Study of the immunologic response of marine-derived collagen and gelatin extracts for tissue engineering applications.

The host immunologic response to a specific material is a critical aspect when considering it for clinical implementation. Collagen and gelatin extracted from marine sources have been proposed as biomaterials for tissue engineering applications, but there is a lack of information in the literature about their immunogenicity. In this work, we evaluated the immune response to collagen and/or gelatin from blue shark and codfish, previously extracted and characterized. After endotoxin evaluation, bone marrow-derived macrophages were exposed to the materials and a panel of pro- and anti-inflammatory cytokines were evaluated both for protein quantification and gene expression. Then, the impact of those materials in the host was evaluated through peritoneal injection in C57BL/6 mice. The results suggested shark collagen as the less immunogenic material, inducing low expression of pro-inflammatory cytokines as well as inducible nitric oxide synthase (encoded by Nos2) and high expression of Arginase 1 (encoded by Arg1). Although shark gelatin appeared to be the material with higher pro-inflammatory expression, it also presents a high expression of IL-10 (anti-inflammatory cytokine) and Arginase (both markers for M2-like macrophages). When injected in the peritoneal cavity of mice, our materials demonstrated a transient recruitment of neutrophil, being almost non-existent after 24 hours of injection. Based on these findings, the studied collagenous materials can be considered interesting biomaterial candidates for regenerative medicine as they may induce an activation of the M2-like macrophage population, which is involved in suppressing the inflammatory processes promoting tissue remodeling. STATEMENT OF SIGNIFICANCE: Marine-origin biomaterials are emerging in the biomedical arena, namely the ones based in marine-derived collagen/gelatin proposed as cell templates for tissue regeneration. Nevertheless, although the major cause of implant rejection in clinical practice is the host's negative immune response, there is a lack of information in the literature about the immunological impact of these marine collagenous materials. This work aims to contribute with knowledge about the immunologic response to collagen/gelatin extracted from blue shark and codfish skins. The results demonstrated that despite some differences observed, all the materials can induce a macrophage phenotype related with anti-inflammation resolution and then act as immuno-modulators and anti-inflammatory inducible materials.

Comparison of real-time PCR methods for quantification of European hake (Merluccius merluccius) in processed food samples.

The quantification of species in commercial products is limited by analytical shortcomings, as most of them provide semiquantitative results. An exception is real-time PCR, which can provide quantitative results using hybridization probes. In the present work, this technique has been applied to the absolute, absolute-relative and relative quantification of the most valued hake species in European markets, Merluccius merluccius (European Hake). The best quantification results for this species in binary mixtures with non-target species (Merluccius capensis) and using a species-specific real-time PCR MMER_VIC system was achieved using a relative quantification approach (MLL as reference system). Absolute quantification using the MLL nuclear system has been demonstrated as appropriate for the quantification of the Merluccius genus in food model samples. This study reveals the impact of different reference systems (MLL and HAKE) in the absolute-relative and relative quantification approaches, showing that the nuclear MLL system performed better than the mitochondrial HAKE system.

Use of restriction fragment length polymorphism to distinguish between salmon species.

Identification of 10 salmon species using DNA-based methodology was investigated. Amplification of DNA was carried out using a primer set which amplified a region of the mitochondrial cytochrome b gene. Sequences of PCR-amplified DNA from the salmon species were used to select six restriction enzymes allowing species to be uniquely classified. RFLP patterns generated following analysis with each enzyme were resolved using polyacrylamide gel electrophoresis and visualized by silver staining. Results indicate that it is possible to differentiate between all 10 salmon species and that the technique could be easily adopted by the food industry for analysis of processed salmon products.

Development of a DNA-based method aimed at identifying the fish species present in food products.

Analysis of restriction fragment length polymorphism (RFLP) profiles of a 464 bp amplicon obtained from the mitochondrial cytochrome b gene was used to differentiate between several different fish species. The method was tested by a collaborative study in which 12 European laboratories participated to ascertain whether the method was reproducible. Each laboratory was required to identify 10 unknown samples by comparison with RFLP profiles from authentic species. From a total of 120 tests performed, unknown samples were correctly identified in 96% of cases. Further work attempting to use the method to analyze mixed and processed fish samples was also performed. In all cases the species contained within mixed samples were correctly identified, indicating the efficacy of the method for detecting fraudulent substitution of fish species in food products.

Identification of flatfish (Pleuronectiforme) species using DNA-based techniques.

Identification of flatfish species using a DNA-based methodology was studied. The polymerase chain reaction was employed to obtain a 464 bp amplicon from mitochondrial cytochrome b gene. The sequences from this fragment belonging to 24 species were analyzed using a genetic distance method, and polymorphic sites were determined. The fragment was found to be highly polymorphic (231 sites), and this permitted the differentiation of most of the species. Phylogenetic tree construction was employed to allow the identification of flatfish species. As a result, each species was grouped in a well-differentiated clade, except for two pairs: Limanda ferruginea and L. limanda, and Solea impar and S. lascaris, which could not be differentiated. On the basis of the sequences obtained, restriction enzymes were selected to provide specific restriction profiles, which allow the differentiation of 21 species of flatfish in a faster and less expensive manner than sequencing. This polymerase chain reaction-restriction fragment length polymorphism methodology (PCR-RFLP) was tested using commercial samples.

Identification of Hake species (Merluccius Genus) using sequencing and PCR-RFLP analysis of mitochondrial DNA control region sequences.

The use of DNA-based methodologies in identification of hake species belonging to the Merluccius genus was shown to be successful. A short fragment of the left hypervariable domain of the mitochondrial control region was amplified, sequenced, and digested from 11 hake species. The hake-specific PCR product, due to its limited size, was obtained in a variety of tissue samples with different levels of DNA concentration and degradation, including sterilized food products. On the basis of this phylogenetically informative 156-bp sequence were selected four restriction enzymes (ApoI, DdeI, DraIII, and MboII) that allow the hake species discrimination. Species identification by phylogenetic analysis of sequences or by PCR-RFLP methodologies is useful in a variety of scenarios including authentication of thermally processed food, detection of food components, and species determination of individuals whose morphological characters are removed.

Identification of European Hake species (Merluccius merluccius) using real-time PCR.

A rapid and precise method for identifying European hake (Merluccius merluccius) based on TaqMan technology is presented. The method can be applied to fresh, frozen, and processed fish products to detect the fraudulent or unintentional mislabeling of this species. Specific primers and a minor groove binding (MGB) TaqMan probe were designed for this purpose based on partial sequences of the mitochondrial DNA control region. Combinations of primers and probe concentrations that gave the lowest Ct value and the highest final fluorescence value were selected to carry out efficiency, specificity, and cross-reactivity assays. The method was successfully tested on 31 commercial hake samples. A Ct value of about 16 was obtained when Merluccius merluccius was present; however, the fluorescence signal was not detected most of the time (Ct value 40) or presented significantly higher Ct values (38.2 +/- 0.96) for the nonhake species.

Identification of shark species in seafood products by forensically informative nucleotide sequencing (FINS).

The identification of commercial shark species is a relevant issue to ensure the correct labeling of seafood products, to maintain consumer confidence in seafood, and to enhance the knowledge of the species and volumes that are at present being captured, thus improving the management of shark fisheries. The polymerase chain reaction was employed to obtain a 423 bp amplicon from the mitochondrial cytochrome b gene. The sequences from this fragment, belonging to 63 authentic individuals of 23 species, were analyzed using a genetic distance method. Nine different samples of commercial fresh, frozen, and convenience food were obtained in local and international markets to validate the methodology. These samples were analyzed, and sequences were employed for species identification, showing that forensically informative nucleotide sequencing (FINS) is a suitable technique for identification of processed seafood containing shark as an ingredient. The results also showed that incorrect labeling practices may occur regarding shark products, probably because of incorrect labeling at the production point.

Denaturation of fish proteins during frozen storage: role of formaldehyde.

Proteins of fish muscle undergo chemical and physical changes during frozen storage which may result in, under certain conditions (i.e. long periods of storage, poor freezing practices, temperature fluctuations, etc), loss of quality, reflected mainly by an unacceptable texture as well as an undesirable flavour, odour and colour. In frozen gadoid fish species, most of these changes are caused by the production of formaldehyde in the muscle. Formaldehyde is produced, along with dimethylamine, by the enzymatic reduction of trimethylamine oxide (TMAO). Many aspects of formaldehyde production by TMAO demethylase (TMAOase) have been studied throughout the last decade. In addition, different approaches have been used to investigate the effect of formaldehyde production on protein denaturation and the associated muscle textural changes. Some insight into the reaction between protein and formaldehyde has clarified the possible mechanism of formaldehyde-mediated denaturation. However, evidence of covalent bonding between proteins and formaldehyde, to form crosslinks, has not explained fully the changes observed in fish proteins during frozen storage. The study of cold-induced denaturation of proteins might give new clues for further investigation of the problem. The implications of formaldehyde in toxicological and nutritional issues is also reviewed, as general concern about the safety of food products is a growing field in food science. Finally, different approaches have been proposed to avoid the detrimental action of formaldehyde during frozen storage of gadoid fish; they are some of the practical applications of the knowledge acquired after years of study of different workers in the field.