Semi-industrial development of nutritious and healthy seafood dishes from sustainable species.

This study aimed to devise innovative, tailor-made, appealing, tasty and semi-industrialized dishes, using sustainable and under-utilized seafood species (bib, common dab, common carp, blue mussel and blue whiting), that can meet the specific nutritional and functional needs of children (8-10-years), pregnant women (20-40-years) and seniors (≥60-years). Hence, contests were organised among cooking schools from 6 European countries and the best recipes/dishes were reformulated, semi-industrially produced and chemically and microbiologically evaluated. The dishes intended for: (i) children and pregnant women had EPA + DHA and I levels that reached the target quantities, supporting the claim as "high in I"; and (ii) seniors were "high in protein" (24.8%-Soup_S and 34.0%-Balls_S of the energy was provided by proteins), "high in vitamin B12", and had Na contents (≤0.4%) below the defined limit. All dishes reached the vitamin D target value. Sausages_C, Roulade_P, Fillet_P and Balls_S had a well-balanced protein/fat ratio. Roulade_P presented the highest n-3 PUFA/n-6 PUFA ratio (3.3), while Sausages_C the lowest SFA/UNS ratio (0.2). Dishes were considered safe based on different parameters (e.g. Hg-T, PBDEs, Escherichia coli). All represent dietary sources contributing to meet the reference intakes of target nutrients (33->100%), providing valuable options to overcome nutritional and functional imbalances of the three groups.

Methodological Approaches for Monitoring Five Major Food Safety Hazards Affecting Food Production in the Galicia-Northern Portugal Euroregion.

The agri-food industry has historically determined the socioeconomic characteristics of Galicia and Northern Portugal, and it was recently identified as an area for collaboration in the Euroregion. In particular, there is a need for action to help to ensure the provision of safe and healthy foods by taking advantage of key enabling technologies. The goals of the FOODSENS project are aligned with this major objective, specifically with the development of biosensors able to monitor hazards relevant to the safety of food produced in the Euroregion. The present review addresses the state of the art of analytical methodologies and techniques-whether commercially available or in various stages of development-for monitoring food hazards, such as harmful algal blooms, mycotoxins, Listeria monocytogenes, allergens, and polycyclic aromatic hydrocarbons. We discuss the pros and cons of these methodologies and techniques and address lines of research for point-of-care detection. Accordingly, the development of miniaturized automated monitoring strategies is considered a priority in terms of health and economic interest, with a significant impact in several areas, such as food safety, water quality, pollution control, and public health. Finally, we present potential market opportunities that could result from the availability of rapid and reliable commercial methodologies.

Detoxification of paralytic shellfish poisoning toxins in naturally contaminated mussels, clams and scallops by an industrial procedure.

Paralytic shellfish poisoning (PSP) episodes cause important economic impacts due to closure of shellfish production areas in order to protect human health. These closures, if are frequent and persistent, can seriously affect shellfish producers and the seafood industry, among others. In this study, we have developed an alternative processing method for bivalves with PSP content above the legal limit, which allows reducing toxicity to acceptable levels. A modification of the PSP detoxifying procedure stablished by Decision 96/77/EC of the European Union in Acanthocardia tuberculata, was developed and implemented for PSP elimination in other bivalves species. The procedure was applied to 6 batches of mussels, 2 batches of clams and 2 batches of scallops, achieving detoxification rates of around 85%. A viable industrial protocol which allows the transformation of a product at risk into a safe product was developed. Although a significant reduction was obtained, in a sample circa 9000 µg STX diHCl equiv/kg, the final toxin level in these highly toxic mussels did not fall below the European limit. The processing protocol described may be applied efficiently to mussels, clams and scallops and it may be a major solution to counteract the closure of shellfish harvesting areas, especially if persistent.

Occurrence of Tetrodotoxin in Bivalves and Gastropods from Harvesting Areas and Other Natural Spaces in Spain.

Tetrodotoxin (TTX) is a potent neurotoxin that is receiving increasing interest in the European Union because it has been found in different fishery products (fish, bivalves and gastropods) captured in European waters. Since available information is scarce, further analytical data regarding the incidence of this toxin in European fishery products is needed in order to perform an appropriate risk assessment devoted to protecting consumers' health. Hence, samples of bivalves and gastropods were collected at different points of the Spanish coast and analyzed by high-performance hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) to evaluate the presence of TTX. None of the analyzed samples showed TTX above an internal threshold of 10 µg/kg or even showed a peak under it. Our results on TTX occurrence obtained in bivalve molluscs and gastropods did not show, at least in the studied areas, a risk for public health. However, taking into account previous positive results obtained by other research groups, and since we did not detect TTX in our samples, a more completed study increasing sampling frequency is needed to ensure proper risk evaluation towards the food safety of these products.

Study of ceramic membrane behavior for okadaic acid and heavy-metal determination in filtered seawater.

The performance of several MF and UF ceramic membranes that filter the seawater surrounding mussel rafts is studied for preventive detection of toxic episodes. The modified fouling index applied to UF membranes (MFI-UF) is used to compare fouling rates and membrane fouling levels. The reduction of several quality parameters such as turbidity, alkalinity, chemical oxygen demand (COD), and chlorophyll content is explained by the higher quality of the UF rather than the MF permeates. Membrane rejection rates of Pb+2 and okadaic acid, the main toxin that provokes toxic episodes due to bloom-forming algae, are measured under different pH and pressures. Measurements are taken particularly at filtration times before and after the formation of stable caking on the membrane surface. The results indicated that trace concentrations of heavy ions were mainly rejected by the membrane charge, until the saturation point was reached, and that no rejections occurred when the pH was lower than the isoelectric point of the membranes. However, most of the okadaic acid was rejected due to the formation of cake on the membrane surface. The rejection of okadaic acid depended on the membrane pore size and transmembrane pressure, yielding negative rejections under specific filtration conditions.

Effect of the industrial canning on the toxicity of mussels contaminated with diarrhetic shellfish poisoning (DSP) toxins.

The effect of canning in pickled sauce and autoclaving on weight, toxin content, toxin concentration and toxicity of steamed mussels was studied. Weight decreased by 25.5%. Okadaic acid (OA) and DTX2 content of mussel meat decreased by 24.1 and 42.5%, respectively. The estimated toxicity of the mussel remained nearly unchanged (increased by 2.9%). A part of the toxins lost by the mussels was leached to the sauce but the remaining part should have been thermally degraded. DTX2 underwent more degradation than OA and, in both toxins, free forms more than conjugated ones. This process, therefore, cannot be responsible for the large increments of toxicity of processed mussels -relative to the raw ones-sometimes detected by food processing companies. The final product could be monitored in several ways, but analysing the whole can content or the mussel meat once rehydrated seems to be the most equivalents to the raw mussel controls.

Tetrodotoxin, an Extremely Potent Marine Neurotoxin: Distribution, Toxicity, Origin and Therapeutical Uses.

Tetrodotoxin (TTX) is a potent neurotoxin responsible for many human intoxications and fatalities each year. The origin of TTX is unknown, but in the pufferfish, it seems to be produced by endosymbiotic bacteria that often seem to be passed down the food chain. The ingestion of contaminated pufferfish, considered the most delicious fish in Japan, is the usual route of toxicity. This neurotoxin, reported as a threat to human health in Asian countries, has spread to the Pacific and Mediterranean, due to the increase of temperature waters worldwide. TTX, for which there is no known antidote, inhibits sodium channel producing heart failure in many cases and consequently death. In Japan, a regulatory limit of 2 mg eq TTX/kg was established, although the restaurant preparation of "fugu" is strictly controlled by law and only chefs qualified are allowed to prepare the fish. Due to its paralysis effect, this neurotoxin could be used in the medical field as an analgesic to treat some cancer pains.

Occurrence of lipophilic marine toxins in shellfish from Galicia (NW of Spain) and synergies among them.

Lipophilic marine toxins pose a serious threat for consumers and an enormous economic problem for shellfish producers. Synergistic interaction among toxins may play an important role in the toxicity of shellfish and consequently in human intoxications. In order to study the toxic profile of molluscs, sampled during toxic episodes occurring in different locations in Galicia in 2014, shellfish were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), the official method for the detection of lipophilic toxins. The performance of this procedure was demonstrated to be fit for purpose and was validated in house following European guidelines. The vast majority of toxins present in shellfish belonged to the okadaic acid (OA) group and some samples from a particular area contained yessotoxin (YTX). Since these toxins occur very often with other lipophilic toxins, we evaluated the potential interactions among them. A human neuroblastoma cell line was used to study the possible synergies of OA with other lipophilic toxins. Results show that combination of OA with dinophysistoxin 2 (DTX2) or YTX enhances the toxicity triggered by OA, decreasing cell viability and cell proliferation, depending on the toxin concentration and incubation time. The effects of other lipophilic toxins as 13-desmethyl Spirolide C were also evaluated in vitro.

Effect of the industrial steaming on the toxicity, estimated by LC-MS/MS, of mussels exposed for a long time to diarrhetic shellfish poisoning (DSP) toxins.

The effect of industrial steaming on mussels that had been naturally exposed to DSP toxins for a long time was studied using LC-MS/MS. The estimated toxicity increased with steaming by a percentage that cannot be explained by weight loss. The estimated toxin content per mussel increased substantially with the treatment, which can only be explained by an incorrect estimation by the technique (at the extraction or analytical level) or by the presence of unknown derivatives or analogues. Direct alkaline hydrolysis of the mussel meat yielded more toxin than the standard hydrolysis (hydrolysis of the methanolic extracts), suggesting that extraction was, at least in part, responsible for the increase of toxin content. In situations as the one described in this work, it can be expected that mussels with toxicities well below the regulatory limit could easily surpass that level after industrial steaming, thus producing important losses for food processors.

lolB gene, a valid alternative for qPCR detection of Vibrio cholerae in food and environmental samples.

In recent years a new genetic target for Vibrio cholerae detection has been reported, but its application was limited to clinical samples. This target, lolB, has never been applied to either food or environmental samples. In the present study the development, as well as the evaluation and pre-validation, of a real-time PCR method based on lolB, is described. The method included a newly designed hydrolysis probe to enhance its specificity. After comparison against other molecular and traditional methods, similar results were obtained regarding relative sensitivity, relative specificity, relative accuracy, positive and negative predictive values and index kappa of concordance (all higher than 91%), as well as a very low limit of detection (2 cfu/25 g). Additionally, after the analysis of more than 160 different food and environmental samples, its applicability in the food industry was completely demonstrated.

In-house validation of a multiplex real-time PCR method for simultaneous detection of Salmonella spp., Escherichia coli O157 and Listeria monocytogenes.

A wide variety of qPCR methods currently exist for Salmonella spp., Escherichia coli O157 and Listeria monocytogenes detection. These methods target several genes and use different detection chemistries, either in simplex or in multiplex formats. However, the majority of these methods have not been carefully validated, and the number of validated methods that use multiplex qPCR is even lower. The aim of the present study was to develop and validate a multiplex qPCR method from previously validated simplex qPCR primers and probes. A modified broth medium was selected and primary and secondary enrichment times were further optimized. Efficiency of the newly combined qPCR system was comprised between 91% and 108%, for simplex and multiplex analyses. A total of 152 food and environmental, natural and spiked samples, were analyzed for the evaluation of the method obtaining values above 91% that were reached for all the quality parameters analyzed. A very low limit of detection (5 cfu/25 g after enrichment) for simultaneous identification of these 3 pathogens was obtained.

Quantitative determination of the okadaic acid toxins group by a colorimetric phosphatase inhibition assay: interlaboratory study.

An interlaboratory collaborative study to validate a colorimetric phosphatase inhibition assay for quantitative determination of the okadaic acid (OA) toxins group in molluscs, OkaTest, was conducted. Eight test materials, including mussels, scallops, clams, and cockles, were analyzed as blind duplicates. Blank samples and materials containing different OA toxin levels ranging from 98 to 275 microg/kg OA equivalents were included. The study was carried out by a total of 16 laboratories from 11 different countries. Values obtained for repeatability relative standard deviations (RSDr) ranged from 5.4 to 11.2% (mean 7.5%). Reproducibility RSD (RSD(R)) values were between 7.6 and 13.2% (mean 9.9%). The Horwitz ratio (HorRat) values ranged between 0.4 and 0.6. A recovery assay was also carried out using a sample spiked with OA. A mean recovery of 98.0% and an RSD of 14.5% were obtained. The results obtained in this validation study indicate that the colorimetric phosphatase inhibition assay, OkaTest, is suitable for quantitative determination of the OA toxins group. OkaTest could be used as a test that is complementary to the reference method for monitoring the OA toxins group.

In vitro approaches to evaluate toxicity induced by organotin compounds tributyltin (TBT), dibutyltin (DBT), and monobutyltin (MBT) in neuroblastoma cells.

The toxic effects of the organotin compounds (OTCs) monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) were evaluated in vitro in a neuroblastoma human cell line. Mechanisms of cell death, apoptosis versus necrosis, were studied by using several markers: inhibition of cell viability and proliferation, F-actin, and mitochondrial membrane potential changes as well as reactive oxygen species (ROS) production and DNA fragmentation. The most toxic effects were detected with DBT and TBT even at very low concentrations (0.1-1 µM). In contrast, MBT induced lighter cytotoxic changes at the higher doses tested. None of the studied compounds stimulated propidium iodide uptake, although the most toxic chemical, TBT, caused lactate dehydrogenase release at the higher concentrations tested. These findings suggest that in neuroblastoma, OTC-induced cytotoxicity involves different pathways depending on the compound, concentration, and incubation time. A screening method for DBT and TBT quantification based on cell viability loss was developed, allowing a fast detection alternative to complex methodology.

Cytotoxic activity of extracts of marine sponges from NW Spain on a neuroblastoma cell line.

Six species of marine sponges collected at intertidal and sublittoral sites of the coast of Galicia (NW Spain) were screened for potential cytotoxic properties on Neuroblastoma BE(2)-M17 cell line. Exposure to Halichondria panicea, Pachymatisma johnstonia, Ophlitaspongia seriata and Haliclona sp. aqueous extracts strongly affected cell appearance, inducing loss of neuron-like morphology and the formation of clumps. Extracts from these species also caused significant rates of cell detachment and decrease of mitochondrial membrane potential. Incubation with P. johnstonia, O. seriata and Suberites massa extracts also decreased the rate of cell proliferation. The increase of incubation time enhanced propidium iodide uptake by neuroblastoma cells. Toxic responses triggered by sponge extracts are compatible with apoptotic phenomena in neuroblastoma cells, even though increasing propidium uptake at long periods of exposure might indicate the induction of secondary necrosis. The cytotoxic properties of the tested extracts suggest the presence of compounds with potential pharmacological or biotechnological applications in the screened sponge species.

Cyclic imines: chemistry and mechanism of action: a review.

In recent years, there has been an increase in the production of shellfish and in global demand for seafood as nutritious and healthy food. Unfortunately, a significant number of incidences of shellfish poisoning occur worldwide, and microalgae that produce phycotoxins are responsible for most of these. Phycotoxins include several groups of small to medium sized natural products with molecular masses ranging from 300 to over 3000 Da. Cyclic imines (CIs) are a recently discovered group of marine biotoxins characterized by their fast acting toxicity, inducing a characteristic rapid death in the intraperitoneal mouse bioassay. These toxins are macrocyclic compounds with imine (carbon-nitrogen double bond) and spiro-linked ether moieties. They are grouped together due to the imino group functioning as their common pharmacore and due to the similarities in their intraperitoneal toxicity in mice. Spirolides (SPXs) are the largest group of CIs cyclic imines that together with gymnodimines (GYMs) are best characterized. Although the amount of cyclic imines in shellfish is not regulated and these substances have not been categorically linked to human intoxication, they trigger high intraperitoneal toxicity in rodents. In this review, the corresponding chemical structures of each member of the CIs and their derivatives are reviewed as well as all the data accumulated on their mechanism of action at cellular level.

Effects of environmental regimens on the toxin profile of Alexandrium ostenfeldii.

Environmental conditions are key factors in the development of marine toxic phytoplankton. Spirolides are marine toxins with a heptacyclic imine ring responsible for the toxicity in mice. Alexandrium ostenfeldii (A. ostenfeldii) is the main producer of these toxins, although this dinoflagellate often produces toxins belonging to the paralytic shellfish poisoning (PSP) group. The present study shows the first evidence that external environmental factors can influence the toxin profile produced by the dinoflagellate A. ostenfeldii. The species investigated is indigenous to the North Atlantic coast, and their cells grew under several environmental parameters. Toxin production was measured by means of liquid chromatography-mass spectrometry (LC-MS) and the chromatograms reflect the presence of two spirolides in all cultures; one in the region m/z 692.5, corresponding to 13-desmethyl spirolide C (13-desMeC) and the other in the region m/z 678.5, which corresponds to 13,19-didesmethyl spirolide C (13,19-didesMeC). The physical parameters studied were salinity, culture media, and photoperiod. The highest amount of toxin per cell was obtained when dinoflagellates grew in F/2 and Walne medium, 28 per thousand salinity, and 24 h of light. However, the highest proportion of 13,19-didesMeC with respect to 13-desMeC was achieved in L1 medium, 33 per thousand salinity, and 14:10 h light:dark. On the contrary, the highest proportion of 13-desMeC in cells was obtained when A. ostenfeldii was cultured in F/2 medium, 28 per thousand salinity, and the same photoperiod. Therefore, from these data the optimum conditions to culture A. ostenfeldii and to obtain the highest amount of spirolide per cell are shown. In addition, these environmental conditions can be considered a tool to predict and avoid A. ostenfeldii blooms.

Comparison between a TaqMan polymerase chain reaction assay and a culture method for ctx-positive Vibrio cholerae detection.

The main objective of the present work was to evaluate a real-time polymerase chain reaction (PCR) method to detect toxigenic Vibrio cholerae in Pangasius hypophthalmus, a freshwater fish cultured mainly in South East Asia. A FDA traditional culture method and a real-time PCR method of the ctx gene were used for detection of V. cholerae in spiked samples of pangasius fish. After an overnight enrichment of samples at 37 degrees C in alkaline peptone water, 2 cfu/25 g of fish was detected with both methods. Although both methods were very sensitive, obtaining results with culture methods may take several days, while real-time PCR takes only a few hours. Furthermore, with traditional methods, complementary techniques such as serotyping, although not available for all serogroups, are needed to identify toxigenic V. cholerae. However, with real-time PCR, toxigenic serogroups are detected in only one step after overnight enrichment.

Comparative evaluation of enzyme-linked immunoassay and reference methods for the detection of shellfish hydrophilic toxins in several presentations of seafood.

A comparative study was conducted to determine the feasibility of enzyme-linked immunosorbent assays (ELISAs) for the detection of amnesic shellfish poisoning (ASP) and paralytic shellfish poisoning (PSP) toxins in nine naturally contaminated species in fresh, frozen, boiled and canned fish and shellfish. PSP and ASP were analyzed in 138 shellfish samples (mussels, clams, barnacles, razor shells, scallops and cockles) and anchovies by mouse bioassay (MBA) and high performance liquid chromatography with ultraviolet detection (HPLC-UV), respectively. Results were compared with toxin concentrations obtained using two commercial competitive ELISAs, saxitoxin and ASP kits. Immunoassays were able to quantify toxins in different matrices showing excellent Pearson's correlation coefficients (r = 0.974 for saxitoxin ELISA and r = 0.973 for ASP ELISA) and to detect PSP and ASP with a lower limit of detection (LOD), namely, 50 microg saxitoxin equivalent/kg shellfish meat for PSP and 60 microg/kg domoic acid in shellfish flesh for ASP, than the reference methods (350 microg saxitoxin equivalent/kg shellfish meat and 1.6 mg/kg domoic acid in shellfish flesh, respectively). These results suggest that the ELISA method could be used as screening systems in a variety of species without matrix interference.

Decrease of marine toxin content in bivalves by industrial processes.

Harmful algal blooms cause important economical losses due to the accumulation of toxins in shellfish. Natural detoxification occurs but this mechanism is very slow in most cases. The achievement of a method for the rapid detoxification of commercial bivalves would be very interesting for the shellfish harvesting sector in order to diminish economical losses due to harvesting areas closure. In this work, four different methods easily applicable in the food industry (freezing, evisceration, ozonization and thermal processing) were studied to gain the detoxification of four species of bivalves (mussels, scallops, clams and cockles) contaminated with the three main types of toxins (ASP, DSP, PSP). Results show that for ASP a significant decrease of the toxin levels below the legal limit (20 microg/g) is achieved by using hepatopancreas ablation or combination of simple steps (evisceration and/or thermal processing/and or freezing). In our hands, PSP toxin levels are sharply decreased under the limit of detection (35 microg STX eq/100g) after a thermal processing, inducing percentages of detoxification higher than 50%. The effect of freezing on the levels of PSP is very dependent on the matrix studied. DSP toxins are not significantly reduced with none of these methods.

Migration of BADGE (bisphenol A diglycidyl-ether) and BFDGE (bisphenol F diglycidyl-ether) in canned seafood.

Migration of potentially toxic materials used for the lining of commercial can goods remains an important issue, especially with respect to certain types of processed foods. Seafood is one type where more information is needed with respect to other ingredients used for adding value to fishery products. Most cans are internally coated with starters of resins such as bisphenol A diglycidyl-ether (BADGE) and bisphenol F diglycidyl-ether (BFDGE), both considered as toxic compounds. Several seafood products, sardines, tuna fish, mackerel, mussels, cod and mackerel eggs, were manufactured in different conditions changing covering sauce, time and temperature of storage and heat-treated for sterilization in cans. Migration kinetics of BADGE and BFDGE from varnish into canned products were evaluated by HPLC in 70 samples after 6, 12 or 18 months of storage. Results showed that there is no migration of BADGE in tuna fish, sardines, mussels or cod. However, migration of BFDGE occurs in all species, in a storage time-dependent way and content of fat, although migration of these compounds is not affected by sterilization conditions. All samples analyzed presented values lower than 9 mg BADGE/kg net product without exceeding European limits. However, concerning BFDGE migration, European legislation does not allow the use and/or the presence of BFDGE. Main migration takes place in mackerel reaching the highest values, 0.74 mg BFDGE/kg and 0.34 mg BADGE/kg net product, in red pepper sauce.