Bertoja, Gianluca (2008) Identificazione di specie dello stoccafisso, nell'ottica dell'ispezione sanitaria di prodotto. [Ph.D. thesis]
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According to regulations in force in the hygiene sector (EC norms 178/2002, 852-853-854/2004, 2073/2005, 333/2007 and 1441/2007 ), this study involved the preparation of a traceability/retraceability model covering the many, varied, hygienic and health problems concerning stockfish, a traditionally important and significant product from the Italian fishing industry. This research was rendered more
salient partly as a result of the modification of August 5 2005 to Italian Ministerial Decree of January 14 2005, regarding the Italian name for fish species of commercial interest. To receive the official denomination of stockfish species, two Gadids were identified: Gadus morhua (Gm) and Gadus macrocephalus. Study of the philogenesis and morphology of the Gadidae - with philogenetics, physiology, reproduction, type of fishing practised, on-board processing of catch (severing of jugular vein, decapitation, evisceration, removal of tail bone, descaling, washing, refrigeration), natural drying, and final classification of the product according to quality and size criteria -involves examination of how a single stage or a few stages may have a considerable effect, positive or negative, on the quality of the
fresh or finished product. These aspects were discussed during two workshops at Tromsø and Høvik, in Norway. Comparisons revealed clearcut experimental guidelines for the primary filière of stockfish, highlighting risk assessment and identifying "Critical Points". Study continued in the field of molecular biology, for definitive identification of the species, both fresh and dried, in a rapid, precise and economical manner. For this purpose, muscle portions from 10 specimens of Gm were sampled, belonging to various populations found off the Norwegian coasts and others belonging to species philogenetically close to Gm, from a similar geographical habitat: Micromesistius poutassou, Melanogrammus aeglefinus, Gadiculus argenteus, Gadus (Merlangius) merlangus, Brosme brosme and Pollachius virens. These species, as regards both their morphological characteristics and their habitat, may enter the production of Norwegian stockfish to replace Gm. After DNA extraction, species identification was ascertained by PCR amplification and determination of nucleotide sequences. The two amplified regions contain a portion of the mitochondrial gene SSU-rRNA16S and one of the mitochondrial gene Cytochrome b, the sequences of which were compared with the GenBank database, allowing correct species identification. This meant that a DNA file could be compiled, useful for preparing a molecular assay for identifying Gm species. For this,
a universal marker for use as an internal amplification check also had to be identified. The mitochondrial gene SSU-rRNA16S, which has the highest conserved nucleotide regions among all the Gadid species, turned out to be the most suitable. Initial difficulties were encountered in identifying a species-specific marker on which to design a PCR assay to amplify only Gm-DNA, and the first assay on mitochondrial gene CoxI turned out to be poorly specific, in that it also amplified a few other neighbouring species. An assay on mitochondrial gene ATP6_8 was thus designed, and later checks showed that it was sufficiently specific to identify Gm species correctly. The two assays (16S-universal and GmATP6_8) were carried out in real-time PCR, a tool which allows rapid, precise and lowcost
analyses, since the whole post-PCR analytical phase can be eliminated. Then, 437 samples of dried Gadus morhua produced in 2006 and 50 produced in 2007 were procured from Italian fish markets, importers and wholesale distribution chains. Identification of each single sample was carried out by both visual and photographic inspection, and tissues from caudal and ventral fins were sampled. DNA was successfully extracted from 306 samples and analysed by 16S-universal and GmATP6_8 (1st screening phase: analysis of ?Ct with Gm ?Ct < 3). Of these two asays, 35 were then submitted to sequence analysis (2nd screening phase), both to verify if the assay was specific even on dried products (15 samples chosen at random) and in order to recheck any samples that had been considered as dubious during the first screening phase (20 samples: 9 with ?Ct > 3 and 11 with ?Ct > 5). For sequence analysis, a
region of gene ATP8_6 was amplified, including the portion used for the real-time PCR assay. Thus, sequencing determined both the correct species and verified the aligned regions of the primers used for the GmATP8_6-assay. The group with ”Ct < 3" that is, definitely identified after the 1st screening phase - contained 278 samples, i.e., 90.85 % of the total. After sequencing of all selected samples, only two of the nine with ?Ct >5 (the threshold identifying Gm), turned out to be non-Gm (Melanogrammus aeglefinus and Pollachius virens, respectively). In parallel, a statistical study was also carried out in support of some characterisation problems
encountered in stockfish, and, after identification of some informational variables associated with specificity, it was possible to compare later batches of product with their declared contents and also to compare groups of different batches, to establish if they did or did not belong to the same population. The variables in question were physical (size, weight, volume) and biochemical (concentration of some biochemical species of interest). The main morphometric measurements were collected and filed, and digital software was then used to create a set of descriptive and inferential statistics. Analysis focused in particular on one subgroup of a stockfish batch/, in which each
subgroup regarded a particular variety of the product, identified by an established series of criteria concerning the qualitative properties of stockfish. Descriptive statistics: for each morphometric measurement and subgroup of interest, position and variability measurements were carried out and frequency histograms and box-plots were prepared. Inferential statistics: for each morphometric measurement and subgroup of interest, the confidence intervals of the mean populations and standard deviations were made for each population. For each subgroup, the simultaneous confidence interval of the means of the two morphometric measurements for the reference populations and the confidence interval of the correlation between them was made. A comparative study among subgroups - for each
morphometric measurement and for each pair of such measurements - is almost completed. After inferential analysis, a map of morphometric characteristics was constructed, showing the following: 1) characterisation of each subpopulation of interest; 2) comparison of the morphometric characteristics of each subpopulation of interest; 3) list of criteria for later implementation of technical control and monitoring statistics for stockfish. With careful attention to "Good Practices in Processing", identification of"Critical Points" during fishing and processing, and the proposed inspection techniques and molecular biology and statistical techniques, it may be
stated that a valid model for the traceability/retraceability of a fish product has been prepared, suitable for examining and safeguarding aspects concerning risk assessment, hygiene and health, and marketing, from the sea to the table.
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