quality and the COI mini-DNA barcoding technique as an effective tool for species identification within Clupeidae. Despite few 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421
labelling inaccuracies concerning a clear highlight of the allergens and the correct indication of the fishing area and of the products origin, the lack of no mislabelled products were observed and the labels were confirmed generally compliant and the general labelling compliance to the current European legislation.. testified. the high level of training and accuracy of food business operators operating in this specific sector of the seafood compart.
In this respect the study also highlighted Therefore, the study revealed, at least for the investigated products, the food operators growing attitude towards the application, on a voluntary basis, of the labeling model described by Reg. EU No. 1379/2013, also for seafood products still laying out of the regulation’s scope.
Acknowledgments
The authors wish to thank the Quality Office of UNICOOP Firenze for its contribution to the research activities.
Figures captions
Figure 1 Comparison of the fragmentation pattern (A) with the amplification success of total DNA belonging to different commercial product types included in the study by the use of the following primer pairs: BA) Handy et al., 2011 (652 bp PCR product); CB) CLUP-FOR9/REV9 (252/205 bp PCR product); DC) CLUP-FOR5/REV4 (159/206 bp PCR product). 1-5 DNA extracted from whole smoked products; 6-10 DNA extracted from smoked fillets; 11-15 DNA extracted from marinated products; 16-20 DNA extracted from canned products; 21-22 total DNA extracted from Clupea harengus fresh tissue; L: Ladder SharpMASS 50® Euroclone S.p.A
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Figure 2 Inferred NJ tree obtained on the 652 bp fragments from Clupea sp. reference COI sequences by the use of kimura 2-parameter model with 1000 nonparametric bootstrap replicates
Figure 3 Inferred NJ tree produced on the 206 bp COI fragments from Clupea sp. reference COI sequences by the use of kimura 2-parameter model with 1000 nonparametric bootstrap replicates
Figure 4 Inferred NJ tree obtained on the 157 bp COI fragments from Clupea sp. reference COI sequences by the use of kimura 2-parameter model with 1000 nonparametric bootstrap replicates
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