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Salmonella spp. is known to survive in intermediate– and low–moisture foods. Bakery products such as cream-filled brioche (aw 0.82–0.84), depending mainly on the aw of the fillings and the baking they receive for food preservation, may support survival of the pathogen. The study aimed to model the inactivation of osmotically adapted and non–adapted Salmonella in cream–fillings (praline and biscuit) and cream-filled brioche at different storage temperatures. All matrices were inoculated with ca. 6.0 log CFU/g of osmotically adapted and non–adapted five–strain cocktail of Salmonella (Typhimurium, Agona, Reading, and Enteritidis) and stored aerobically in 120 mL screw-capped containers at 15, 20, and 30 °C. Adaptation of Salmonella was induced in cream–fillings (praline and biscuit) with aw adjusted to 0.88, by adding sterile water to each of the original fillings (aw 0.78–0.83) and incubating at 37 °C for 1 h. Survival of Salmonella was assessed at regular time intervals throughout storage using thin layer agar method to enhance the recovery of injured cells (n = 4). Inactivation curves were fitted best with the Weibull model using the freeware GInaFit tool and the estimated δ and β values were used to calculate the time for 4D reduction–t4D. Results showed that inactivation of Salmonella increased with temperature, while osmotic adaptation enhanced its survival in a food matrix–related manner. Higher survival rates of adapted cells were observed in cream–fillings (t4D: 79.9 ± 27.1 days on biscuit and 150.3 ± 19.6 days on praline) compared to brioche (t4D: 61.3 ± 0.9 days on biscuit and 52.5 ± 4.6 days on praline) at 20 °C. Secondary (linear) modelling of t4D showed that the survival of Salmonella was affected by temperature and osmotic adaptation. Model simulation of pathogen inactivation in independent trials on cream–fillings agreed well with observed data. In conclusion, the present data could be used as a means to identify areas for improving the performance of existing models quantifying the survival of Salmonella in bakery-confectionary products with intermediate aw.

The scope of the present study was to assess the spoilage potential of different Alicyclobacillus spp. in commercial pasteurized (ambient-stable) plant-based dairy beverages mixed with fruit juices at different inoculation levels and storage temperatures. Different products (coconut and berry [CB]; almond, mango, and passionfruit [AMP]; and oat, strawberry, and banana [OSB]) were inoculated with 10 or 2 × 103 spores per mL of either Alicyclobacillus acidoterrestris, Alicyclobacillus fastidiosus, or Alicyclobacillus acidocaldarius strain composites, whereas noninoculated samples served as controls. Samples inoculated with A. acidoterrestris and A. fastidiosus were stored at 30 and 45°C, whereas A. acidocaldarius storage took place at 50°C for 240 days. Gas composition, Alicyclobacillus spp. populations, total viable counts, pH, water activity, color, and guaiacol off-taste were monitored. CB and AMP supported growth of A. acidoterrestris and A. fastidiosus, reaching populations of 4.0 to 5.0 log CFU/mL. In OSB, populations of A. fastidiosus remained close to the initial inoculation levels during storage at 30°C, whereas at 45°C, the populations declined

The coexistence and interactions among Listeria monocytogenes strains in combination with the structural characteristics of foods, may influence their growth capacity and thus, the final levels at the time of consumption. In the present study, we aimed to evaluate the effect of oxygen availability in combination with substrate micro-structure on growth and inter-strain interactions of L. monocytogenes. L. monocytogenes strains, selected for resistance to different antibiotics (to enable distinct enumeration), belonging to serotypes 4b (C5, ScottA), 1/2a (6179) and 1/2b (PL25) and were inoculated in liquid (Tryptic Soy Broth supplemented with Yeast Extract — TSB-YE) and solid (TSB-YE supplemented with 0.6% and 1.2% agar) media (2–3 log CFU/mL, g or cm2), single or as two-strain cultures (1:1 strain-ratio). Aerobic conditions (A) were achieved with constant shaking or surface inoculation for liquid and solid media respectively, while static incubation or pour plated media corresponded to hypoxic environment (H). Anoxic conditions (An) were attained by adding 0.1% w/v sodium thioglycolate and paraffin overlay (for solid media). Growth was assessed during storage at 7 °C (n = 3 × 2). Inter-strain interactions were manifested by the difference in the final population between singly and co-cultured strains. Τhe extent of suppression increased with reduction in agar concentration, while the impact of oxygen availability was dependent on strain combination. During co-culture, in liquid and solid media, 6179 was suppressed by C5 by 4.0 (in TSB-YE under H) to 1.8 log units (in solid medium under An), compared to the single culture, which attained population of ca. 9.4 log CFU/mL or g. The growth of 6179 was also inhibited by ScottA by 2.7 and 1.9 log units, in liquid culture under H and An, respectively. Interestingly, in liquid medium under A, H and An, ScottA was suppressed by C5, by 3.3, 2.4 and 2.3 log units, respectively, while in solid media, growth inhibition was less pronounced. Investigating growth interactions in different environments could assist in explaining the dominance of L. monocytogenes certain serotypes.

The objectives of the study were to evaluate: (i) the efficacy of Na-alginate films immersed in 40% v/v distillery ethanol and traditional Greek alcoholic beverages (ABs), namely “tsipouro”, “raki”, and “ouzo” to inhibit Listeria monocytogenes growth on frankfurters and ham slices and (ii) the impact of edible films-ABs on heat sensitivity of the pathogen after microwave (MW) reheating of frankfurters in water during storage at 4 and 10 °C. Samples without edible films (controls) or with edible films but free of distillery ethanol or ABs were also tested. Two pieces of frankfurters were placed between two edible films, while every ham slice was covered by a film from both sides. On ham, all edible films immersed in ABs maintained levels of L. monocytogenes at least 5.0 log CFU/cm 2 lower (p L. monocytogenes population remained 4.0–4.5 log CFU/cm 2 lower (p 2 was observed after 15 days at 10 °C, at all assays except for samples packaged with raki. However, the results from reheating experiments revealed that subsequent inadequate MW reheating of frankfurters packaged with edible films-ABs would not lead to remarkable inactivation of the pathogen, since cross-protection against thermal stress is been acquired as a result of products' exposure to sub-lethal stresses such as low pH formed by metabolic products, indigenous microbiota, and ethanol exposure ( ca. 2.4–2.7% w/w) during cold storage. Our findings suggest that food safety regulations enabling antimicrobial interventions for the safety of ready to eat meat products should ensure both low levels of L. monocytogenes during storage and minimum tolerance to recommended consumer practices, such as MW reheating.

The present study aimed to develop a commercial active packaging system of ground beef, by exploiting the antimicrobial and antioxidant properties of a traditional Greek alcoholic distillate called &ldquo, tsipouro&rdquo, Commercial packages (500 g) were used and 40 mL of &ldquo, was added in absorbent pads placed underneath the ground beef, while 10 mL was also mounted under the packaging film, facing the headspace. Samples were packaged in 80% O2: 20% CO2 and stored at 0, 4, 8, and 12 &deg, C. Total Viable Counts, pseudomonads, Brochothrix thermosphacta, lactic acid bacteria, yeasts-moulds, pH, colour (L*, a*, b*), odour (buttery and acidic), and ethanol migration to ground beef (SPME/GC-FID) were determined. Moreover, mathematical models (square root and Arrhenius) describing the effect of temperature on determinant indicators of spoilage and quality deterioration like growth of dominant microorganisms and red colour reduction were developed and validated under non-isothermal conditions. B. thermosphacta dominated the microbial association of ground beef, while LAB were second in dominance, revealing a high growth potential at all assays. a* value (redness) was gradually decreased in controls, while samples treated with &ldquo, showed more stable red colour during storage. Although ethanol was organoleptically detectable, especially at low storage temperatures (0&ndash, 4 &deg, C), it was rather perceived as a pleasant cool odour. Prediction by both models for microbial growth as well as those of Arrhenius model for reduction of a* value showed good agreement with the observations under non-isothermal storage. Overall, our study showed that the developed antimicrobial active packaging of ground beef based on &ldquo, combined with high oxygen MAP lead to an almost 2-fold shelf-life extension compared with controls during storage at chill and abuse temperatures.

Fungi are common spoilers of intermediate moisture foods such as bakery products. Brioche are bakery products prone to fungal spoilage due to their pH (5.8-6.2) and water activity (aw) (0.82-0.84). The aims of the present study were: (i) the identification of fungal species occurring in brioche products, (ii) the in vitro assessment of their growth potential, and (iii) the development of a validated growth model following the gamma concept. A total of 102 fungal strains were isolated, with Penicillium sp., Cladosporium sp., and Aspergillus sp. being the main genera, representing 90% of the isolates. Given the isolation frequency, any potential fungal prevalence throughout the bakery processs and/or the results of in vitro assessment of fungal growth potential under conditions mimicking brioche (pH, aw, temperature), Aspergillus flavus, Aspergillus fumigatus, and Penicillium sp. were selected for the development of the gamma model. According to in vitro validation, the model successfully predicted fungal growth, while on in situ experiments, the intrinsic parameters (aw and/or level of used preservative) of brioche in combination with packaging conditions (modified atmosphere) did not allow fungal growth.

The objective of this study was to correlate the relative transcription of Listeria monocytogenes (strains C5, 6179) stress- (gad2, sigB) and virulence- (prfA) associated genes following habituation at twenty-five pH (4.8, 5.0, 5.2, 5.5, 6.4) and NaCl (2, 4, 6, 8, 10% w/v) combinations at 7 °C, with the survival against subsequent exposure to severe acid stress (pH 2.0 at 37 °C). Our findings pointed out the inter-strain variation governing growth inhibiting conditions (pH ≤5.0 and NaCl ≥6%), where C5 was less affected (a reduction of 2.0-3.0 log CFU/mL) than 6179 which was reduced by 4.0-6.0 log CFU/mL at the end of storage. Nevertheless, the higher the habituation at the growth permitting (pH ≥5.5; NaCl ≤4% w/v) or growth inhibiting conditions, the higher the acquired acid resistance or sensitization, respectively. At day 2, gad2 increased relative transcriptional levels are more related to elevated acid resistance, while at day 6 both gad2 transcriptional levels and upregulation of sigB were correlated to low log reductions and high DpH:2.0-values against severe acid stress. Regarding virulence, the increased transcriptional levels of prfA at day 2 were correlated to adverse pH and NaCl combinations, while prolonged stay in suboptimal conditions as well as exposure to severe acid stress resulted in general activation of the virulence regulator. Such data could definitely contribute in designing safe intervention strategies and additionally integrate -omics aspects in quantitative microbial risk assessment. (Less)

The present study aimed to evaluate the effect of AB vapors on microbial, physicochemical, and sensory profile of pork meat stored in different MAP conditions. Pork pieces (10g) and cotton/cellulose absorbent cloths (2×2cm) were placed into compartmentalized Petri-dishes in two sections. Aliquots (1mL) of water (control), 30% v/v and 40% v/v ethanol, whisky, brandy, tsipouro, raki, and ouzo were added separately to the cotton/cellulose absorbent cloths. Each pork sample was placed in one compartment and cotton/cellulose absorbent cloths supplemented with different ABs were placed in a separate compartment of each Petri-dish. Samples were packaged in 40% CO2: 30% O2: 30% N2 and 80% O2: 20% CO2 and stored at 4 and 10°C. Total viable counts, Pseudomonas sp., Brochothrix thermosphacta, lactic acid bacteria, yeasts and molds, and Enterobacteriaceae, were enumerated during storage. Changes in pH, color (L*, a*, b*), odor, taste, and overall appearance of pork meat were also evaluated along with changes in organic acid levels via HPLC. At 4°C, lactic acid bacteria and B. thermosphacta were the dominant organisms under 40% CO2: 30% N2: 30% O2 and 80% O2: 20% CO2, respectively, while at 10°C, lactic acid bacteria dominated in both MAP conditions. All applied ABs were effective (p

Analysis for Listeria monocytogenes by ISO11290-1 is time-consuming, entailing two enrichment steps and subsequent plating on agar plates, taking five days without isolate confirmation. The aim of this study was to determine if a polymerase chain reaction (PCR) assay could be used for analysis of the first and second enrichment broths, saving four or two days, respectively. In a comprehensive approach involving six European laboratories, PCR and traditional plating of both enrichment broths from the ISO11290-1 method were compared for the detection of L. monocytogenes in 872 food, raw material and processing environment samples from 13 different dairy and meat food chains. After the first and second enrichments, total DNA was extracted from the enriched cultures and analysed for the presence of L. monocytogenes DNA by PCR. DNA extraction by chaotropic solid-phase extraction (spin column-based silica) combined with real-time PCR (RTi-PCR) was required as it was shown that crude DNA extraction applying sonication lysis and boiling followed by traditional gel-based PCR resulted in fewer positive results than plating. The RTi-PCR results were compared to plating, as defined by the ISO11290-1 method. For first and second enrichments, 90% of the samples gave the same results by RTi-PCR and plating, whatever the RTi-PCR method used. For the samples that gave different results, plating was significantly more accurate for detection of positive samples than RTi-PCR from the first enrichment, but RTi-PCR detected a greater number of positive samples than plating from the second enrichment, regardless of the RTi-PCR method used. RTi-PCR was more accurate for non-food contact surface and food contact surface samples than for food and raw material samples especially from the first enrichment, probably because of sample matrix interference. Even though RTi-PCR analysis of the first enrichment showed less positive results than plating, in outbreak scenarios where a rapid result is required, RTi-PCR could be an efficient way to get a preliminary result to be then confirmed by plating. Using DNA extraction from the second enrichment broth followed by RTi-PCR was reliable and a confirmed result could be obtained in three days, as against seven days by ISO11290-1.

Different physicochemical and microbiological characteristics of cheeses may affect Listeria monocytogenes potential to grow, survive, or exhibit an acid adaptive response during storage and digestion. The objectives of the present study were to assess: i) the survival or growth potential of L.monocytogenes on various cheeses during storage, ii) the effect of initial indigenous microbiota on pathogen growth in comparison to expected growth curves retrieved by existing predictive models, and iii) the impact of habituation on/in cheeses surfaces on the subsequent acid resistance during simulated gastric digestion. Portions of cream (Cottage and Mascarpone), soft (Anthotyros, Camembert, Mastelo®, Manouri, Mozzarella, Ricotta), and semi-hard (Edam, Halloumi, Gouda) cheeses were inoculated with ca. 100CFU/g or cm2 of L.monocytogenes and stored under vacuum or aerobic conditions at 7°C (n=4). The impact of varying (initial) levels of starter culture or indigenous spoilage microbiota on pathogen growth was evaluated by purchasing cheese packages on different dates in relation to production and expiration date (subsequently reflecting to different batches) mimicking a potential situation of cheese contamination with L.monocytogenes during retail display. Values of pH and aw were also monitored and used to simulate growth of L. monocytogenes by existing models and compare it with the observed data of the study. Survival in simulated gastric fluid (SGF) (pH1.5; HCl; max. 120min) was assessed at three time points during storage. Mascarpone, Ricotta, Mozzarella, Camembert, and Halloumi supported L.monocytogenes growth by 0.5-0.8logCFU/g or cm2per day, since low initial levels of total viable counts (TVC) (1.8-3.8logCFU/g or cm2) and high pH/aw values (ca. 6.23-6.64/0.965-0.993) were recorded. On Cottage, Anthotyros, Manouri, Mastelo®, Edam, and Gouda, the pathogen survived at populations similar or lower than the inoculation level due to the high reported competition and/or low pH/aw during storage. L. monocytogenes growth was significantly suppressed (p

Five composites of yeast and six of bacterial isolates from fermented products were studied, in order to assess their ability to inhibit Aspergillus carbonarius growth and reduce OTA concentration in culture media and beverages. The antagonistic effect of the above composites against A. carbonarius growth was studied in synthetic grape medium of pH 3.5 and a w 0.98, 0.95, 0.92 after incubation at 25 °C. Different combinations of initial inocula of bacteria or yeast composites and fungi were used (10 2 cfu/mL vs 10 5 spores/mL; 10 5 cfu/mL vs 10 2 spores/mL; and 10 5 cfu/mL vs 10 5 spores/mL). Regarding the OTA reduction experiment, 10 3 and 10 7 cfu/mL of the bacteria and yeast composites were inoculated in liquid media of different pH (3.0, 4.0, 5.0, and 6.1 or 6.5) and initial OTA concentration (50 and 100 μg/L) and incubated at 30 °C. Moreover, grape juice, red wine, and beer were supplemented with 100 μg/L of OTA and inoculated with composites of 16 yeasts (16YM) and 29 bacterial (29BM) strains (10 7 cfu/mL) to estimate the kinetics of OTA reduction at 25 °C for 5 days. Fungal inhibition and OTA reduction were calculated in comparison to control samples. None of the bacterial composites inhibited A. carbonarius growth. The high inoculum of yeast composites (10 5 cfu/mL) showed more efficient fungal inhibition compared to cell density of 10 2 cfu/mL. All yeast composites showed higher OTA reduction (up to 65%) compared to bacteria (2–25%), at all studied assays. The maximum OTA reduction was obtained at pH 3.0 by almost all yeast composites. For all studied beverages the decrease in OTA concentration was higher by yeasts (16YM) compared to bacteria (29BM). The highest OTA reduction was observed in grape juice ( ca 32%) followed by wine ( ca 22%), and beer ( ca 12%). The present findings may assist in the control of A. carbonarius growth and OTA production in fermented foodstuffs by the use of proper strains of technological importance.

The present study aimed: (i) to develop models for the combined effect of water activity (0.99, 0.94 and 0.90), microstructure expressed as 0, 5, 10 and 20% w/v gelatin, and temperature (15, 20 and 25 °C), on growth and OTA production rates by Aspergillus carbonarius; and (ii) to evaluate the performance of the developed models on food matrices (jelly, custard and marmalade) of different viscosity at pH 5.5. The square root of biomass increase rate (fungal growth rate) and OTA production rate were determined by the Baranyi model and were further modeled as a function of temperature, gelatin concentration and aw by applying polynomial models. Time for visible growth and the upper asymptote of the OTA production curve were also determined by the Baranyi model. Increase in gelatin concentration resulted in a significant delay in all parameters describing fungal growth and OTA production rates, at all temperatures and aw. The effect of microstructure on fungal growth and OTA production rates was less evident at stress conditions of aw and temperature. Detection time for visible fungal growth was markedly influenced by aw and temperature. Coefficients of determination were 0.899 and 0.887 for the models predicting the square root ( μ max ) of growth and OTA production rate, respectively. Predictions of growth rate agreed well with the recorded data of custard and marmalade, while observations of OTA production rate indicated low agreement with model predictions, in all food matrices except for marmalade. The present findings may provide a basis for reliable assessment of the risk of fungal growth and OTA production in foods of different structural and rheological properties.