Gut microbiota fingerprinting as a potential tool for tracing the geographical origin of farmed mussels (Mytilus galloprovincialis).

Identifying the provenance of seafood is critical to combat commercial fraud, enforce food safety regulations and ensure consumers' confidence. Hence, the current study aimed to determine if the bacterial composition present in the digestive gland and stomach of M. galloprovincialis mussels could be used as traceability approach to discriminate their geographic origin. The microbiota of 160 mussels collected seasonally in 2019 from five different mussel farms located in three regions in Spain (Galicia, Basque Country and Catalonia) was characterized using 16S rRNA targeted amplicon sequencing. Results showed that the bacterial community composition/fingerprint was significantly different between harvesting locations and seasons, with the effect prompted by the origin exceeding the seasonal variability. To further evaluate the stability and potential of this traceability approach, the bacterial fingerprint of 20 new individuals collected from the Basque Country in autumn 2020 were compared to the profiles obtained in 2019. Results showed that mussels collected from the Basque Country in two consecutive years cluster together, even matching the season of harvesting. The findings of this preliminary study support that this methodological approach has the potential to trace the geographical origin of unprocessed mussels and could have potential uses in seafood traceability and food safety.

Characterization of the heteropolysaccharides produced by Liquorilactobacillus sicerae CUPV261 and Secundilactobacillus collinoides CUPV237 isolated from cider.

Some lactic acid bacteria (LAB) strains isolated from alcoholic beverages are able to produce exopolysaccharides (EPS). The present work focuses on the physico-chemical characterization of the heteropolysaccharides (HePS) produced by Liquorilactobacillus sicerae CUPV261T (formerly known as Lactobacillus sicerae) and Secundilactobacillus collinoides CUPV237 (formerly known as Lactobacillus collinoides) strains isolated from cider. Genome sequencing and assembly enabled the identification of at least four putative HePS gene clusters in each strain, which correlated with the ability of both strains to secrete EPS. The crude EPS preparation from CUPV261T contained glucose, galactose and rhamnose, and that of CUPV237 was composed of glucose, galactose and N-acetylglucosamine. Both EPS were mixtures of HePS of different composition, with two major soluble components of average molecular weights (Mw) in the range of 106 and 104 g.mol-1. These HePS were resistant to gastric stress conditions in an in vitro model, and they significantly reduced zebrafish larvae mortality in an in vivo model of inflammatory bowel disease.

Evidence of IgE-Mediated Cross-Reactions between Anisakis simplex and Contracaecum osculatum Proteins.

Fish consumers may develop allergic reactions following the ingestion of fish products containing nematode larvae within the genus Anisakis. Sensitized patients may cross-react with proteins from insects, mites and mollusks, leading to allergic reactions even in the absence of the offending food. Potential cross-reactivity in Anisakis-allergic patients with larval proteins from other zoonotic parasites present in freshwater and sea fish should be investigated due to an increasing occurrence in certain fish stocks, particularly Contracaecum osculatum. In this work, we evaluated IgE-cross reactions by in vivo (skin prick tests with parasites extracts) and in vitro methods (IgE-ELISA and IgE-immunoblot). In vivo skin prick tests (SPT) proved the reactivity of Anisakis-sensitized patients when exposed to C. osculatum antigens. Sera from Anisakis-sensitized patients confirmed the reaction with somatic antigens (SA) and excretory/secretory proteins (ES) from C. osculatum. Only anecdotal responses were obtained from other freshwater worm parasites. Consequently, it is suggested that Anisakis-sensitized humans, especially patients with high levels of specific anti-Anisakis antibodies, may react to C. osculatum proteins, possibly due to IgE-mediated cross-reactivity.

Negligible risk of zoonotic anisakid nematodes in farmed fish from European mariculture, 2016 to 2018.

BackgroundThe increasing demand for raw or undercooked fish products, supplied by both aquaculture and fisheries, raises concerns about the transmission risk to humans of zoonotic fish parasites. This has led to the current European Union (EU) Regulation No 1276/2011 amending Annex III of Regulation (EC) No 853/2004 and mandating a freezing treatment of such products. Zoonotic parasites, particularly anisakid larvae, have been well documented in wild fish. Data on their presence in European aquaculture products, however, are still scarce, except for Atlantic salmon (Salmo salar), where the zoonotic risk was assessed as negligible, exempting it from freezing treatment.AimTo evaluate the zoonotic Anisakidae parasite risk in European farmed marine fish other than Atlantic salmon.MethodsFrom 2016 to 2018 an observational parasitological survey was undertaken on 6,549 farmed fish including 2,753 gilthead seabream (Sparus aurata), 2,761 European seabass (Dicentrarchus labrax) and 1,035 turbot (Scophthalmus maximus) from 14 farms in Italy, Spain and Greece. Furthermore, 200 rainbow trout (Oncorhynchus mykiss) sea-caged in Denmark, as well as 352 seabream and 290 seabass imported in Italy and Spain from other countries were examined. Fish were subjected to visual inspection and candling. Fresh visceral organs/fillet samples were artificially digested or UV pressed and visually examined for zoonotic anisakid larvae.ResultsNo zoonotic parasites were found in any of the fish investigated.ConclusionsThe risk linked to zoonotic Anisakidae in the examined fish species from European mariculture appears negligible. This study laid the groundwork for considerations to amend the current EU regulation.

Standalone direct pumping photovoltaic system or energy storage in batteries for supplying irrigation networks. Cost analysis.

Solar photovoltaic systems have become one of the most popular topics in the water management industry. Moreover, irrigation networks are water- and energy-hungry, and utility managers are likely to adapt water consumption (and consequently energy demand) to the hours in which there is energy availability. In countries such as Spain (with high irradiance values), solar energy is an available green alternative characterised by zero electricity costs and significantly lower environmental impact. In this work, several types of irrigation scheduled programmes (according to different irrigation sectors) that minimise the number of photovoltaic solar panels to be installed are studied; moreover, the effects of the variable costs linked to energy (energy and emissions costs) are presented. Finally, the effect of incorporating batteries for storing energy to protect the system against emergencies, such as unfavourable weather, is proposed. The irrigation hours available to satisfy water demands are limited by sunlight; they are also limited by the condition that the irrigation schedule type has to be rigid (predetermined rotation) and that the pressure at any node has to be above minimum pressure required by standards. A real case study is performed, and the results obtained demonstrate that there is no universal solution; this is because the portfolio of alternatives is based on investments for purchasing equipment at present and also on future energy savings (revenues). Apart from these two values, there is an economic value (equivalent discontinuous discount rate), which also influences the final results.

Dextran production by Lactobacillus sakei MN1 coincides with reduced autoagglutination, biofilm formation and epithelial cell adhesion.

In this work we have investigated two dextran-producing lactic acid bacteria, Lactobacillus sakei MN1 and Leuconostoc mesenteroides RTF10, isolated from fermented meat products. These bacteria synthesise dextran when sucrose, but not glucose, is present in the growth medium. The influence of dextran on bacterial aggregation, adhesion and biofilm formation was investigated in cultures challenged with sucrose or glucose. For Lb. sakei MN1, the synthesis of the dextran drastically impaired the three processes; in contrast it had no effect on Lc. mesenteroides RTF10. Therefore, the influence of dextran on probiotic properties of Lb. sakei MN1 was tested in vivo using gnotobiotic zebrafish models. The bacterium efficiently colonised the fish gut and inhibited the killing activity of Vibrio anguillarum NB10[pOT11]. Furthermore, under conditions of dextran synthesis, the adhesion of Lb. sakei MN1 to the epithelial cells decreased, without greatly affecting its anti V. anguillarum activity.

Development of a multiplex PCR-ELISA method for the genetic authentication of Thunnus species and Katsuwonus pelamis in food products.

In the present work a PCR-ELISA technique for the authentication of Thunnus species was developed. This method is composed by four systems that can be used in a hierarchical way allowing the identification of several scombroids species; or each individual system independently. The hierarchical strategy, proposes a first step, to assign one sample to the Thunnus genus. Next, if the result is positive, several tests can be applied to assign the sample to some particular species of the Thunnus genus. In the case that the result is negative (absence of Thunnus species), it is possible to verify if Katsuwonus pelamis is included in the sample. The method even allows the detection of mixtures of these species in relatively low amounts (up to 1%). Finally, this method was applied to 11 commercial samples to verify the labelling status of tuna products in the market, detecting that 18% were mislabelling.

Evaluation of a dual-probe real time PCR system for detection of mandarin in commercial orange juice.

A dual-probe real time PCR assay, based on the simultaneous detection of two TaqMan® probes, was evaluated for the detection of mandarin in orange juice. A single conserved polymorphism, located at the 314 position of intron belongs to chloroplast trnL gene, was confirmed by sequencing in 30 mandarin, 28 orange cultivars and 13 hybrids. The assay was also successfully evaluated in a blind trial against analysing 60 samples from different industrial processes in different countries around the world. The detection limit of the assay was established in 1% presence of mandarin detectable in processed orange juice and with a 100% precision. The quantitative application of the assay on citrus mixtures was also investigated, pointing out that the number of chloroplast DNA copies is too variable for its possible use as quantitative analysis. This assay can be employed as a routine methodology to control the accidental mixing during industrial processes and to deter intentional fraud.

Use of gnotobiotic zebrafish to study Vibrio anguillarum pathogenicity.

We evaluated the use of the gnotobiotic zebrafish system to study the effects of bacterial infection, and analyzed expression of genes involved in zebrafish innate immunity. Using a GFP-labeled strain of Vibrio anguillarum, we fluorescently monitored colonization of the zebrafish intestinal tract and used gene expression analysis to compare changes in genes involved in innate immunity between nongnotobiotic and gnotobiotic larvae. The experiments performed with the gnotobiotic zebrafish reveal new insights into V. anguillarum pathogenesis. Specifically, an alteration of the host immune system was detected through the suppression of a number of innate immune genes (NFKB, IL1B, TLR4, MPX, and TRF) during the first 3 h post infection. This immunomodulation can be indicative of a "stealth mechanism" of mucus invasion in which the pathogen found a sheltered niche, a typical trait of intracellular pathogens.

Toxic effects of colloidal nanosilver in zebrafish embryos.

A variety of consumer products containing silver nanoparticles (Ag NPs) are currently marketed. However, their safety for humans and for the environment has not yet been established and no standard method to assess their toxicity is currently available. The objective of this work was to develop an effective method to test Ag NP toxicity and to evaluate the effects of ion release and Ag NP size on a vertebrate model. To this aim, the zebrafish animal model was exposed to a solution of commercial nanosilver. While the exposure of embryos still surrounded by the chorion did not allow a definite estimation of the toxic effects exerted by the compound, the exposure for 48 h of 3-day-old zebrafish hatched embryos afforded a reliable evaluation of the effects of Ag NPs. The effects of the exposure were detected especially at molecular level; in fact, some selected genes expressed differentially after the exposure. The Ag NP toxic performance was due to the combined effect of Ag(+) ion release and Ag NP size. However, the effect of NP size was particularly detectable at the lowest concentration of nanosilver tested (0.01 mg l(-1)) and depended on the solubilization media. The results obtained indicate that in vivo toxicity studies of nanosilver should be performed with ad hoc methods (in this case using hatched embryos) that might be different depending on the type of nanosilver. Moreover, the addition of this compound to commercial products should take into consideration the Ag NP solubilization media.

Multiple SNP markers reveal fine-scale population and deep phylogeographic structure in European anchovy (Engraulis encrasicolus L.).

Geographic surveys of allozymes, microsatellites, nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) have detected several genetic subdivisions among European anchovy populations. However, these studies have been limited in their power to detect some aspects of population structure by the use of a single or a few molecular markers, or by limited geographic sampling. We use a multi-marker approach, 47 nDNA and 15 mtDNA single nucleotide polymorphisms (SNPs), to analyze 626 European anchovies from the whole range of the species to resolve shallow and deep levels of population structure. Nuclear SNPs define 10 genetic entities within two larger genetically distinctive groups associated with oceanic variables and different life-history traits. MtDNA SNPs define two deep phylogroups that reflect ancient dispersals and colonizations. These markers define two ecological groups. One major group of Iberian-Atlantic populations is associated with upwelling areas on narrow continental shelves and includes populations spawning and overwintering in coastal areas. A second major group includes northern populations in the North East (NE) Atlantic (including the Bay of Biscay) and the Mediterranean and is associated with wide continental shelves with local larval retention currents. This group tends to spawn and overwinter in oceanic areas. These two groups encompass ten populations that differ from previously defined management stocks in the Alboran Sea, Iberian-Atlantic and Bay of Biscay regions. In addition, a new North Sea-English Channel stock is defined. SNPs indicate that some populations in the Bay of Biscay are genetically closer to North Western (NW) Mediterranean populations than to other populations in the NE Atlantic, likely due to colonizations of the Bay of Biscay and NW Mediterranean by migrants from a common ancestral population. Northern NE Atlantic populations were subsequently established by migrants from the Bay of Biscay. Populations along the Iberian-Atlantic coast appear to have been founded by secondary waves of migrants from a southern refuge.

Characterization and feasibility of a miniaturized stirred tank bioreactor to perform E. coli high cell density fed-batch fermentations.

The use of small scale bioreactors that are mechanically and functionally similar to large scale reactors is highly desirable to accelerate bioprocess development because they enable well-defined scale translations. In this study, a 25-mL miniaturized stirred tank bioreactor (MSBR) has been characterized in terms of its power input, hydrodynamics, and volumetric oxygen transfer coefficient (k(L)a) to assess its potential to grow high cell density (HCD) cultures using adequate scale-down criteria. Engineering characterization results show scale down, based on matched specific power input (P(G)/V), is feasible from a 20-L pilot scale stirred tank bioreactor. Results from fed-batch fermentations performed using Fab' producing E. coli W3110 at matched (P(G)/V) in the MSBR and 20-L STR demonstrated that the MSBR can accurately scale down the 20-L fermentation performance in terms of growth and Fab' production. Successful implementation of a fed-batch strategy in the MSBR resulted in maximum optical density of ca. 114 and total Fab' concentration of 940 µg/mL compared with ca. 118 and 990 µg/mL in 20-L STR. Furthermore, the use of the MSBR in conjunction with primary recovery scale-down tools to assess the harvest material of both reactors showed comparable shear sensitivity and centrifugation performance. The conjoint use of the MSBR with ultra scale-down (USD) centrifugation mimics can provide a cost-efficient manner in which to design and develop bioprocesses that account for good upstream performance as well as their manufacturability downstream.

Growth and productivity impacts of periplasmic nuclease expression in an Escherichia coli Fab' fragment production strain.

Host cell engineering is becoming a realistic option in whole bioprocess strategies to maximize product manufacturability. High molecular weight (MW) genomic DNA currently hinders bioprocessing of Escherichia coli by causing viscosity in homogenate feedstocks. We previously showed that co-expressing Staphylococcal nuclease and human Fab' fragment in the periplasm of E. coli enables auto-hydrolysis of genomic DNA upon cell disruption, with a consequent reduction in feedstock viscosity and improvement in clarification performance. Here we report the impact of periplasmic nuclease expression on stability of DNA and Fab' fragment in homogenates, host-strain growth kinetics, cell integrity at harvest and Fab' fragment productivity. Nuclease and Fab' plasmids were shown to exert comparable levels of growth burden on the host W3110 E. coli strain. Nuclease co-expression did not compromise either the growth performance or volumetric yield of the production strain. 0.5 g/L Fab' fragment (75 L scale) and 0.7 g/L (20 L scale) was achieved for both unmodified and cell-engineered production strains. Unexpectedly, nuclease-modified cells achieved maximum Fab' levels 8-10 h earlier than the original, unmodified production strain. Scale-down studies of homogenates showed that nuclease-mediated hydrolysis of high MW DNA progressed to completion within minutes of homogenization, even when homogenates were chilled on ice, with no loss of Fab' product and no need for additional co-factors or buffering.

A phage display system for the identification of novel Anisakis simplex antigens.

Anisakis simplex has been recognized as an important cause of disease in man and as a foodborne allergen source. Actually, this food-borne was recently identified as an emerging food safety risk including allergenic symptoms. This parasite contains a large variety of allergenic proteins enforcing the necessity to detect new allergens. Commonly, these efforts have been focused on the developing of cDNA libraries, where virtually all expressed mRNAs are present, by using immunoreactive patient serum or polyclonal antibodies. Phage display system is an alternative strategy which permits the physical binding of the genotype with the phenotype, since the products are expressed by the phage on its surface, thereby allowing more efficient selection. In this work we have constructed two libraries in the pJuFo phage, obtaining a primary titer of around 103 cfu/ml and an amplified titer of the order of 1013 cfu/ml whereas the insert sizes varied from 0.35 to 1.2kb. Both libraries were subsequently analyzed by enrichment with polyclonal antibodies to an A. simplex extract and immunoreactive sera from patients with a clinical history of allergy to this parasite. Finally, 30 clones were scrutinized detecting several Anisakis candidate antigens. Actually, one protein, belongs to the fructose-1,6-bisphosphatase family, was found in 34% of scrutinized clones revealing as a promising novel A. simplex allergen. Phage display technology has to date not yet been applied to the identification of new A. simplex allergens, and the present work opens up new avenues to the understanding of the Anisakis allergenic process.

Evaluation of a real-time polymerase chain reaction (PCR) assay for detection of anisakis simplex parasite as a food-borne allergen source in seafood products.

Anisakis simplex has been recognized as an important cause of disease in humans and as a food-borne allergen source. Actually, this food-borne parasite was recently identified as an emerging food safety risk. An A. simplex -specific primer-probe system based on a real-time polymerase chain reaction (PCR) detection assay has been successfully optimized and validated with seafood samples. In addition, a DNA extraction procedure has been optimized to detect the presence of the nematode in food samples. The assay is a very reliable, specific, and sensitive methodology to detect the presence of traces of this parasite in seafood products, including highly processed samples. As a result, 13 sequences of cytochrome c oxidase II gene were obtained and scrutinized to calculate intra- and interspecific variabilities of 0 and 35-67%, respectively. Finally, an efficiency of 2.07 +/- 0.14 of the assay was calculated, and a limit of detection of 40 ppm parasite in 25 g of sample was also optimized. Actually, the presence of this parasite in several seafood products has been demonstrated, enforcing the necessity of a design for a good manufacturing practice protocol for the processing industry to minimize the presence of this parasite as a food-borne allergen source in seafood products.

Innate immune gene expression in individual zebrafish after Listonella anguillarum inoculation.

Zebrafish were intraperitoneally injected with 10(6)CFU (LD50) Listonella anguillarum. Three inoculated and control fish were collected at 1, 2, 4, 6 and 22h post infection (hpi) and the expression of genes related to the immune response (il1b, cebpb, tfa, mpx, tnfa, nitr9, tlr22, hsc70, cp, mrlp1, c3b and lyz) in each fish was monitored by means of real-time RT-PCR. A similar experiment was performed considering an intermediate time point at 15 hpi. Different relative levels of expression were found among genes. Also, wide interindividual variation in gene expression for most genes was detected among fish, inoculated or not. A steady increase of expression starting from the initial stages of the interaction was found for interleukin-1beta. An initial increase in levels of gene expression was found for the genes coding for the CCAAT/Enhancer Binding Protein subunit beta and the Novel Immune-Type Receptor 9, although their levels decreased later on and were indistinguishable from the controls at 22 hpi. Finally, some genes (Transferrin, Myeloid-specific Peroxidase and Tumour Necrosis Factor alpha) were upregulated at 22 hpi. Taken together, our results show an induction in gene expression of genes involved in the inflammatory and immune response upon L. anguillarum infection but also reveal the existence of a wide variation in the levels of expression of the studied genes in the zebrafish population.

Application of relative quantification TaqMan real-time polymerase chain reaction technology for the identification and quantification of Thunnus alalunga and Thunnus albacares.

A novel one-step methodology based on real-time Polymerase Chain Reaction (PCR) technology has been developed for the identification of two of the most valuable tuna species. Nowadays, species identification of seafood products has a major concern due to the importing to Europe of new species from other countries. To achieve this aim, two specific TaqMan systems were devised to identify Thunnus alalunga and Thunnus albacares. Another system specific to Scombroidei species was devised as a consensus system. In addition, a relative quantification methodology was carried out to quantify T. alalunga and T. albacares in mixtures after the relative amount of the target was compared with the consensus. This relative quantification methodology does not require a known amount of standard, allowing the analysis of many more samples together and saving costs and time. The utilization of real-time PCR does not require sample handling, preventing contamination and resulting in much faster and higher throughput results.