[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Wild juvenile eels or “glass eels” are caught and used as “seed” in aquaculture production. Eel farming is responsible for over 90 % of all Anguilla production worldwide. Prior to 1990, eel farming was almost exclusively carried out using species of local provenance. However, the exploitation of some species, as A. japonica, has resulted in an increased of eels from European trade, as well as glass eels and other life stages, to supply the aquaculture industry. All eel aquaculture is still based on wild source seed, and this primary resource for aquaculture should be controlled, knowing at all times the origin of the seed that is acquired for farming. Morphological authentication of glass eels is very difficult due to the few diagnostic characters in early stages. For this reason, in the present study a fast real-time PCR methodology has been developed for A. anguilla authentication, Europe’s most valuable species. The methodology developed was performed and validated in adult and glass eels. The results show that this methodology is specific and efficient, and it can be apply to adult and glass freshwater eel authentication.
No preview · Article · Aug 2015 · European Food Research and Technology
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The identification of species presents in food is important due to many reasons. Some of them are economic
fraud and/or sanitary problems, which could arise from inaccurate identifications of the composition of food
products. Adulteration of processed meat generally occurs when there is a considerable price differential between raw materials from different species. Also, it occurs too when trying to introduce raw material of dubious origin, or even raw materials prohibited in some countries for cultural reasons.
For example, dog traces were discovered in pet food in Spain in early 2013. This work describes development
and validation of two real-time PCR methods, one for dog detection and other one for cat in foodstuffs. The method is based on specific primers/TaqMan probe sets that amplify a fragment of the Cytochrome oxidase subunit I sequences.
TaqMan probes allow rapid and accurate assessment of even very small quantities of contaminants, even in thermally treated products. The detection limit was 2 and 0.2 pg of the target DNA from dog and cat, respectively. The methodology was validated to check how the degree of food processing affects the applicability of this technique. Additionally, a total of 100 samples from pet food, farm animal products
and raw material were analyzed to apply the developed methodology to commercial samples. Possible applications of these methods are as process control, food security, traceability verification in commercial trade and the correct application of food labelling regulations. In conclusion, the real-time PCR methodology described herein represents a useful tool for the detection of dog and cat traces in all kind of products, including processed products that have undergone aggressive treatments (canned meals and feeds).
No preview · Article · Mar 2015 · European Food Research and Technology
[Show abstract][Hide abstract] ABSTRACT: This chapter revises current knowledge on marine biotoxins that are known nowadays, considering all the distinct groups based on chemical structure and lipophilic or hydrophilic characteristics. Diarrheic shellfish poisoning (DSP ), paralytic shellfish poisoning (PSP), amnesic shellfish poisoning (ASP ), azaspiracid shellfish poisoning (AZP ), ciguatera fish poisoning (CFP ) are some of the groups that will be reviewed in this chapter. The recent development and application of advanced technologies from the generically defined -omics sciences coupled with bioinformatics platforms has been included in this chapter in order to understand the ecology and evolution of phytoplankton species and bloom dynamics. Dinoflagellate toxins are structurally and functionally diverse, and many present unique biological activities. The literature and information regarding the biological activities and the potential application of these phycotoxins has been gathered in this book section.
[Show abstract][Hide abstract] ABSTRACT: Real-Time PCR is a very powerful tool with multiple applications in food microbiology, being pathogen detection the most important for food safety. In early January 2013 an increase in the incidence of Listeria monocytogenes was observed in a mussel-processing facility in the north-west of Spain. Then, a previously in-house validated qPCR method was applied to detect the contamination origin in the processing line. By the end of the same month a total of 62 samples from different spots in the processing plant were analyzed, obtaining 25 positive results by qPCR. All the isolates, identified as L. monocytogenes, presented the same biochemical profile and belonged to the same molecular typing group (Group 3). After the identification and elimination of the contamination source, 25 additional samples were analyzed over the following nine months, without any positive sample. Results obtained showed that the qPCR method is a suitable technique to identify the exact source of contamination that could appear in food processing plants, saving time respect to traditional culture methods.
[Show abstract][Hide abstract] ABSTRACT: Consumption of Lepidocybium flavobrunneum and Ruvettus pretiosus is related with the gastrointestinal disease called Keriorrhea. Sometimes, intentionally or not, these species are mislabelled as other harmless species, causing severe disruptions to consumers. The correct identification of these species helps to avoid food fraud and health problems. For this reason, a multiplex Real Time-PCR method based on TaqMan technology for the correct authentication of L. flavobrunneum and R. pretiosus has been developed. The method is based on a species-specific set of primers and TaqMan probe which amplifies a 276 bp fragment of the cytochrome oxidase I (COI) mitochondrial DNA region. This methodology allows your application to any type of product, regardless of the degree of processing it has undergone with high specificity, sensitivity and rapidity. Also, it might be a useful tool in monitoring and verifying labelling regulation and protect consumer rights.
[Show abstract][Hide abstract] ABSTRACT: Harmful algal blooms (HABs) are excessive accumulation of phytoplankton that produce biotoxins oradversely affect humans and animals. Among all microalgae, the genus Prorocentrum biosynthesizes toxins responsible for the human syndrome known as diarrhetic shellfish poisoning (DSP), one of the most frequent in Spanish coasts. HABs impact on the public health, fisheries, ecosystem and tourism have increased over the past few decades being an important and widespread problem. Therefore, the development of control methods to minimize the risks derived from these events is a matter of vital importance.
The use of clay for removing microalgae has been previously explored. Clay flocculation is based on the physical and chemical properties of the mineral particles in aqueous suspensions. In this preliminary work various types of clay were investigated to evaluate the adsorption and flocculation properties in seawater containing Prorocentrum lima dinoflagellates.
We set up water columns with cells of Prorocentrum lima from an established in vitro culture. Then we added different types of clays to a final concentration of 0.2%. The experimental results confirmed the cell removal through the flocculation of algal and mineral particles leading to the formation of aggregates, which rapidly settle and further entrain cells during their descent. The reduction in the number of cells was time dependent and noticeable within several minutes exposure to clays.Moreover, the microscopy images of the samples enable to observe the clays in aggregates of two or more cells where the mineral particles were bound to the outer membranes of the dinoflagellates.
Therefore, this preliminary data offers promising results to use these clays for the mitigation of HABs.
[Show abstract][Hide abstract] ABSTRACT: Food allergy is recognised as an important human health problem. Fish represent one of the most important causes of food hypersensitivity reaction. Small amounts of the allergen can cause severe reactions in sensitive individuals, so correct labelling is essential to ensure the protection of consumers. The objective of the present work was to develop a reliable, sensitive and specific real-time PCR method for the detection of fish and traces of fish in all kind of products included those that have undergone aggressive treatments such as high temperature or pressure. This methodology was validated simulating products likely to contain this allergen and spiking them with fish cooking water. In addition, a comparison between the performance of in-house methodology and a commercial kit, both of them based on real-time PCR, was carried out. This work is relevant because it is the first, rapid real-time PCR method developed to date for the detection of fish in processed food products. The results obtained confirm the present assay is a useful tool in detecting fish and, therefore, minimising exposure and reducing incidences of allergic reaction to fish in contaminated products.
[Show abstract][Hide abstract] ABSTRACT: Kadaic acid (OA) and its analogues, the dinophysis toxins DTX1, DTX2, and DTX3 together form the group of OA-toxins. These compounds are potent inhibitors of type 1 and type 2A serine/threonine phosphatases. It is well-known that protein phosphorylation is one of the main mechanisms by which cell functioning and proliferation are controlled. Modifications of cellular proteins phosphorylation, eventually leads to collapse of regulatory processes; in fact, several molecular alterations have been reported in cells challenged with OA. The process by which OA induces diarrhea in animals and humans is well known, however, the knowledge on the precise toxicological mechanisms of OA-toxins group is limited and comprises mostly studies on their acute toxicity whereas long-term toxicity remains relatively unknown. It is considered that OA acts as a direct cytotoxic and genotoxic agent for human cells and its effect depends on the cell model and the concentration. In addition, this toxin is known to act as a potent neurotoxin for cultured neurons, to induce apoptotic events in various cell lines, and to exert tumour promoting activity in various organs. The extent of the OAinduced injuries and the toxin organotrophicity are dose related and may be determined by the administration route. After intravenous administration, OA acts as a hepatotoxin with undetectable effects on the intestine but also has an impact on cytoskeletal elements at sublethal doses. In this paper the state-of-the-art on mechanisms of action of OA and its analogues, and the effects on chronic toxicity, carcinogenicity or genotoxicity are reviewed. The effect on cellular functions and the impact on human health, as well as theapplications of OA as a tool to investigate cellular metabolic processes providing new insights on strategies and targets in different pathologies are summarized.
[Show abstract][Hide abstract] ABSTRACT: Marine biotoxins or shellfish toxins are mainly produced by microalgae. Based on their solubility, they have been classified in lipophilic and hydrophilic. Most of these toxins accumulate in the digestive gland of filter-feeding shellfish and pose a health risk to humans when these molluscs are consumed. Other concern in shellfish is chemical contamination with heavy metals such as cadmium (Cd), lead (Pb) or mercury (Hg). Human activities, as agriculture and mining have added to naturally-occurring sources of heavy metals in the environment, including marine ecosystems. Influence of shellfish thermal processing on the levels and redistribution of these contaminants is not very well known. It has been described that processing, cooking, steaming or autoclaving can lead to an increase in the level of contaminants in shellfish meat correlated with water loss during cooking. Commission Regulation (EC) No 1881/2006 of 19 December 2006  setting maximum levels for certain contaminants in foodstuffs, allows to apply maximum levels of contaminants to dried, diluted, processed and compound foodstuffs, where no specific community maximum levels were established. Many contaminants are included in this regulation as mycotoxins, metals, dioxins, PCBs. In this context, food business operators should provide the specific concentration and dilution factors to be taken into account when legislation is applied. Regulation (EC) No 853/2004 of the European Parliament and of the Council of 29 April 2004  laying down specific hygiene rules for food or animal origin establishes maximum levels for marine biotoxins in live bivalve molluscs. Nevertheless, the initial level of lipophilic toxins can be modified by heat treatment, since it can increase after the processing. It was previously reported that concentrations increased correlated with water loss during steaming and it was observed distribution of OA and DTX2 from the digestive gland to the remainder tissues . This chapter will compile information related to the influence of shellfish processing on heavy metals and lipophilic marine biotoxins levels. Experimental data regarding the effect of shellfish heat treatment on these contaminants will be presented. Moreover, a discussion considering if it is appropriate to set maximum levels for marine biotoxins in cooked, steamed or canned shellfish will be included.
[Show abstract][Hide abstract] ABSTRACT: Part of the new IFST Advances in Food Science Series, Seafood Processing: Technology, Quality and Safety covers the whole range of current processes which are applied to seafood, as well as quality and safety aspects. The first part of the book (‘Processing Technologies’) covers primary processing, heating, chilling, freezing, irradiation, traditional preservation methods (salting, drying, smoking, fermentation, etc), frozen surimi and packaging. The subjects of waste management and sustainability issues of fish processing are also covered. In the second part (‘Quality and Safety Issues’), quality and safety analysis, fish and seafood authenticity and risk assessment are included.
[Show abstract][Hide abstract] ABSTRACT: Enteromyxum scophthalmi is a myxozoan parasite that causes severe parasitic diseases in cultured turbot affecting mainly the intestine of the host. It is characterized by producing acute enteritis, starvation and eventually death. Current diagnosis of E. scopthalmi use traditional techniques, based on the identification of the morphology of the parasite. These techniques take extended time to be carried out and do not favour the adoption of control measure at turbot farms and require the sacrifice of fish. This study develops a fast real-time PCR molecular tool for the detection of E. scophthalmi in infected farmed turbot. This methodology is applicable for routine controls on the farm at every stage of the parasite infection. Results of the study demonstrate the robustness, specificity, efficiency and reliability of the technique. In addition, this study also provides a non-invasive procedure of sampling through swaps. This allows control, prevention and diagnosis of the parasite infection at turbot farms while maintaining the welfare of the cultivated fish and avoiding sacrifice of the fish sampled.
No preview · Article · Jan 2014 · Aquaculture Research
[Show abstract][Hide abstract] ABSTRACT: The EU FP7 funded project \“Bio-engineered micro Encapsulation of Active
agents Delivered to Shellfish (BEADS)” is focused on mitigating the impact of
marine biotoxins (ASP/DSP), microbial contamination (bacteria/norovirus) and
the parasitic protozoan Bonamia ostreae on shellfish aquaculture.
Purpose: to develop probiotic diets and a microencapsulated delivery system
in the digestive tract of shellfish to improve depuration.
Feeding experiments were performed to identify the optimum size of alginate
microcapsules, testing three different sizes and colours containing nondegradable
fluorescent dye microbeads.
Oysters and mussels were placed in tanks containing filtered seawater. Three
sizes of microcapsules were mixed and shellfish were fed for 3 hours. Shellfish
were removed during the feeding period at 0.5, 1, 2 and 3 hours and dissected.
Any capsules remaining in the water and in the digestive organs of shellfish were
extracted and measured using a fluoroskan analyzer.
A higher concentration of the smaller capsules was found in the digestive
gland, indicating that the smaller capsules were preferentially ingested. The
ingestion increased during the three-hour period. Fluorescent beads of different
colours embedded in alginate capsules were observed bound to the mucus string.
Mussels were more efficient than oysters in incorporating alginate beads that
were observed by light microscopy as intact in the digestive gland of mussels
and oysters during feeding period. Broken alginate beads were found in faeces.
During passage through the intestine, active agents embedded by alginate
capsules are released into the digestive gland providing a useful tool to transport
[Show abstract][Hide abstract] ABSTRACT: In recent years the incidence of vibriosis has greatly increased, raising the concern among consumers about the innocuity of certain food products. Previous studies demonstrated various advantages of molecular methods, including qPCR, for the screening of food-borne pathogens. The new method developed in the present study allows fast and reliable detection of the main human pathogenic Vibrio species (Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus). Specificity of the combination of primers and probes was successfully tested against several bacterial species and strains (44 different strains). Evaluation of the qPCR efficiency reported a value of 94% with the simultaneous amplification of the internal amplification control. The evaluation of the quality of the method was based on six parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values as well as Kappa index of concordance. Each of the values obtained were higher than 96%. Additionally a very low limit of detection was determined for the developed method (less than 10 cfu/25 g). All the parameters of the method analyzed were obtained from the analysis of a wide variety of foodstuffs, water samples and reference material from proficiency tests, and compared against the culture reference method.