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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 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)

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.

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 objectives of this study were: (a) to evaluate the potential of the ELISA method in the determination of the produced OTA by Aspergillus ochraceus and Aspergillus carbonarius in malt extract agar (MEA) at different pH (3.9, 5.1, 5.9, 6.8), water activity (aw) (0.87, 0.93, 0.99), and temperature (10, 15, 20, 25, 30, 40 °C) levels, providing a rapid screening for the optimum and marginal conditions of OTA production, (b) to comparatively evaluate the performance of ELISA and HPLC method, and (c) to evaluate the ability of A. ochraceus to produce OTA in rehydrated Corinth raisins during storage for 36 days. Two independent experiments were carried out to estimate OTA production on MEA and Corinth raisins. The produced OTA was evaluated qualitatively by the ELISA method and selected cases were verified by HPLC. The levels of OTA decreased with water activity, whereas pH seemed to have no specific effect. Furthermore, A. ochraceus produced maximum amounts of OTA on raisins at the 24th day of incubation, indicating that the endogenous microflora may restrictively affect OTA production. The knowledge of optimal and marginal levels of ecological factors in order to optimise post-harvest and storage of food products may significantly affect the production of OTA. Moreover, endogenous microflora of certain foodstuffs may cause OTA detoxification and consequently reduction of OTA levels; a fact that has to be taken into account in food commodities such as raisins, grapes, and wine.

The ubiquitous nature of microbes renders their presence inevitable in the food-processing chain as well as in domestic kitchens. Their introduction into a sterile or well-disinfected environment and their establishment on an abiotic or biotic surface is termed contamination. The major contamination source in such environments derives from the incoming raw materials, which may be carriers of a large and diverse association of spoilage bacteria, such as pseudomonads, enterobacteria, lactic acid bacteria, enterococci, yeasts, and molds. From a food safety aspect, these raw materials may also serve as vehicles of pathogenic microorganisms, including Listeria monocytogenes, Salmonella spp., Escherichia coli, Vibrio parahaemolyticus, Shigella, and Campylobacter. These microorganisms may be part of the indigenous microora of the food (of plant or animal origin), retrieved from the direct contact with the growth environment or introduced to the food during harvest and subsequent handling in the eld and/or during slaughter and further-processing and storage. Similarly, the working staff in a food-processing environment or the cook in a domestic kitchen may also assist in the introduction and spreading of undesirable6.1 Routes and Vehicles of Bacterial Immigration in the Food Industry and the Domestic Environment 89 6.1.1 Food Handlers 91 6.1.2 Cleaning Sponges, Cloths, and Towels 92 6.1.3 Kitchen Utensils and Storage Containers 92 6.1.4 Other Vehicles of Cross-Contamination 936.2 Factors Affecting Attachment of Pathogens on Food Contact Surfaces-Biolm Formation ..... 93 6.2.1 Type of Surface 94 6.2.2 Microbial Strain 95 6.2.3 Temperature 96 6.2.4 Surface Conditioning 97 6.2.5 Mixed-Species Biolm Formation 97 6.2.6 Persistence, Survival, and Stress Hardening of Attached Populations 976.3 Stochastic Description of Bacterial Transfer 99 6.3.1 Transfer between Abiotic Surfaces and Foods of Plant Origin 100 6.3.2 Transfer between Abiotic Surfaces and Foods of Animal Origin 101 6.3.3 Modeling Bacterial Transfer 1036.4 Concluding Remarks 105 References 105microbes, especially of pathogenic nature. In particular, food handlers may be potential disease vectors without experiencing any obvious symptoms. For instance, Staphylococcus aureus is commonly encountered on human skin and skin wounds, nose, or throat, and it can be easily transferred to foods or food-related surfaces. Other sources of undesirable contamination in the food industry or a kitchen environment include the water used for washing of the equipment, utensils, or the pets and pests that have access to a food preparation site, which may excrete a variety of food-poisoning microorganisms. For these reasons, it is of high importance that the hygienic level of the incoming raw materials as well as that of the food handlers should be strictly maintained and regularly validated, while all the food-related environments should also be protected from animals and pests.

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.