A stochastic approach for modelling the in-vitro effect of osmotic stress on growth dynamics and persistent cell formation in Listeria monocytogenes.

Persistent bacteria (or persisters) can be defined as a microbial subpopulation that, exposed to bactericidal treatment, is killed more slowly than the rest of the population they are part of. They stochastically originate in response to environmental stressors or spontaneously without external signals. When transferred into a fresh medium, persisters can resume active replication although they spend more time adapting to the new conditions remaining in the lag phase longer. They were studied for decades for their ability to survive antibiotic treatments while studies on their formation in food and potential impact on their safety are lacking. The most common food preservation techniques may act as stressors that trigger the formation of persistent bacteria able to survive bactericidal treatments and grow later in foods during storage. This study aimed to investigate a possible relationship between exposure to different salt concentrations (osmotic stress) and the amount of persisters triggered in a strain of Listeria monocytogenes. Furthermore, we described this phenomenon from a mathematical perspective through predictive microbiology models commonly used in the food field. The lag time distribution of a L. monocytogenes ATCC 7644 strain grown in broth with additional 2 %, 4 % and 6 % NaCl was evaluated using the software ScanLag. It uses office scanners to automatically record the colony growth on agar plates and evaluate the frequency distribution of their appearance times (lag phase) by automated image analysis. The same broth cultures were diluted to equalize salt concentration and transferred into a fresh broth to evaluate how the previous salt exposure impacted their growth kinetics. The observed growth curves were reproduced using predictive models in which the mean duration of the lag phase of the whole population took into account the occurrence of persisters with a longer lag phase. The models were solved first using a deterministic approach and then a stochastic one introducing a stochastic term that mimics the variability of lag phase duration due to the persisters occurrence. Results showed that the growth of L. monocytogenes in broth with additional NaCl might trigger the formation of persistent cells whose number increased consistently with salt concentrations. The proposed predictive approach reproduced the observed real curves in strong agreement, especially through the stochastic resolution of the models. Persistence is currently a neglected bacterial defence strategy in the food sector but the persisters' formation during production cannot be excluded; therefore, further insights on the topic are certainly desirable.

Quantitative Detection of Viable but Nonculturable Vibrio parahaemolyticus in Frozen Bivalve Molluscs.

Vibrio parahaemolyticus is a foodborne pathogen diffusely distributed in the marine environment and often isolated from raw seafood belonging to different species, mostly shellfish. Ingestion of under- or uncooked seafood contaminated by V. parahaemolyticus can cause severe gastrointestinal symptoms in humans. Due to its ability to withstand low temperatures, Vibrio spp. could survive in frozen seafoods for long periods by entering the viable but nonculturable state (VBNC) and may constitute an unrecognized source of food contamination and infection. In the present study, seventy-seven frozen bivalve molluscs (35 mussels; 42 clams) were subjected to the detection and enumeration of viable V. parahaemolyticus using standard culture methods. VBNC forms were detected and quantified by applying an optimized protocol based on Propidium Monoazide (PMA) and Quantitative PCR (qPCR). All samples were negative for both the detection and enumeration of V. parahaemolyticus by the standard culture methods. VBNC forms were detected in 11.7% of the samples (9/77), with values ranging from 1.67 to 2.29 Log CFU/g. Only clam samples were positive for the detection of VBNC forms. The results of this study highlighted that VBNC V. parahaemolyticus may be present in frozen bivalve molluscs. Further data on the prevalence of VBNC V. parahaemolyticus in frozen seafood are needed in order to perform a robust risk assessment.

Chemical Composition and Comprehensive Antimicrobial Activity of an Ethanolic Extract of Propolis from Tunisia.

In the present study, the chemical composition and the in vitro antimicrobial and antibiofilm activity of an ethanolic extract of propolis (EEP) from Tunisia against different ATCC and wild bacterial strains were evaluated. In situ antimicrobial activity and sensory influence of different EEP concentrations (0.5% and 1%), also in combination with 1% vinegar, were evaluated in chilled vacuum-packed salmon tartare. Furthermore, a challenge test was performed on salmon tartare experimentally contaminated with Listeria monocytogenes and treated with the different EEP formulations. The in vitro antimicrobial and antibiofilm activity was observed only against Gram-positive bacteria, such as L. monocytogenes and S. aureus, both ATCC and wild. Results of the in situ analyses revealed significant antimicrobial activity against aerobic colonies, lactic acid bacteria, Enterobacteriaceae and Pseudomonas spp. only when the EEP was used at 1% and in combination with 1% vinegar. The 1% EEP in combination with 1% vinegar was the most effective treatment also against L. monocytogenes, although 0.5% and 1% EEP used alone also showed antilisterial effects. After 7 days of storage, the sensory influence on odor, taste and color of salmon tartare was negligible for all EEP formulations. In this background, results obtained confirmed the antimicrobial efficacy of propolis which could be proposed as a suitable biopreservative to ensure safety and improve the quality of food.

Effects of osmotic stress on Listeria monocytogenes ATCC 7644: persistent cells and heat resistance.

Persistent bacteria are a microbial subpopulation that, exposed to bactericidal treatment, is killed at a slower rate than the rest of the population they are part of. They can be triggered either following stress or stochastically without external signals. The hallmark of persistent bacteria is the biphasic killing curve, a sign that, within a microbial population, two subpopulations are inactivated at a different rate. Furthermore, when plated into a fresh medium and in the absence of stressors, persistent bacteria typically remain in the lag phase longer before resuming active replication. This study aims to evaluate in vitro whether the formation of persistent cells in a strain of Listeria monocytogenes can be triggered by exposure to osmotic stress and if this phenomenon can increase heat resistance in the bacterial population. In a first experiment, the lag time distribution of a L. monocytogenes strain grown in a 6% NaCl broth was evaluated using the software ScanLag. A stationary phase broth culture was inoculated on agar plates placed on an office scanner inside an incubator at 37°C. The plates were scanned every 20' for 4 days and the acquired images were automatically elaborated with the aid of MatLab software in order to evaluate the appearance times of every single colony. The experiment was also carried out on a control culture obtained by growing the strain in the broth without salt. In a second experiment, the same broth cultures, after proper dilutions to rebalance NaCl concentration, were subjected to a heat treatment at 51°C and the death curves obtained were parameterized using the GinaFit system. Results showed that the lag phase of 31.40% of the salt culture colonies was long enough to suppose the formation of persistent bacteria. Analyses of the thermal survival curves showed that the shoulder and tail model was the one that best represented the inactivation trend of the salt culture, unlike the control culture, whose trend was essentially linear. Results of the present study show how exposure to salt could induce the formation of persistent bacteria in a L. monocytogenes strain. The last raises concerns as persistent cells may not only be undetected with common analytical techniques but they even show a greater heat resistance.

Temperature fluctuations along food supply chain: A dynamic and stochastic predictive approach to establish the best temperature value in challenge tests for Listeria monocytogenes.

This study aims to evaluate the behaviour of Listeria monocytogenes under fluctuating temperature comparing the efficacy of deterministic and stochastic methods for its prediction. In the first part of the study, a strain of L. monocytogenes was maintained at two different fluctuating temperature regimes both from 2 to 8°C and regularly sampled for the quantitative determination. The first temperature regime lasted 204 hours with a fluctuation length of 12 hours whereas the second lasted 167 hours with a fluctuation length of 24 hours. A dynamic predictive model was implemented for the reproduction of the observed data. Model resolution has been carried out by using values of the recorded temperature as well as the value of the mean temperature, the kinetic mean temperature, the 75th and 95th percentile of the temperature. A stochastic resolution was also performed considering the mean temperature and Standard Deviation as stochastic variable. In the second part of the study, a temperature mean curve was constructed by monitoring temperature of 8 refrigerated conveyances, 10 display cabinet and 15 domestic refrigerators. This curve was used to obtain predictive scenarios for L. monocytogenes based on the above and also considering temperature regime suggested by the EURL Lm TECHNICAL GUIDANCE DOCUMENT on challenge tests and durability studies for assessing shelf-life of ready-to-eat foods related to Listeria monocytogenes (Version 4 of 1 July 2021). All predicted behaviours were compared to the observed ones through the Root Mean Squared Error. Firstly, dynamic predictive model as well as the stochastic one, provided the best level of reproducibility of the observed data. The kinetic mean temperature reproduced the observed data better than the mean temperature for the 12 hoursregime while for the 24 hours-regime was the opposite. The 75th and 95th percentile overestimated the observed growths. Secondary, predictions obtained with the mean temperature, kinetic temperature and stochastic approach well fitted the observed data. The 75th and 95th percentile of Temperature and the "Eurl LM" temperature regimes overestimated the observed prediction. Dynamic approach as well as the stochastic one allowed to obtain the lowest values of Root Mean Squared Error. The mean temperature and kinetic mean temperature appeared the most representative values in a deterministic "single-point" approach.

Development of a predictive model for the shelf-life of Atlantic mackerel (Scomber scombrus).

Despite its commercial value, the shelflife of the Atlantic mackerel (Scomber scombrus) during refrigerated storage was poorly investigated. In this regard, the Quality Index Method (QIM) was proposed as a suitable scoring system for freshness and quality sensorial estimation of fishery products. This study aims to develop a deterministic mathematical model based on dynamic temperatures conditions and a successive statistical analysis of the results obtained. This model will be exploited to predict the shelf-life of the Atlantic mackerel based on specific storage temperatures. A total of 60 fresh fishes were subdivided into two groups and respectively stored in ice for 12 days at a constant temperature of 1±0.5°C (Group A) and a fluctuating temperature ranging between 1 and 7°C (Group B). Microbiological analysis and sensory evaluation through the QIM were performed on each fish at regular time intervals. A critical value of 6 Log cfu/g of spoilage bacteria (mainly psychotropic) associated with a significant decay of the sensorial characteristics was exceeded after 9 days of storage for Group A and 3 days for Group B. A reliable prediction of fish freshness was obtained by modelling the QIM as a function of the spoilage bacteria behaviour. A coefficient ß of correlation was determined to convert the spoilage bacteria load into a Quality Index score. The adoption of mathematical predictive models to assess microbial behaviour under different environmental conditions is an interesting tool for food industries to maximize production and reduce waste.

Microplastics in vacuum packages of frozen and glazed icefish (Neosalanx spp.): A freshwater fish intended for human consumption.

It is widely accepted that human is exposed to microplastics through food consumption, however data occurrence in foodstuffs are still little and basically limited to seafood. In this study, the presence of microplastics was investigated in icefish (Neosalanx spp.) samples sourced from various mass-market retailers in Italy, supplied as frozen, glazed and vacuum-packed product. Icefish is a small freshwater fish widely imported in Europe from China as surrogate of other fish species subjected to commercial restriction, consumed whole after cooking in several culinary preparation. The samples (~10 g of icefish from each of the 40 packs tested) were digested using a solution of 10% potassium hydroxide and filtered through a 5 µm pore-size filter. Filters of the samples were observed under a stereomicroscope and the chemical composition of the items detected were analysed by FTIR spectroscopy. A total of 163 items were counted in 37 (92.5%) samples with a mean value of 0.42±0.28 items/g w.w. Fibers were the most detected morphotype and several plastic polymers, such as polypropylene, polyethylene, polyethylene terephthalate and polystyrene, were identified by FT-IR analysis. As store-bought samples, the sources of microplastics could be substantially related to contamination during food processing. However, an intravital exposure to microplastics present in the surroundings waters cannot be ruled out. More foodstuffs need to be investigated for microplastic presence. In this study, microplastic occurrence was reported in freshwater biota intended for human consumption sampled directly from supermarket contributing to the risk assessment of human exposure to microplastics via food consumption.

Characterization of the temperature fluctuation effect on shelf life of an octopus semi-preserved product.

The aim of this work is to study the effect of temperature fluctuations on spoilage microbial flora behaviour of a semi-preserved seafood product in modified atmosphere packaging (MAP) as well as to find correct interpretation criteria for simulating temperature fluctuations during storage tests. The study concerned 54 packages of "Octopus carpaccio" that were grouped in three batches and stored at 3 different temperature profiles: the first (16 packages - Group 4°C) was stored at 4±0.5°C; the second (16 packages - Group 8°C) was stored at 8±0.5°C; the third (16 packages - Group F) was stored under a fluctuating temperature regime between 2°C and 14°C. Spoilage microflora, pH and AW has been monitored, at regular intervals, along the storage period (44 days). A predictive model was constructed according to the accredited scientific literature and validated against the observed growth curves of the above three groups. Afterwards, the predictive model has been used setting the temperature at the mean value of fluctuations (6.72°C), at the kinetic mean value of fluctuations (7.80°C) and at the 75th percentile value of fluctuations (11.14°C). The best fitting to the observed data was obtained with the kinetic mean temperature value and this result shows that this parameter can be proposed to reproduce the temperature fluctuation along the distribution and the domestic storage when a storage test has to be carried out.

Quality assessment of Zeus faber (Peter's fish) ovaries regularly commercialized for human consumption.

In the last few years, the consumption of fish eggs has increased rapidly, finding widespread use also in mass catering. This increase has involved also those of the Peter's fish (Zeus faber). Females of this species, by their reproductive characteristics, have highly developed gonads in different periods of the year, making the raw material easy to find. The aim of the present study was to perform a quality assessment of Zeus faber ovaries regularly commercialized for human consumption. A total number of 34 samples, divided in fresh (11) and frozen (23), were processed for microbiological characterization, parasitological and histological evaluations. Fresh and frozen samples have significant (P<0.01) differences in total bacterial charge, with values of 4.75±0.5 Log CFU/g and 3.65±0.7 Log CFU/g respectively. The mean value of Enterobacteriaceae was 2.58±0.7 Log CFU/g in fresh products, while 52.17% (12) of frozen samples reported loads of <1 Log CFU/g. No Salmonella spp. and Listeria monocytogenes were found. Aeromonas spp. was detected in two frozen sample (with loads of 2.2 and <1 Log CFU/g) and in 5 fresh ovaries with value ranged from 1.70 to 3.48 Log CFU/g. Vibrio spp. was found in 4 (36.36%) and 3 (13.04%) of fresh and frozen samples respectively, with loads always <1 Log CFU/g. All 31 Vibrio strains isolated, were identified as Vibrio alginolyticus, and 61.29% (19) of them was positive for the ToxRS factor and 6.45% (2) for ToxR. The 47.06% (16) of total samples showed infestations by larvae of Anisakis Type 1 in the serous and inside the ovary. In this last case, histologically it was found to be free larvae. This study attested satisfactory hygiene conditions for Zeus faber ovaries currently marked for human consumption. The presence of potentially pathogenic strains of V. alginolyticus and Aeromonas spp., but above all the frequent infestation by Anisakis larvae, represent a potentially hazard for the consumer.

Study on microbial communities in domestic kitchen sponges: Evidence of Cronobacter sakazakii and Extended Spectrum Beta Lactamase (ESBL) producing bacteria.

Domestic environment, in particular, kitchen setting is a well-established source of microbial contamination. Kitchen sponges represent an important vehicle of microbial transmission and maintenance of spoilage bacteria and pathogenic strains responsible for food borne diseases. The aim of this study was to evaluate the microbial communities of 100 'in-use' kitchen sponges, improving the knowledge on their role in cross-contamination in domestic environment and transmission of ESBLproducing strains. Sponges were processed for: aerobic mesophilic bacteria (AMB), Enterobacteriaceae (EB), yeasts and molds (YM), coagulase-positive staphylococci (CPS), micrococci (MCC), anaerobic sulfite reducing bacteria (ASR), and for the detection of Listeria monocytogenes, Salmonella spp. and Yersinia enterocolitica. A total of 309 enterobacteria strains were identified and then processed for ESBL (Extended Spectrum Beta Lactamase) phenotypical expression. A high contamination level of kitchen sponges was observed (mean value AMB 8.25±1.1; EB 5.89±1.2; YM 5.57±1.1; MCC 4.82±0.1 log CFU/g). Identified enterobacteria strains revealed several opportunistic and pathogenic agents such as Enterobacter cloacae (28%), Citrobacter freundii (23.3%), Cronobacter sakazakii (14.6%) and other strains in lower percentage. Listeria monocytogenes was found in only one sponge (1%). A total of 69 (22.3%) enterobacteria resulted ESBL+, with the following prevalence: P. rettgeri (50%), L. adenocarboxilata (30%), K. pneumoniae (25%), K. oxytoca (25%), C. sakazakii (20%), E. cloacae (20.7%), C. freundii (20.1%). Results confirm the potential role of kitchen sponges as vehicle for food-borne pathogens such as, C. sakazakii for the first time, infectious agents and spoilage microorganisms. The observed high contamination level and the presence of several ESBLs opportunistic pathogens, stresses the necessity to improve a proper education of the consumers on the effective treatment to reduce their microbial loads.

Use of Tunisian flavored olive oil as anisakicidal agent in industrial anchovy marinating process.

BACKGROUND: Natural compounds are more frequently used against Anisakis, responsible for the important fish-borne disease anisakidosis. The aim of the study was to evaluate the effectiveness of enriched Tunisian olive oil with different spices (cumin, turmeric, clove, thyme, and lemon) against Anisakis larvae type 1. RESULTS: In vitro experiment: larvae were submerged separately in the aforementioned oils and then examined to check viability. For each oil, LT50 and LT100 were calculated. Turmeric and cumin oils are the most effective against the parasites; followed by lemon, thyme and clove oils. For the in vivo experiment, turmeric and cumin oils were tested in anchovy fillets previously artificially parasitized with L3 larvae. Cumin was the most effective against parasites (dead after 5 days) compared with turmeric (8 days). For the two oils, the resulting odor was pleasant, as was the taste, while changes in color were much more evident in turmeric fillets. CONCLUSION: All the flavored oils demonstrated a good nematodical action against Anisakis. Cumin oil was the most effective against encysted larvae. Turmeric oil showed the best activity in the in vitro experiment. The use of flavored oils in the marinating process could represent an efficient strategy to devitalize Anisakis. © 2017 Society of Chemical Industry.

Exposure to Anisakis extracts can induce inflammation on in vitro cultured human colonic cells.

Anisakis spp. is a parasitic nematode whose infective third-stage larvae may be found within the flesh of fish species commonly consumed by humans. Thorough cooking or freezing should render the fish safe for consumption; furthermore, marinating solutions containing biocidal agents might have a significant action against Anisakis larvae. Some studies suggest a relationship between some parasitic infections and development of inflammatory bowel disorders, and Anisakis infection might be a risk factor for stomach or colon cancer. The aim of our study was to investigate if crude extracts (CEs) obtained from Anisakis larvae marinated in a solution with added allyl isothiocyanate (ACE-AITC) and frozen, or from frozen only Anisakis larvae (ACE), can induce an inflammatory effect on in vitro differentiated colonic Caco-2 cells exposed or not to LPS. Caco-2 exposure to the two CEs induced a marked COX-2 expression and potentiated LPS-induced COX-2 overexpression, confirming that substances present in Anisakis larvae can induce an inflammatory response in the intestinal epithelium, possibly also exacerbating the effects of other inflammatory stimuli. ACE induced a marked decrease in caspase-3 activation, while AITC-ACE increased its activation. However, LPS-induced caspase-3 activation appeared lower in cells treated with ACE and with the lower concentration of AITC-ACE. Thus, it is evident that Anisakis CEs may affect various cell pathways crucial not only in the inflammatory process but also in cell growth and death. Thus, CEs obtained from nonviable Anisakis larvae retain or are otherwise provided with noxious properties able to induce a strong inflammation response in intestinal epithelial cells. Furthermore, their influence may persist also following pretreatment with the biocidal agent AITC, indicating that the harmful substances contained in crude extracts from Anisakis larvae are resistant to the thermal or biocidal agent treatments.

A New Approach to Predict the Fish Fillet Shelf-Life in Presence of Natural Preservative Agents.

Three data sets concerning the behaviour of spoilage flora of fillets treated with natural preservative substances (NPS) were used to construct a new kind of mathematical predictive model. This model, unlike other ones, allows expressing the antibacterial effect of the NPS separately from the prediction of the growth rate. This approach, based on the introduction of a parameter into the predictive primary model, produced a good fitting of observed data and allowed characterising quantitatively the increase of shelf-life of fillets.

Activity of Tagetes minuta Linnaeus (Asteraceae) essential oil against L3 Anisakis larvae type 1.

OBJECTIVE: To evaluate in vitro effects of Tagetes minuta L. essential oil (TEO) on L3 Anisakis larvae type 1. METHODS: In order to evaluate the potential use of Tagetes minuta essential oil against L3 Anisakis larvae three different media were tested: 1) a saline solution (SS); 2) an industrial marinating solution (MS); 3) sunflower seeds oil (SO). For each media and concentrations of TEO (0.1%, 0.5%, 1.0% and 5.0% v/v), 20 parasites were introduced into plastic Petri dishes (diameter 90 mm) and maintained at room temperature. As controls, larvae were maintained without TEO under identical experimental conditions in SS, MS and SO. A total of 900 larvae were tested. The normalized mean viability, LT100, LT50 and the percentage of inactivation at 24 h were calculated. RESULTS: In vitro tests revealed a complete inactivation of parasites in saline solution after 2 h with 5% and 1% of TEO. In marinating solution, a complete inactivation of parasites was observed after 4 h at all concentrations used. A slower activity for all TEO concentration was reported in SO. CONCLUSIONS: The results obtained, showing a strong activity against Anisakis larvae, confirm TEO as a larvicidal agent in the treatment of human anisakidosis and in the industrial marinating process.

Prevalence and quantification of thermophilic Campylobacter spp. in Italian retail poultry meat: Analysis of influencing factors.

Retail poultry meat is a crucial vehicle for consumers' exposure to Campylobacters, but no official controls are currently applied in Italy. The aim of this study was the evaluation of Campylobacter contamination of a wide range of poultry meats marketed in Italy. N. 472 chicken and turkey meat samples (sectioned meats, offal, meat preparations and products) were taken from slaughterhouses, deboning plants and different retailers and submitted to detection/enumeration of Campylobacter spp. The isolates were identified by phenotypic and biomolecular techniques. Campylobacter spp. was detected in 34.1% of the samples, with general low counts. Higher values were observed in offal (especially liver) and sectioned meats, with significantly higher rates in skin-on samples (86.8% vs 32.7%). Minced meat preparations showed lower prevalence (22.4% vs 58.3%) and counts than whole pieces. Decreasing rates were observed among slaughterhouses (80%), deboning plants (49%), butcher's shops (37%) and large scale retailers (25%). Sectioned chicken meats were significantly more contaminated than turkey meats. Almost all the isolates were identified as C. jejuni or C. coli, with similar prevalences (18.4% and 20.5%, respectively); C. jejuni was predominant only in samples from slaughterhouses/deboning plants. For setting future control programs, meat typology should be considered the main critical factor.

Activity of R(+) limonene on the maximum growth rate of fish spoilage organisms and related effects on shelf-life prolongation of fresh gilthead sea bream fillets.

R(+)limonene (LMN) is the major aromatic compound in essential oils obtained from oranges, grapefruits, and lemons. The improvement of preservation techniques to reduce the growth and activity of spoilage microorganisms in foods is crucial to increase their shelf life and to reduce the losses due to spoilage. The aim of this work is to evaluate the effect of LMN on the shelf life of fish fillets. Its effectiveness was preliminarily investigated in vitro against 60 strains of Specific Spoilage Organisms (SSOs) and then on gilt-head sea bream fillets stored at 2±0.5°C for 15days under vacuum. LMN showed a good inhibitory effect against tested SSOs strains. On gilt-head sea bream fillets, LMN inhibited the growth SSOs effectively, and its use resulted in a shelf-life extension of ca. 6-9days of treated fillets, compared to the control samples. The LMN addition in Sparus aurata fillets giving a distinctive smell and like-lemon taste to fish fillets that resulted pleasant to panellists. Its use contributed to a considerable reduction of fish spoilage given that the fillets treated with LMN were still sensory acceptable after 15days of storage. LMN may be used as an effective antimicrobial system to reduce the microbial growth and to improve the shelf life of fresh gilt-head sea bream fillets.

Evaluation of the Antibacterial Activity of Bergamot Essential Oils on Different Listeria Monocytogenes Strains.

Essential oils are aromatic and volatile substances extracted from plants and characterized by antimicrobial activity. The aim of the present study was to evaluate the antibacterial activity (agar disc-diffusion method) of seven different bergamot essential oils (BEOs) on eight Listeria monocytogenes strains. Minimal inhibitory concentration (MIC) of most efficient BEOs was estimated. Extremely variable results for agar disc-diffusion method for L. monocytogenes strains were reported. One of the tested microorganisms resulted insensible to all the BEOs; 3 strains showed an inhibition from weak to null and the remaining 4 a variable susceptibility. Among the BEOs tested, one showed a strong activity against four pathogenic strains. Four BEOs revealed weak, moderate or null activity in all the 7 sensitive strains, while for two oils only a weak or no activity was reported. MIC values were 0.625 µL/mL for the most efficient BEO, 2.5 and 5 µL/mL for the other samples that showed moderate inhibition. Experiment results are significantly related to the strains tested (P<0.01), rather than the BEO employed (P>0.01). In conclusion, we can consider BEO as a natural technological hurdle for Listeria monocytogenes in combination with other preservation strategies. Finally, this study underlines the necessity to evaluate the antimicrobial activity of EOs on a significant strains number of the same bacteria.

Preliminary Investigation on the Use of Allyi Isothiocyanate to Increase the Shelf-Life of Gilthead Sea Bream (Sparus Aurata) Fillets.

The aim of this work is to evaluate the activity of allyl isothiocyanate (AITC) against fish spoilage bacteria (specific spoilage organisms; SSOs) as well as its possible use in gilthead sea bream (Sparus aurata) fillets to extend their shelf-life. In this regard, in vitro tests are carried out in order to evaluate the inhibitory activity of AITC and its vapours on several strains of SSOs. The AITC effect on the shelf-life of sea bream fillets was made by putting them in plastic trays hermetically closed with the addition AITC. Microbiological and sensorial evaluations were made on fish fillets during storage. Treated fillets maintained microbial populations at a significantly lower level compared with the control samples during storage, showing better sensorial characteristics. Therefore, the use of AITC's vapours seems to be a new and interesting alternative way to increase fish product shelf-life.

Activity of R(+) Limonene Against Anisakis Larvae.

The aim of this work is to evaluate the activity of R(+) limonene of against Anisakidae larvae. Its effectiveness was tested in vitro. The results obtained showed a significant activity of the compound against Anisakis larvae, suggesting further investigation on its potential use in the industrial marinating process. In this regard, the use of R(+) limonene in seafood products could be interesting, also due the sensory attributes resulting from its use and its relatively safe status.