Panagiota Katikou

Effect of rosemary extract, chitosan and α-tocopherol on microbiological parameters and lipid oxidation of fresh pork sausages stored at 4 °C

The effect of rosemary extract, chitosan and α-tocopherol, added individually or in combination, on microbiological parameters and lipid oxidation of fresh pork sausages stored for 20days at 4°C was investigated. Microbiological determinations included the enumeration of Enterobacteriaceae, Pseudomonas spp., total viable count, yeasts and moulds and lactic acid bacteria, while pH values were also determined. Lipid oxidation was evaluated through measurement of primary (peroxide value) and secondary (malondialdehyde concentration) oxidation products. Chitosan addition resulted in significant (P⩽0.05) inhibition of microbial growth, while the lowest microbial counts were obtained in the samples containing both chitosan and rosemary, indicating a possible synergistic effect. Chitosan and its combinations with either α-tocopherol or rosemary, and especially the latter combination, showed also the most intense antioxidative effect (P⩽0.05), at least when compared to α-tocopherol alone or the controls, while use of rosemary extract alone had a comparable (P>0.05) antioxidative effect with chitosan and its combinations. Shelf life of samples containing chitosan was almost doubled compared to the remaining samples. In conclusion, the best antimicrobial and antioxidative effects were obtained from the combination of chitosan with the rosemary extract.

Occurrence of palytoxin-group toxins in seafood and future strategies to complement the present state of the art.

Palytoxin (PlTX) and palytoxin-like (PlTX-like) compounds in seafood have been raising scientific concern in the last years. The constant increase in record numbers of the causative dinoflagellates of the genus Ostreopsis together with the large spatial expansion of this genus has led to intensification of research towards optimization of methods for determination of PlTX presence and toxicity. In this context, identification of seafood species which could possibly contain PlTXs constitutes an important issue for public health protection. In the present paper, worldwide occurrence of PlTX-like compounds in seafood is reviewed, while potential future strategies are discussed. PlTX has been reported to be present in several species of fish, crustaceans, molluscs and echinoderms. In one occasion, PlTX has been identified in freshwater puffer fish whereas all other records of PlTXs refer to marine species and have been recorded in latitudes approximately between 43°N and 15°S. PlTX determination in seafood has relied on different methodologies (mainly LC-MS, mouse bioassay and hemolysis neutralization assay) that have evolved over time. Future recommendations include systematic screening of PlTX in those species and areas where PlTX has already been recorded implementing updated methodologies.

First report on toxicity assessment of the Lessepsian migrant pufferfish Lagocephalus sceleratus (Gmelin, 1789) from European waters (Aegean Sea, Greece)

According to the current European Union legislative requirements (Regulation 853/2004/EC; Regulation 854/2004/EC, poisonous fish of the family Tetraodontidae and products derived from them must not be placed on the European markets. Following the increased publicity regarding the presence of the pufferfish species Lagocephalus sceleratus in Greek waters, this study was undertaken in order to confirm its toxicity and assess the risk of poisoning in case of accidental consumption. Acidic extracts from tissues of L. sceleratus specimens of different sizes were examined by means of the official mouse bioassay for tetrodotoxin, while some of the extracts were also tested for the presence of Paralytic Shellfish Poisoning (PSP) toxins with a commercial ELISA kit. Toxicity in mice, with symptomatology indicative of tetrodotoxin, was confirmed in a number of samples and indicated a correlation with fish size. Toxicity of certain tissues (liver, gonads, gastrointestinal tract) in larger individuals, expressed as microg/g tetrodotoxin equivalents, was largely above levels required to cause death in human adults. On the other hand, all tested extracts provided a positive reaction in the ELISA test for PSP toxins. This constitutes the first report for presence of toxicity in L. sceleratus caught in European coastal waters.

First Detection of Tetrodotoxin in Greek Shellfish by UPLC-MS/MS Potentially Linked to the Presence of the Dinoflagellate Prorocentrum minimum

During official shellfish control for the presence of marine biotoxins in Greece in year 2012, a series of unexplained positive mouse bioassays (MBA) for lipophilic toxins with nervous symptomatology prior to mice death was observed in mussels from Vistonikos Bay–Lagos, Rodopi. This atypical toxicity coincided with (a) absence or low levels of regulated and some non-regulated toxins in mussels and (b) the simultaneous presence of the potentially toxic microalgal species Prorocentrum minimum at levels up to 1.89 × 103 cells/L in the area’s seawater. Further analyses by different MBA protocols indicated that the unknown toxin was hydrophilic, whereas UPLC-MS/MS analyses revealed the presence of tetrodotoxins (TTXs) at levels up to 222.9 μg/kg. Reviewing of official control data from previous years (2006–2012) identified a number of sample cases with atypical positive to asymptomatic negative MBAs for lipophilic toxins in different Greek production areas, coinciding with periods of P. minimum blooms. UPLC-MS/MS analysis of retained sub-samples from these cases revealed that TTXs were already present in Greek shellfish since 2006, in concentrations ranging between 61.0 and 194.7 μg/kg. To our knowledge, this is the earliest reported detection of TTXs in European bivalve shellfish, while it is also the first work to indicate a possible link between presence of the toxic dinoflagellate P. minimum in seawater and that of TTXs in bivalves. Confirmed presence of TTX, a very heat-stable toxin, in filter-feeding mollusks of the Mediterranean Sea, even at lower levels to those inducing symptomatology to humans, indicates that this emerging risk should be seriously taken into account by the EU to protect the health of shellfish consumers.

Detection of okadaic acid and related esters in mussels during diarrhetic shellfish poisoning (DSP) episodes in Greece using the mouse bioassay, the PP2A inhibition assay and HPLC with fluorimetric detection.

An approach involving chemical, functional and biological techniques was taken for the detection and quantification of the marine toxin okadaic acid (OA) in mussels from Thermaikos and Saronikos Gulfs, Greece, during DSP episodes that occurred in 2006-2007. Samples were analyzed using the mouse bioassay, high performance liquid chromatography with fluorimetric detection (HPLC-FLD), using l-bromoacetylpyrene (BAP), as a precolumn derivatisation reagent, and the protein phosphatase 2A inhibition assay (PP2AIA) using a commercially available kit. Okadaic acid (OA) and its polar and non-polar esters were detected and quantified by HPLC-FLD, after hydrolysis of the samples during preparation. The detection limit of the HPLC method for OA was 5.86 microg OA/kg, which permits this method to be used for the regulatory control of these toxins in shellfish. Comparison of the results by all three methods revealed excellent consistency.

Specific and dynamic detection of palytoxins by in vitro microplate assay with human neuroblastoma cells

Palytoxin is one of the most complex and biggest molecules known to show extreme acute toxicity. The dinoflagellate Ostreopsis spp., the producer organism of palytoxin, has been shown to be distributed worldwide, thus making palytoxin an emerging toxin. Rat-derived hepatocytes (Clone 9) and BE (2)-M17 human neuroblastoma cells were used to test palytoxin or palytoxin-like compounds by measuring the cell metabolic rate with Alamar Blue. The dose-dependent decrease in viability was specifically inhibited by ouabain in the case of BE (2)-M17 neuroblastoma cells. This is a functional, dynamic and simple test for palytoxins with high sensitivity (as low as 0.2 ng/ml). This method was useful for toxin detection in Ostreopsis extracts and naturally contaminated mussel samples. A comparative study testing toxic mussel extracts by LC (liquid chromatography)-MS/MS (tandem MS), MBA (mouse bioassay), haemolysis neutralization assay and a cytotoxicity test indicated that our method is suitable for the routine determination and monitoring of palytoxins and palytoxin-like compounds.

Determination of okadaic acid, dinophysistoxin-1 and related esters in Greek mussels using HPLC with fluorometric detection, LC-MS/MS and mouse bioassay.

An approach involving both chemical and biological methods was undertaken for the detection and quantification of the marine toxins okadaic acid (OA), dinophysistoxin-1 (DTX-1) and their respective esters in mussels from different sampling sites in Greece during the period 2006-2007. Samples were analyzed by means of a) high performance liquid chromatography with fluorometric detection (HPLC-FLD), using 9-athryldiazomethane (ADAM), as a pre-column derivatization reagent, b) liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and c) the mouse bioassay. Free OA and DTX-1 were determined by both HPLC-FLD and LC-MS/MS, while their respective esters were determined only by LC-MS/MS after alkaline hydrolysis of the samples. The detection limit (L.O.D.) and quantification limit (L.O.Q.) of the HPLC-FLD method were 0.015 microg/g HP and 0.050 microg/g HP, respectively, for OA. The detection limit (L.O.D.) and quantification limit (L.O.Q.) of the LC-MS/MS method were 0.045 microg/g HP and 0.135 microg/g HP, respectively, for OA. Comparison of results between the two analytical methods showed excellent agreement (100%), while both HPLC-FLD and LC-MS/MS methods showed an agreement of 97.1% compared to the mouse bioassay.

Effect of Lactobacillus-protective cultures with bacteriocin-like inhibitory substances¿ producing ability on microbiological, chemical and sensory changes during storage of refrigerated vacuum-packaged sliced beef

Aims:  To investigate the effect of applying two different Lactobacillus‐protective cultures, with bacteriocin‐like inhibitory substances’ (BLIS) producing ability, individually or in combination, on microbiological, chemical and sensory changes during storage of refrigerated vacuum‐packaged sliced beef meat.

The association of bacterial C9-based TTX-like compounds with Prorocentrum minimum opens new uncertainties about shellfish seafood safety

In 2012, Tetrodotoxin (TTX) was identified in mussels and linked to the presence of Prorocentrum minimum (P. minimum) in Greece. The connexion between TTX and P. minimum was further studied in this paper. First, the presence of TTX-producer bacteria, Vibrio and Pseudomonas spp, was confirmed in Greek mussels. In addition these samples showed high activity as inhibitors of sodium currents (INa). P. minimum was before associated with neurotoxic symptoms, however, the nature and structure of toxins produced by this dinoflagellate remains unknown. Three P. minimum strains, ccmp1529, ccmp2811 and ccmp2956, growing in different conditions of temperature, salinity and light were used to study the production of toxic compounds. Electrophysiological assays showed no effect of ccmp2811 strain on INa, while ccmp1529 and ccmp2956 strains were able to significantly reduce INa in the same way as TTX. In these samples two new compounds, m/z 265 and m/z 308, were identified and characterized by liquid chromatography tandem high-resolution mass spectrometry. Besides, two TTX-related bacteria, Roseobacter and Vibrio sp, were observed. These results show for the first time that P. minimum produce TTX-like compounds with a similar ion pattern and C9-base to TTX analogues and with the same effect on INa.

Warm Seawater Microalgae: Growth and Toxic Profile of Ostreopsis Spp. from European Coasts

Palytoxin (PLTX) is a complex marine toxin synthesized by the soft coral Palythoa toxica and by species of the benthic dinoflagellate Ostreopsis spp. The toxin binds to the active Na,K-ATPase pump in the cellular membrane. This interaction changes the protein conformation and produces a non-specific cation channel. Using the Fluorescent Polarization (FP) technique to quantify the PLTX concentration, the production of PLTX-like products was measured in several cultures of Ostreopsis ovata and Ostreopsis siamensis. The cultures were grown under different conditions to study the optimal parameters to grow and to produce toxins. Serious difficulties were found to quantify the number of cells at the end of the exponential phase. In order to avoid any toxin loss, the weight of the pellet obtained after careful filtration was used as reliable parameter to calculate the growth of the cultures. Also, this parameter was used to refer the results of toxin concentration. In addition the toxicity of the cultures was measured by mouse bioassay. In these conditions, the optimal parameters to grow these strains are 24°C, 37% of salinity and 16:8 h light-dark photoperiod. Within these parameters high amounts of PLTX-like compounds with different toxin profiles were obtained.