Vida Šimat

Molecular identification and population dynamic of Anisakis pegreffii (Nematoda: Anisakidae Dujardin, 1845) isolated from the European anchovy (Engraulis encrasicolus L.) in the Adriatic Sea

Anchovy Engraulis encrasicolus (L.) is a coastal pelagic and euryhaline species that represents the only European species of the family Engraulidae, with a widespread distribution. In Croatia, it is marketed fresh, frozen, salted or marinated and mainly exported to Italy and Spain, however Anisakis sp. larval infection is frequently the reason for border rejection. Since it is known that the prevalence and intensity of Anisakis infection varies with fish species, fishing area and season, the aim of our study was to identify Anisakis sp. parasitizing European anchovy and infer its population dynamic through a 2.5-year period. Larvae were found coiled and encysted on the external wall of intestine (94%) and reproductive organs (6%), rarely in fillets. Prevalence was 76.1% (95% confidence limits 74.51-77.56%), mean abundance 6.59 (bootstrap 95% confidence limits 5.81-7.26) and mean intensity 8.67 (bootstrap 95% confidence limits 7.82-9.35). The partial CO2 mitochondrial DNA sequence of the isolated anisakids confirmed clustering of the anchovy parasite within A. pegreffii sister group. Parasite population structure showed plasticity inferred by fishing ground, sampling year and fish gender and size. Compared to anisakid prevalence/abundance in other fish, the European anchovy in the Adriatic Sea represents a moderately high-infected paratenic host, although in the Mediterranean and Atlantic waters, anchovies have shown strikingly lesser values of prevalence. Since this host represents one of the most attractive Mediterranean fisheries products traditionally consumed without thermal preparation that in any case would not disrupt larval antigenicity and prevent human allergies, and given the high prevalence of the anisakid within the host, it is necessary to include anchovy into more firm risk assessment frames in order to develop measures that will support the safe alimentary production and consumption of seafood.

As, Cd, Hg and Pb in four edible shellfish species from breeding and harvesting areas along the eastern Adriatic Coast, Croatia.

Four species of shellfish (Mytilus galloprovincialis, Ostrea edulis, Chlamis varia and Venus verrucosa) were collected during the autumn 2011 and spring 2012 along the eastern Adriatic coast from six shellfish harvesting areas (all species) and 13 breeding sampling areas (mussels) to assess As, Cd, Hg and Pb levels and the human risks of shellfish consumption. The mean metal concentrations (wet weight) in the examined species ranged from 1.420 to 9.575mg/kg for As, 0.034 to 1.270 for Cd, 0.005 to 0.680 for Hg and 0.140 to 2.072 for Pb. Examination of the spatial distribution of As, Cd, Hg and Pb revealed statistically significant differences among the studied areas. Since the concentrations were below the maximum prescribed by the laws of the EU and Croatia (the concentrations slightly exceeded the upper limits for three samples; Pb, Cd and Hg) and the hazard index, (HI) for Cd, Hg and Pb were below 1 and the target cancer risk (TR) for As was lower than 1×10(-6), there is no human health risk of consumption of shellfish from Croatian waters.

Seasonal Variations of Phenolic Compounds and Biological Properties in Sage (Salvia officinalis L.)

The aim was to investigate the phenolic content, antioxidant capacity, and antibacterial activity of Dalmatian sage (Salvia officinalis L.) leaves collected during different vegetation periods. Separation and quantification of the individual phenols were performed by reversed‐phase (RP)‐HPLC coupled with a PDA (photodiode array) detector and using an internal standard, while the contents of total phenols, flavonoids, flavones, and flavonols were determined spectrophotometrically. The antioxidant properties of the sage leaf extracts were evaluated using five different antioxidant assays (FRAP, DPPH, ABTS, BriggsRauscher reaction, and β‐carotene bleaching). The antimicrobial activity of the extracts was tested against two Gram‐positive (Bacillus cereus and Staphylococcus aureus) and two Gram‐negative (Salmonella Infantis and Escherichia coli) bacterial reference strains. All extracts were extremely rich in phenolic compounds, and provided good antioxidant and antibacterial properties, but the phenophase in which the leaves were collected affected the phenolic composition of the sage extracts and consequently their biological activity. The May Extract, the richest in total flavonoids, showed the best antioxidant properties and the highest antimicrobial activity. Thus, collection of the plants during May seems the best choice for further use of them in the pharmaceutical and food industry.

Role of biogenic amines in the post-mortem migration of Anisakis pegreffii (Nematoda: Anisakidae Dujardin, 1845) larvae into fish fillets.

Infective third-stage larvae (L3) of nematode Anisakis spp. have been recognized as one of the major food-borne threats in lightly processed fish products in Europe, particularly in the Mediterranean region. Therefore, the effect of different storage temperatures of fish on larval post-mortem migration from visceral cavity into fillets is an important parameter to take into account when evaluating the risk for consumer safety. The European anchovy (Engraulis encrasicolus) were caught during fishing season, a subsample of fillets was checked for the presence of Anisakis larvae at capture (mean abundance=0.07), and the rest was stored at four different temperatures (-18, 0, 4 and 22°C) in order to count migrating larvae and measure the production of biogenic amines over a period of time. Larvae were identified by morphological features and molecular tools. Post-mortem migration was observed in fillets stored at 0 and 4°C after three and five days, respectively, but not at 22 and -18°C. In case of storage at 22°C for two days, at the onset of putrefaction of the visceral organs, larvae migrated out of the visceral cavity towards the fish surface. Measured pH and biogenic amine profile during storage indicated that certain biochemical conditions trigger larval migration into fillets. Likewise, migration was observed at pH ~6.4 when sensory degradation of the fish was markedly visible. Although larval migration was delayed for approximately four days at a temperature of <4°C the correlation between pH and abundance of A. pegreffii larvae in the fillet was high and statistically significant at both 0 (r=0.998, p<0.01) and 4°C (r=0.946, p<0.05). Out of eight biogenic amines measured, cadaverine and putrescine levels correlated the most with the post-mortem migration at 4°C, while tyramine levels were significant at both temperatures.

First report on TTX levels of the yellow spotted pufferfish (Torquigener flavimaculosus) in the Mediterranean Sea

ABSTRACT The differences of tetrodotoxin (TTX) levels in various parts of pufferfish (Torquigener flavimaculosus) were examined in conjunction with the seasonal and sexual variations. The TTX levels in gonads, liver, intestines, skin and muscle tissue were determined using the Q‐TOF LC/MS. Instrumental analysis revealed that all examined tissues from T. flavimaculosus contained high TTX concentrations. TTX levels in the gonads, liver, intestines, skin and muscle tissue of pufferfish were within the range of 5.03–100.71, 7.04–106.80, 12.59–86.30, 33.95–139.88 and 15.88–86.07 (&mgr;g/g), respectively. It was determined that in all seasons, except for summer, female individuals had higher TTX levels than males. Among all seasons, the highest level of TTX was found in winter and the lowest in autumn. Consequently, T. flavimaculosus is a highly toxic pufferfish that is dangerous for human consumption and should not be consumed under any circumstances. HIGHLIGHTSThe first report is on the TTX content of T. flavimaculosus (pufferfish) in the Mediterranean Sea.TTX content in pufferfish was determined by using the Q‐TOF LC/MS analysis.All seasons except for summer, female individuals had higher TTX levels than males.Regardless of gender, this species is a highly toxic pufferfish that is dangerous for human consumption.

Effect of Natural Zeolite (Clinoptilolite) on in vitro Biogenic Amine Production by Gram Positive and Gram Negative Pathogens

The effect of two levels of clinoptilolite (1 and 5%) on the production of biogenic amines (BA) and ammonia (AMN) by Gram positive (Staphylococcus aureus, Enterococcus faecalis, and Listeria monocytogenes) and Gram negative bacteria (Aeromonas hydrophila, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Salmonella Parathypi A), in tyrosine decarboxylase broth (TDB) was studied. A. hydrophila and E. coli produced the highest amounts of amines which were 1223.06 and 2627.90 mg/l, respectively. All strains were able to decarboxylate tyrosine to tyramine (TYR) with E. coli being the highest (1657.19 mg/l). A. hydrophila formed >50 mg/l histamine (HIS) while the other strains produced none or very low concentrations (<4 mg/l). Among Gram-positive pathogens, E. faecalis was characterized as the main amine producer (478.23 mg/l). Although dependent on bacterial strain and level used, the natural zeolite clinoptilolite can be used to decrease BA and AMN production by bacterial strains that are of health concern. Practical Applications: Uses of natural prodcuts for biogenic amines inhibition. Clinoptilolite was used to reduce the amounts of amines such as spermine, putrescine, and dopamine produced by pathogenic and spoilage bacteria.