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Introduction: The endorsement of circular economy, zero-waste, and sustainable development by the EU and UN has promoted non-thermal technologies in agro-food and health industries. While northern European countries rapidly integrate these technologies, their implementation in Mediterranean food-supply chains remains uncertain. Aims: We evaluated the usefulness of hydrodynamic cavitation (HC) for valorizing orange peel waste in the fresh orange juice supply chain of the Maltese Islands. Method: We assessed: a) the effectiveness of HC in extracting bioactive compounds from orange peels (Citrus sinensis) in water (35°C) and 70% (v/v) ethanol (-10°C) over time, compared to conventional maceration, and b) the potato sprouting-suppression and biosorbent potential of the processed peel for copper, nitrate, and nitrite binding. Results: Prolonged HC-assisted extractions in water (high cavitation numbers), damaged and/or oxidized bioactive compounds, with flavonoids and ascorbic acid being more sensitive, whereas cold ethanolic extractions preserved the compounds involved in radical scavenging. HC-processing adequately modified the peel, enabling its use as a potato suppressant and biosorbent for copper, nitrate, and nitrite. Conclusion: Coupling HC-assisted bioactive compound extractions with using leftover peel for potato-sprouting prevention and as biosorbent for water pollutant removal offers a straightforward approach to promoting circular economic practices and sustainable agriculture in Malta., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Psakis, Lia, Valdramidis and Gatt.)

This study aimed to explore the diversity of fifty-four Photobacterium strains isolated from muscle tissue of European plaice (Pleuronectes platessa) caught at different fishing seasons and stored 14-days under various conditions. Single phylogenetic markers (16S rRNA, gapA, gyrB and recA) and multilocus sequence analysis (MLSA) were employed to classify isolates at species level. Furthermore, intra- and interspecies variability in the phenotypic traits, maximum specific growth rate (μ max ) and spoilage potential of the Photobacterium isolates were investigated. The isolates were classified into the P. iliopiscarium (53.7 %), P. phosphoreum (40.7 %) and P. piscicola (5.6 %) clades using MLSA. Two housekeeping genes, gyrB and recA, exhibited a consistent phylogenetic relationship with MLSA, suggesting that they might be used as individual phylogenetic markers for the Photobacterium genus. Intra- and interspecies variability in the expression of phenotypic characteristics and the production of trimethylamine (TMA), inosine (HxR), and hypoxanthine (Hx) were observed. A growth optimum temperature for P. iliopiscarium was approximately 20 °C, while those for P. phosphoreum and P. piscicola were closer to 15 °C. All isolates exhibited the highest growth density at 1.5 % NaCl, followed by 0.5 %, 3 %, and 6 % NaCl. However, P. phosphoreum demonstrated a higher NaCl tolerance than the other two species. Although, the high CO 2 atmosphere significantly inhibited the growth of all strains at 4 °C, P. phosphoreum and P. piscicola showed higher growth density at 15 °C than P. iliopiscarium. Notably, all strains demonstrated H 2 S production. The μ max varied considerably within each species, highlighting the significance of strain-level variability. This study demonstrates that P. iliopiscarium and P. piscicola, alongside P. phosphoreum, are efficient TMA-, HxR-, Hx-, and H 2 S-producers, suggesting their potential contribution to synergistic off-odour generation and spoilage. Moreover, the Photobacterium isolates seem to exhibit diverse adaptations to their environments, resulting in fluctuated growth and spoilage potential. Understanding intra- and interspecies variability will facilitate modelling seafood spoilage in microbial risk assessments and developing targeted hurdles to prolong products' shelf-life., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The EU-FORA programme 'Quantitative tools in microbial and chemical risk assessment' was dedicated to training on predictive microbiology fundamentals, implementation of different modelling strategies, design of experiments and software tools such as MATLAB, GInaFiT and DMFit. The fellow performed MATLAB training on maximum specific growth rate (μ max ) determination according to the Ratkowsky model. GInaFiT training on different models for bacterial inactivation and DMFit training on growth parameters of Vibrio parahaemolyticus were also carried out. Optical density measurements of V. parahaemolyticus bacterial cultures were performed. The obtained kinetics of optical density measurements were used to estimate μ max . Hereafter, Minimum inhibitory concentrations and non-inhibitory concentrations of aminoglycoside antibiotics were estimated based on the quantification of the fractional areas of the optical density vs time. It can be concluded that the results of the quantitative characterisation of V. parahaemolyticus are reliable and can be used for exposure assessments. Also, the turbidimetric assay can be applied for successful estimation of minimum inhibitory concentrations and non-inhibitory concentrations., (© 2023 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

The popularity of biological origin food protection substances is driven by demands from consumers for natural and clean label product, increasing various food-related safety and health concerns and sustainability issues. Lactic acid bacteria (LAB) are most promising because they are a large group of beneficial microorganisms commonly used in food protection due to their ability to inhibit the growth of pathogenic bacteria and enhance food safety. Extensive scientific research has been conducted to understand the mechanisms by which LAB exert their protective effects in various food systems. Even though LAB activity against various food pathogens and spoilers is distinguished, use of cell-free supernatant (CFS) is still under investigation. This report is dedicated to present how qualitative measures can elaborate in new bacteria-origin food additive investigation. As part of the EU-FORA programme, the fellow was involved in the risk assessment tasks and projects which include gaining basic knowledge in predicative microbiology fundamentals, including different types of modelling strategies; delivering essential understanding about experimental design, knowledge in three specific software tools (MATLAB, GInaFiT and DMFit) and gained overall understanding what are the main differences while modelling growth or inactivation models. Secondary activities were included as a way to expand competences beyond qualitative measures to overall all activities done regarding risk assessment and build a strong network of food safety experts and professionals to continue engaging in risk assessment beyond fellowship programme., (© 2023 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

An investigation into the mechanisms of action on bacteria involving exposure to stress factors was conducted in this study. The effects of ultrasound on Escherichia coli K-12 MG1655 and its isogenic mutant, ∆ gadW , under high power ultrasound treatments (26 kHz) were screened and identified by analysing their transcriptome differences between primary and secondary sequential treatments using RNA-Seq. This also helped to assess any developed protection for cells between different generations. According to our results, 1825 genes of all tested conditions were expressed, playing different roles in the cell. The expression of these genes is associated with DNA damage, cell membrane integrity, and also metabolic effects. The studied strains also showed different differential expressed genes (DEGs), with some genes being directly responsible for defence mechanisms, while others play an indirect effect due to cell damage. A gradual decrease in the expression of the genes, as we moved from just one cycle of ultrasound treatment to sequential treatment, was evident from a heat map analysis of the results. Overall, E. coli K-12 builds a self-protection mechanism by increasing the expression of genes involved in the respiration for increased growth, and production of flagellum and pili. It can be concluded that high power ultrasound is a technology that triggers several different defence mechanisms which directly link to E. coli .

Aims: The global level of carbon dioxide and temperature in the atmosphere is expected to increase, which may affect the survival of the stress-adapted bacteria. In this study, the effect of temperature and dissolved carbon dioxide on the growth rate of Escherichia coli-eGFP tagged strain was studied, thus assessing its response to induced environmental stress factors., Methods and Results: A kinetic assay has been performed using a microplate reader with a spectrofluorometer to determine the specific growth rates. Polynomial models were developed to correlate the environmental conditions of temperature and carbon dioxide with Escherichia coli BL21 (DE3) growth in culture media and dairy by-products. At a temperature of 42°C, as the dissolved CO2 increased, a decrease in μmax by 0.76 h-1 was observed. In contrast, at 27°C, this increase led to an increase in μmax by 0.99 h-1. Moreover, a correction factor was added when applying the model to dairy whey samples., Conclusions: The application of this developed model can be considered a useful tool for predicting the growth of Escherichia coli using climate projections., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Sprouts are particularly prone to microbial contamination due to their high nutrient content and the warm temperatures and humid conditions needed for their production. Therefore, disinfection is a crucial step in food processing as a means of preventing the transmission of bacterial, parasitic and viral pathogens. In this study, a dielectric coplanar surface barrier discharge (DCSBD) system was used for the application of cold atmospheric plasma (CAP), plasma activated water (PAW) and their combination on mung bean seeds. Germination assessments were performed in a test tube set-up filled with glass beads and the produced irrigation water. Overall, it was found that the combined seed treatment with direct air CAP (350 W) and air PAW had no negative impact on mung bean seed germination and growth, nor the concentration of secondary metabolites within the sprouts. These treatments also reduced the total microbial population in sprouts by 2.5 log CFU/g. This research reports for first time that aside from the stimulatory effect of plasma discharge on seed surface disinfection, sustained plasma treatment through irrigation of treated seeds with PAW can significantly enhance seedling growth. The positive outcome and further applications of different forms, of plasma i.e., gaseous and aqueous, in the agro-food industry is further supported by this research., (© 2021. The Author(s).)

The application of non-destructive process analytical technologies in the area of food science got a lot of attention the past years. In this work we used hyperspectral imaging to detect mould on milk agar and cheese. Principal component analysis is applied to hyperspectral data to localise and visualise mycelia on the samples' surface. It is also shown that the PCA loadings obtained from a set of training samples can be applied to hyperspectral data from new test samples to detect the presence of mould on these. For both the agar and cheeselets, the first three principal components contained more than 99 % of the total variance. The spatial projection of the second principal component highlights the presence of mould on cheeselets. The proposed analysis methods can be adopted in industry to detect mould on cheeselets at an early stage and with further testing this application may also be extended to other food products., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Aims: Alicyclobacillus acidoterrestris is a sporulating, acidophilic bacterial species which spoils acidic beverages such as fruit juices. This work aims to quantify the heat resistance of A. acidoterrestris spores and their recovery potential as a function of heating and recovery media pH., Methods and Results: The heat treatments were carried out with the strain of A. acidoterrestris Ad 746 in Bacillus acidoterrestris thermophilic medium. The pH of the heating medium from pH 7 to pH 2 nonsignificantly reduced the heat resistance. However, the pH levels of the recovery media strongly affected the apparent heat resistance of this strain. The maximum heat resistance was found when the pH was 4·70 and decreased when the pH decreased to pH 2·8, close to the minimum growth pH and when the recovery medium pH increased to pH 5·3., Conclusion: The heating medium pH has a slight effect on the spore heat resistances of this acidophilic species. However, the pH of the recovery media strongly affected the apparent heat resistance of this strain., Significance and Impact of the Study: The obtained parameters quantifying the heat resistance of A. acidoterrestris spores are tools to optimize the heat treatments and to control its development., (© 2020 The Society for Applied Microbiology.)

The dairy industry is of great importance to the European economy contributing towards € 8.7 billion of the total trade surplus. Caprine and ovine milk amount to 3.1% of the 152 million tonnes of milk produced in Europe, 95% of which is transformed into dairy products such as cheese. This cheese is mostly produced in small holdings from untreated milk, making it a high-risk dairy product for human consumption. A total of 49 foodborne disease outbreaks caused by dairy products were registered in 2017 in Europe. Therefore, these products remain a serious health risk. This meta-analysis examined 30 studies assessing bacterial or fungal contamination of caprine or ovine milk cheeses. The significantly contaminating microbes were found to be Acremonium spp. (19%), Aspergillus spp. (23%), Bacillus spp. (2%), Brucella spp. (34%), Enterobactericae spp. (36%), Enterococcus spp. (28%), Escherichia spp. (15%), Fusarium spp. (21%), Geotrichum spp. (22%), Listeria spp. (11%), Mucor spp. (15%), Penicillium spp. (25%), Phoma spp. (20%), Rhizopus spp. (15%), Salmonella spp. (3%), Scopulariopsis spp. (19%) and Staphylococcus spp. (25%) in caprine and ovine cheese, indicating a variety of food pathogens as well as spoilers. Raw milk is nutritious hence prone to contamination. However, since traditional cheese is often made from untreated milk, it is important to educate cheesemakers of key safety measures and good manufacturing practice allowing for the safe production of these food items., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Highly concentrated sugar solutions are known to be effective antimicrobial agents. However, it is unknown whether this effect is solely the result of the collective osmotic effect imparted by a mixture of sugars or whether the type of carbohydrate used also has an individual chemical effect on bacterial responses, that is, inhibition/growth. In view of this, in this work, the antimicrobial properties of four sugars, namely, glucose, fructose, sucrose and maltose against three common food pathogens; Staphylococcus aureus, Escherichia coli and Salmonella enterica, were investigated using a turbidimetric approach. The results obtained indicate that the type of sugar used has a significant effect on the extent of bacterial inhibition which is not solely dependent on the water activity of the individual sugar solution. In addition, while it was shown that high sugar concentrations inhibit bacterial growth, very low concentrations show the opposite effect, that is, they stimulate bacterial growth, indicating that there is a threshold concentration upon which sugars cease to act as antimicrobial agents and become media instead. SIGNIFICANCE AND IMPACT OF THE STUDY: In this work, an analysis on the antimicrobial properties of glucose, fructose, sucrose and maltose in solution was conducted using a turbidimetric approach. Our findings indicate that while, as expected, all of these sugars exhibit significant antimicrobial effects at high concentrations, at low concentrations they appear to act as substrates for the bacteria which results in enhanced microbial growth instead of inhibition. In addition, the results obtained also suggest that the resultant osmotic stress imparted by the sugar solutions is not the only factor which determines their antimicrobial activity and that other chemical factors may be playing a significant role., (© 2020 The Society for Applied Microbiology.)

Abstract: Fungal pathogens cause surface contamination and potential premature fruit spoilage of bambinella, a fruit endemic to the Maltese islands, leading to the loss of fruit during the postharvest phase. The objective of this study was to isolate, quantify, and characterize fungal contaminants of the small Maltese June Pear and describe their growth kinetics. In total, 284 fungicide-free fruits were collected over three consecutive summers (2014, 2015, 2016). The isolated fungi were identified by using forward and reverse colonial morphology. Species identification was determined using PCR-based methods. The number of CFU per square centimeter of bambinella outer skin was calculated. Mycelium diameter growth rate studies of the isolates were also carried out at seven different temperatures, ranging from 5 to 35°C. Fungi isolated from bambinella included Cladosporium ramotenellum, Alternaria arborescens, Penicillium lanosum, Penicillium expansum, and Aspergillus sydowii, listed from the most abundant to the least abundant. The Rosso model was fitted to the growth kinetic data and showed that the optimal temperatures for growth of all five fungi were in the range of 20 to 22°C, whereas growth was slower at temperatures below 10°C and above 30°C. As observed in the diameter studies, the order of highest to lowest germination rate was found to be P. expansum, A. sydowii, P. lanosum, C. ramotenellum, and A. arborescens. Germination studies showed that the highest germination rate was observed for P. lanosum, followed by A. arborescens, C. ramotenellum, P. expansum, and A. sydowii, in descending order. The highest germination lag time was observed for A. arborescens, followed by C. ramotenellum, P. expansum, P. lanosum, and A. sydowii, in ascending order., (Copyright ©, International Association for Food Protection.)

Image 1., Competing Interests: The authors declare that there was no conflict of interest to declare., (© 2020 The Author(s).)

A geographical and seasonal assessment of indigenous lactic acid bacteria (LAB) in Maltese cow milk was conducted in this study. To investigate this, milk was collected from different regions of Malta during winter and summer seasons. Total viable counts (TVC) and LAB population were enumerated. Afterwards, LAB were isolated and identified by molecular methods. According to the results, similar TVC were enumerated on winter and summer samples, while highest LAB population was detected on summer samples. LAB isolates were grouped in seven different clusters which were assigned to Lactobacillus casei , Pediococcus pentosaceus , Lactobacillus plantarum , Weissella paramesenteroides , Lactobacillus rhamnosus , Lactococcus lactis , and Lactococcus garvieae . In addition, Enterococcus and Streptococcus species were also isolated. Season seemed to affect the genus / species of LAB since Lactobacill us were mainly isolated from winter samples, while Lactococcus and Enterococcus species were the main genera identified in summer samples. Regarding the geographical distribution, the majority of the Lactobacillus spp. were isolated from the South-eastern region in both seasons. In conclusion, through this study the diversity of indigenous LAB in the Maltese cow milk was monitored for the first time and highlighted that the microbial communities are affected by seasonality and geographical distribution of the farms., Competing Interests: The authors declare no conflict of interest.

Highlights: CAPP technology has high application potential for decontamination of berries. Impacts of CAPP in aspects of food safety and security still need to be addressed. Optimized treatment parameters need to be investigated for each berry type.

Coupling microbial dynamics with the complete dynamics of the packaging gases is still a challenge. In this work the microbial growth kinetic parameters for Pseudomonas and Lactic Acid Bacteria (LAB) in MAP are identified based on accurate estimation of diffusivity of gases and parameter scaled sensitivity approaches. The microbial dynamics are also compared with those estimated based on partial pressure measurement. Scaled sensitivity coefficient analysis using dissolved gases as variable inputs, shows that in most cases the only coefficients large enough for estimation were those for CO 2max-diss , and for μ max . The current data showed that dissolved gases led significant differences on the microbial parameter of CO 2max values when compared with the headspace gases. On the other hand, the (so-called) dissolved specific growth rate follows a clear trend down for both microorganisms in relation to the increase of the initial headspace CO 2 . Finally, current results indicate a possible correlation between CO 2max-diss , CO 2max-headspace , and μ max as functions of CO 2init ., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The effect of temperature on the mycelium growth kinetics of four postharvest fungal isolates (i.e., Penicillium expansum, Alternaria alternata, Botrytis cinerea and Rhizopus stolonifer) was assessed. A cardinal model with inflection (CMI) was used to describe the effect of the temperature on the growth rate (μ) and the lag time (λ) of each isolate. Cardinal temperature values such as T min , T max and T opt were estimated and isolates were sorted according to their growth rate and lag time duration. Additionally, model validation was performed on a medium prepared from mashed pear pulp and on artificially wound-inoculated pear fruits. P. expansum was shown to be the most psychotrophic fungus with the lowest estimated T min  = -8.78. Model validation on pear pulp agar showed growth rate over-prediction in the case of R. stolonifer and B. cinerea but a good correlation in the case of P. expansum and A. alternata. In vivo experiments on pear fruits showed discrepancies from the synthetic and the simulated counterparts for all the fungi with the only exception of P. expansum., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Rapid assessment of fungal growth and screening antifungal compounds, such as nanoparticles (NPs), for effectiveness is a challenging procedure because no primary standards exist as they do for yeasts and bacteria. Because fungi do not grow as single cells, but as hyphal filaments, they cannot be quantified by the usual enumeration techniques used in bacteriology. The growth of three postharvest fungal isolates ( Alternaria alternata, Rhizopus stolonifer, and Botrytis cinerea) was investigated at different inoculum concentrations and in three nutrient media (Sabouraud dextrose agar, potato dextrose agar, and yeast extract dextrose agar [YED]) with a turbidimetric assay. Sequential measurements were performed to generate optical density versus time plots, whereas the growth responses were expressed quantitatively as the generated trapezoidal area. YED medium showed the lowest variation among replicated experiments; potato dextrose agar showed the next lowest, but there was no significant difference. The inoculum size had a minimal effect on the variation of the fungal dynamics. Microscopic assessment of the fungal growth confirmed that YED medium allowed the most homogeneous development of the studied fungi. Therefore, we developed a rapid and reliable technique to evaluate the efficacy of novel antifungal compounds such as zinc oxide NPs. Turbidimetric assessment showed that these NPs were able to inhibit the growth of all three isolates. A. alternata and B. cinerea did not show a significant difference in the level of inhibition at 15 mM, whereas R. stolonifer showed the highest inhibition at the same concentration.

Aims: Foods and drinks are major contributors (17%) to the greenhouse gas (GHG) emissions caused by private consumption in Europe. The carbon footprint (CF) of a certain product expresses the total GHG emissions over its whole life cycle, and its calculation for foodstuff is a necessary first step to reduce their contribution to global warming. The calculation of the CF of Maltese food products is especially relevant for two reasons: the economic characteristics of the island, whose food sector is highly dependent on imports, implying longer transport distances; and the Maltese electricity production mix, based almost exclusively on oil combustion., Methods and Results: The CF of ten multi-fruit juices marketed in Malta has been determined, covering all the processes from the agricultural stage to the distribution of the final products. As a functional unit (FU), a 250 ml bottle of packaged product arriving at the retailer has been considered. The Maltese orange juice, the only final product in which only local ingredients are used, presents the lowest CF (0.50 kgCO 2 /FU), while the remaining ones range from 0.67 kgCO 2 /FU to 0.80 kgCO 2 /FU. The major contributor to all the CFs is juice processing at the Maltese plant (0.42 kgCO 2 /FU), mainly due to the use of electricity (78%)., Conclusions: The influence of both the electricity mix and the Maltese supply chain in the CF of the final products has been demonstrated. Alternatives to reduce the impacts of the final products have been proposed and evaluated that could lower the average CF of the juices by 32%., Significance and Impact of the Study: The calculation of the CF of Maltese juices represents an innovative case study due to the characteristics of the island, and it is expected to act as a first step to lower their environmental impacts., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Increasing concerns continue to be expressed about health hazards and environmental pollution resulting from the use of conventional fungicides for postharvest disease control. Nanoparticles represent an alternative solution for postharvest disease management. The objective of this work was to assess the physiological effects and the antifungal efficiency of ZnO nanoparticles (ZnO NPs) against a number of fungal contaminants. The efficacy of ZnO NPs was qualitatively and quantitatively assessed against: Penicillium expansum, Alternaria alternata, Botrytis cinerea and Rhizopus stolonifer. Mycelium growth diameters were measured onto Potato Dextrose Agar (PDA) plates loaded with different ZnO NPs concentrations (from 0mM to 15mM). Hereafter, the rate of the fungal diameter increase was quantified by linear regression modelling. Microscopic analysis was performed by scanning electron microscopy (SEM) images of agar plugs excised from plates with 0mM and 12mM ZnO. All the fungi were inhibited by ZnO NPs at concentrations higher than 6mM. SEM images showed clear morphological aberrations in the fungal structures of all the isolates grown in presence of ZnO. Additionally, knowing that the chelating agent EDTA sequesters metal ions, it was added to fungal inoculated PDA plates with ZnO to study the NPs' mode of action. Cultures where ZnO was mixed with EDTA showed a decrease in the antifungal effect of the nanoparticles. In conclusion, ZnO NPs are therefore a good candidate as an effective postharvest disease control antifungal agent., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Ultrasound creates cavitation phenomena, resulting in the formation of several free radicals, namely OH˙ and H˙, due to the breakdown of the H 2 O molecule. These radicals affect the cellular integrity of the bacteria, causing the inactivation of several processes, and thus it is important to unravel the mechanism of action of this technology. This research looks into the application and mechanism of action of ultrasound technology as a means of disinfection by acoustic cavitation. Sterile water and synthetic waste water were inoculated with different mutants of Escherichia coli K12 strains containing deletions in genes affecting specific functional properties of E. coli. These were: dnak soxR, soxS, oxyR, rpoS, gadA/gadB, gadC and yneL. Escherichia coli K-12 ΔoxyR appeared to be more resistant to the treatment together with gadW, gadX, gabT and gabD, whereas the mutant K-12 ΔdnaK was more sensitive with c. 2·5 log (CFU per ml) reduction in comparison to their isogenic wild-type E. coli K-12. This indicates that the dnaK gene participates in general stress response and more specifically to hyperosmotic stress. The other E. coli deleted genes tested (soxS, rpoS, gadB, gadC, yneL) did not appear to be involved in protection of microbial cells against ultrasound., Significance and Impact of the Study: This study looks at the mechanism of action of ultrasound technology for the disinfection of wastewater. Different mutants with deleted genes were used to study the respective sensitivity or resistance to this treatment. This is essential to characterize changes at the molecular level, which might be occurring during treatment, resulting in bacterial adaptation., (© 2017 The Society for Applied Microbiology.)

Public concerns about food safety have triggered a worldwide implementation of new legislations aimed at banning many of the most popular food conventional antifungal treatments. There is therefore an urgent need to identify novel and safer solutions to prevent fungal contamination of food. The antifungal effect of zinc oxide nanoparticles (ZnO NPs) against the postharvest pathogenic fungus Penicillium expansum has been investigated in this study. An automated turbidimetric assay, with a standard 96-well microplate, has been developed and optimised regarding the selection of the inoculum size in order to collect sequential optical density measurements. Data were processed by the updated version of the Lambert Pearson model to estimate the minimum inhibitory concentration and the non-inhibitory concentration values which were found to be 9.8 and 1.8 mM (i.e. 798 and 147 ppm), respectively. The current results show that turbidimetry is a reliable technique for assessing the antifungal activity of metal nanoparticles and that zinc oxide (ZnO) is an effective fungicide which can be potentially used to control food safety.

Air filters support fungal growth, leading to generation of conidia and volatile organic compounds, causing allergies, infections and food spoilage. Filters that inhibit fungi are therefore necessary. Zinc oxide (ZnO) nanoparticles have anti-fungal properties and therefore are good candidates for inhibiting growth. Two concentrations (0.012 M and 0.12 M) were used to coat two types of filters (melt-blown and needle-punched) for three different periods (0.5, 5 and 50 min). Rhizopus stolonifer and Penicillium expansum isolated from spoiled pears were used as test organisms. Conidial suspensions of 10 5 to 10 3 spores ml -1 were prepared in Sabouraud dextrose agar at 50°C, and a modified slide-culture technique was used to test the anti-fungal properties of the filters. Penicillium expansum was the more sensitive organism, with inhibition at 0.012 M at only 0.5 min coating time on the needle-punched filter. The longer the coating time, the more effective inhibition was for both organisms. Furthermore, it was also determined that the coating process had only a slight effect on the Young's Moduli of the needle-punched filters, while the Young's Moduli of the melt-blown filters is more susceptible to the coating method. This work contributes to the assessment of the efficacy of filter coating with ZnO nanopaticles aimed at inhibiting fungal growth., Competing Interests: The authors have no competing interests.

The large potential of cold atmospheric plasma (CAP) for food decontamination has recently been recognized. Room-temperature gas plasmas can decontaminate foods without causing undesired changes. This innovative technology is a promising alternative for treating fresh produce. However, more fundamental studies are needed before its application in the food industry. The impact of the food structure on CAP decontamination efficacy of Salmonella Typhimurium and Listeria monocytogenes was studied. Cells were grown planktonically or as surface colonies in/on model systems. Both microorganisms were grown in lab culture media in petri dishes at 20°C until cells reached the stationary phase. Before CAP treatment, cells were deposited in a liquid carrier, on a solid(like) surface or on a filter. A dielectric barrier discharge reactor generated helium-oxygen plasma, which was used to treat samples up to 10min. Although L. monocytogenes is more resistant to CAP treatment, similar trends in inactivation behavior as for S. Typhimurium are observed, with log reductions in the range [1.0-2.9] for S. Typhimurium and [0.2-2.2] for L. monocytogenes. For both microorganisms, cells grown planktonically are easily inactivated, as compared to surface colonies. More stressing growth conditions, due to cell immobilization, result in more resistant cells during CAP treatment. The main difference between the inactivation support systems is the absence or presence of a shoulder phase. For experiments in the liquid carrier, which exhibit a long shoulder, the plasma components need to diffuse and penetrate through the medium. This explains the higher efficacies of CAP treatment on cells deposited on a solid(like) surface or on a filter. This research demonstrates that the food structure influences the cell inactivation behavior and efficacy of CAP, and indicates that food intrinsic factors need to be accounted when designing plasma treatment., (Copyright © 2016. Published by Elsevier B.V.)

Bacillus sporothermodurans produces highly heat-resistant endospores, that can survive under ultra-high temperature. High heat-resistant sporeforming bacteria are one of the main causes for spoilage and safety of low-acid foods. They can be used as indicators or surrogates to establish the minimum requirements for heat processes, but it is necessary to understand their thermal inactivation kinetics. The aim of the present work was to study the inactivation kinetics under both static and dynamic conditions in a vegetable soup. Ordinary least squares one-step regression and sequential procedures were applied for estimating these parameters. Results showed that multiple dynamic heating profiles, when analyzed simultaneously, can be used to accurately estimate the kinetic parameters while significantly reducing estimation errors and data collection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Atmospheric cold plasma (ACP) is a non-thermal technology, effective against a wide range of pathogenic microorganisms. Inactivation efficacy results from plasma generated reactive species. These may interact with any organic components in a test matrix including the target microorganism, thus food components may exert a protective effect against the antimicrobial mode of action. The effect of an in-package high voltage ACP process applied in conjunction with common meat processing MAP gas compositions as well as bacteria type and meat model media composition have been investigated to determine the applicability of this technology for decontamination of safety challenges associated with meat products. E. coli, L. monocytogenes, and S. aureus in PBS were undetectable after 60 s of treatment at 80 kVRMS in air, while ACP treatment of the contaminated meat model required post-treatment refrigeration to retain antimicrobial effect. The nutritive components in the meat model exerted a protective effect during treatment, where 300 s ACP exposure yielded a maximum reduction of 1.5 log using a high oxygen atmosphere, whilst using air and high nitrogen atmospheres yielded lower antimicrobial efficacy. Furthermore, an ROS assay was performed to understand the protective effects observed using the meat model. This revealed that nutritive components inhibited penetration of ROS into bacterial cells. This knowledge can assist the optimization of meat decontamination using ACP technology where interactions with all components of the food matrix require evaluation.

Steam surface pasteurization is a promising decontamination technology for reducing pathogenic bacteria in different stages of food production. The effect of the artificial inoculation type and initial microbial load, however, has not been thoroughly assessed in the context of inactivation studies. In order to optimize the efficacy of the technology, the aim of this study was to design and validate a model system for steam surface pasteurization, assessing different inoculation methods and realistic microbial levels. More specifically, the response of Listeria innocua, a surrogate organism of Listeria monocytogenes, on a model fish product, and the effect of different inoculation levels following treatments with a steam surface pasteurization system was investigated. The variation in the resulting inoculation level on the samples was too large (77%) for the contact inoculation procedure to be further considered. In contrast, the variation of a drop inoculation procedure was 17%. Inoculation with high levels showed a rapid 1-2 log decrease after 3-5 s, and then no further inactivation beyond 20 s. A low level inoculation study was performed by analysing the treated samples using a novel contact plating approach, which can be performed without sample homogenization and dilution. Using logistic regression, results from this method were used to model the binary responses of Listeria on surfaces with realistic inoculation levels. According to this model, a treatment time of 23 s will result in a 1 log reduction (for P = 0.1)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Ultrasound requires high power and longer treatment times to inactivate micro-organisms when compared to ultrasound combined with other technologies. Previous reports have shown that the effectiveness of ultrasound as a decontamination technology can be increased by combining it with another treatment such as pressure, heat and antimicrobial solutions. Assisted ultrasound, the combination of ultrasound with another technology, is more energy efficient, and it has less impact on the food properties. In this review paper, the power ultrasound antimicrobial mechanisms of action, the antimicrobial effects of ultrasound in combination with other physical processes and antimicrobial solutions are comprehensively discussed. Furthermore, the present interest on using these technologies as alternative processing and decontamination methods is presented. Research outputs on the application of ultrasound combined with physical processes are showcased including applications of thermosonication, manosonication, manothermosonication and osmosonication. Antimicrobial efficacy, energy requirements and optimal operation conditions of the different assisted ultrasound technologies are critically discussed, and their impact on the food industry for future applications is presented. Overall, this review paper highlights the importance and recent developments of assisted ultrasound for enhancing food safety., (© 2014 The Society for Applied Microbiology.)

The growth dynamics of Listeria monocytogenes strains isolated from salmon or a salmon processing environment and two reference Listeria innocua strains were investigated at refrigerated and close-to-optimal growth temperatures. Estimates for the growth rates and the lag-phase duration at 4, 8, 12, and 30°C were obtained for optical density measurements by using different growth parameter estimation methods, i.e., the serial dilution (SD) method and the relative rate to detection (RRD) method. Both single L. innocua and L. monocytogenes strains and mixtures of L. monocytogenes strains (cocktails) were studied. Both methods show an increase in maximum growth rate (μ(max)) of Listeria with increasing temperatures. Generally, single-strain growth rate estimates were quite similar for both species, although L. monocytogenes showed slightly higher μ(max) estimates at 4°C. The SD method gave the highest estimates for the growth rate, i.e., the estimates from the RRD method were 10 to 20% lower. This should lead to caution when using the latter method for Listeria, particularly at lower temperatures. Overall, the SD method is preferred as this method yields μ(max) estimates close to the biological value and provides estimates for the duration of lag time (λ). For discrimination between different strains, λ appeared to be a more suitable parameter than μ(max). This effect was most prominent for L. innocua. Significant differences were observed between μ(max) and/or λ of L. monocytogenes cocktails and single strains at all temperatures investigated. At 4°C, the average growth rate of cocktails was higher than that of single strains. At 8 and 30°C, this trend was reversed. The average λ of single strains were more than twice as long as those of cocktails at 4°C. At 8 and 30°C, the λ of cocktails were significantly slower than those of single strains, but the variation was considerably less and the differences were less pronounced.

Aims: To investigate the effect of the oxidative stress of ozone on the microbial inactivation, cell membrane integrity and permeability and morphology changes of Escherichia coli., Methods and Results: Escherichia coli BW 25113 and its isogenic mutants in soxR, soxS, oxyR, rpoS and dnaK genes were treated with ozone at a concentration of 6 μg ml⁻¹ for a period up to 240 s. A significant effect of ozone exposure on microbial inactivation was observed. After ozonation, minor effects on the cell membrane integrity and permeability were observed, while scanning electron microscopy analysis showed slightly altered cell surface structure., Conclusions: The results of this study suggest that cell lysis was not the major mechanism of microbial inactivation. The deletion of oxidative stress-related genes resulted in increased susceptibility of E. coli cells to ozone treatment, implying that they play an important role for protection against the radicals produced by ozone. However, DnaK that has previously been shown to protect against oxidative stress did not protect against ozone treatment in this study. Furthermore, RpoS was important for the survival against ozone., Significance and Impact of the Study: This study provides important information about the role of oxidative stress in the responses of E. coli during ozonation., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

This research investigated the efficacy of gaseous ozone on the inactivation of Escherichia coli ATCC 25922 and NCTC 12900 strains in apple juice of a range of pH levels, using an ozone bubble column. The pH levels investigated were 3.0, 3.5, 4.0, 4.5 and 5.0. Apple juice inoculated with E. coli strains (10(6)CFU/mL) was treated with ozone gas at a flow rate of 0.12L/min and ozone concentration of 0.048 mg/min/mL for up to 18 min. Results show that inactivation kinetics of E. coli by ozone were affected by pH of the juice. The ozone treatment duration required for achieving a 5-log reduction was faster (4 min) at the lowest pH than at the highest pH (18 min) studied. The relationship between time required to achieve 5log reduction (t(5d)) and pH for both strains was described mathematically by two exponential equations. Ozone treatment appears to be an effective process for reducing bacteria in apple juice and the required applied treatment for producing a safe apple juice is dependant on its acidity level., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Traditionally, ozone processing within the food industry has focused on solid foods by either gaseous treatment or washing with ozonized water. However, with the FDA's approval of ozone as a direct additive to food, the potential for liquid applications has emerged. This study investigates the effect of ozone processing on microbial inactivation (E. coli ATCC 25922 and NCTC 12900) and quality parameters (color, phenolic content) of cloudy apple juice. Apple juice samples were ozonated at room temperature (20 ± 1.5 °C) with a generated ozone concentration of 0.048 mg O(3) at a constant flow rate of 0.12 L/min and treatment time of 0 to 10 min. E. coli inactivation kinetics in apple juice were described quantitatively by using the Shoulder log-linear and the Weibull model. Ozone treatment of E. coli in apple juice demonstrate that a desired 5 log reduction can be achieved within 5 min. Apple juice color (L*, a*, and b*) and total phenols were significantly affected by ozone concentration and treatment time.

Many of the documented outbreaks of Cronobacter sakazakii have been linked to infant formula. The aims of this work are to monitor the inactivation kinetics of C.sakazakii NCTC 08155 and ATCC 11467 and to determine quantitatively the effectiveness of ultrasonic treatments as an alternative to heat processing of reconstituted infant milk formula before feeding of infants at highest risk. Inactivation studies of C. sakazakii inoculated in reconstituted infant formula were performed at the combined conditions of temperature, i.e., 25 degrees C, 35 degrees C, 50 degrees C and amplitude, i.e., 24.4, 30.5, 42.7, 54.9, 61 microm and the kinetics were described by a range of inactivation models. The dependency of the specific inactivation rate with respect to the product of temperature and amplitude was described by a modified Bigelow type model. Ultrasound combined with temperature was efficient to reduce significantly the microbial levels of C. sakazakii. C. sakazakii strain NCTC 08155 was at the same range of temperature and amplitude resistance as strain ATCC 11467. Application of ultrasound is an alternative process for the production of safe reconstituted infant formula. This study contributes on the quantitative assessment of the resistance of C. sakazakii., (Copyright 2010 Elsevier B.V. All rights reserved.)

In this review, antimicrobials from a range of plant, animal, and microbial sources are reviewed along with their potential applications in food systems. Chemical and biochemical antimicrobial compounds derived from these natural sources and their activity against a range of pathogenic and spoilage microorganisms pertinent to food, together with their effects on food organoleptic properties, are outlined. Factors influencing the antimicrobial activity of such agents are discussed including extraction methods, molecular weight, and agent origin. These issues are considered in conjunction with the latest developments in the quantification of the minimum inhibitory (and noninhibitory) concentration of antimicrobials and/or their components. Natural antimicrobials can be used alone or in combination with other novel preservation technologies to facilitate the replacement of traditional approaches. Research priorities and future trends focusing on the impact of product formulation, intrinsic product parameters, and extrinsic storage parameters on the design of efficient food preservation systems are also presented.

In this study dynamic microbial inactivation experiments are exploited for performing parameter identification of a non-linear microbial model. For that purpose microbial inactivation data are produced and a differential equation exhibiting a shoulder and a loglinear phase is employed. The derived parameter estimates from this method were used to perform predictions on an independent experimental set at fluctuating temperature. Joint confidence regions and asymptotic confidence intervals of the estimated parameters were compared with previous studies originating from parameter identification under isothermal conditions. The developed approach can provide more reliable estimates for realistic conditions compared to the usual or standard two step approach.

Aims: In previous studies the microbial kinetics of Escherichia coli K12 have been evaluated under static and dynamic conditions (Valdramidis et al. 2005, 2006). An acquired microbial thermotolerance following heating rates lower than 0.82 degrees C min(-1) for the studied micro-organism was observed. Quantification of this induced physiological phenomenon and incorporation, as a model building block, in a general microbial inactivation model is the main outcome of this work., Methods and Results: The microbial inactivation rate observed (k(obs)) under time-varying temperature conditions is studied and expressed as a function of the heating rate (dT/ dt). Hereto, a model building block related to the microbial physiology (k(phys)) under stress conditions is developed. Evaluation of the performance of the developed mathematical approach depicts that physiological adaptation is an essential issue to be considered when modelling microbial inactivation., Conclusions: Consideration, at a mathematical level, of microbial responses resulting in physiological adaptations contribute to the reliable quantification of the safety risks during food processing., Significance and Impact of the Study: By taking into account the physiological adaptation, the microbiological evolution during heat processing can be accurately assessed, and overly conservative or fail dangerous food processing designs can be avoided.

In a recent study we demonstrated that a high-hydrostatic-pressure-tolerant isolate of Listeria monocytogenes lacks a codon in the class 3 heat shock regulator gene ctsR. This mutation in the region that encodes four consecutive glycines was directly responsible for the observed piezotolerance, increased stress resistance, and reduced virulence. The aim of the present study was to determine whether mutations in ctsR are frequently associated with piezotolerance in L. monocytogenes. Wild-type cultures of L. monocytogenes were therefore exposed to 350 MPa for 20 min, and the piezotolerance of individual surviving isolates was assessed. This rendered 33 isolates with a stable piezotolerant phenotype from a total of 84 survivors. Stable piezotolerant mutants were estimated to be present in the initial wild-type population at frequencies of >10(-5). Subsequent sequencing of the ctsR gene of all stable piezotolerant isolates revealed that two-thirds of the strains (i.e., n = 21) had mutations in this gene. The majority of the mutations (16 of 21 strains) consisted of a triplet deletion in the glycine-encoding region of ctsR, identical to what was found in our previous study. Interestingly, 2 of 21 mutants contained a codon insertion in this repeat region. The remaining three stable piezotolerant strains showed a 19-bp insertion in the glycine repeat region, a 16-bp insertion downstream of the glycine repeat area (both leading to frameshifts and a truncated ctsR), and an in-frame 114-bp deletion encoding a drastically shortened carboxy terminus of CtsR. In four instances it was not possible to generate a PCR product. A piezotolerant phenotype could not be linked to mutations in ctsR in 8 of 33 isolates, indicating that other thus-far-unknown mechanisms also lead to stable piezotolerance. The present study highlights the importance of ctsR in piezotolerance and stress tolerance of L. monocytogenes, and it demonstrates that short-sequence repeat regions contribute significantly to the occurrence of a piezotolerant and stress-tolerant subpopulation within L. monocytogenes cultures, thus playing an important role in survival.

This contribution focuses on the presentation of GInaFiT (Geeraerd and Van Impe Inactivation Model Fitting Tool), a freeware Add-in for Microsoft Excel aiming at bridging the gap between people developing predictive modelling approaches and end-users in the food industry not familiar with or not disposing over advanced non-linear regression analysis tools. More precisely, the tool is useful for testing nine different types of microbial survival models on user-specific experimental data relating the evolution of the microbial population with time. As such, the authors believe to cover all known survivor curve shapes for vegetative bacterial cells. The nine model types are: (i) classical log-linear curves, (ii) curves displaying a so-called shoulder before a log-linear decrease is apparent, (iii) curves displaying a so-called tail after a log-linear decrease, (iv) survival curves displaying both shoulder and tailing behaviour, (v) concave curves, (vi) convex curves, (vii) convex/concave curves followed by tailing, (viii) biphasic inactivation kinetics, and (ix) biphasic inactivation kinetics preceded by a shoulder. Next to the obtained parameter values, the following statistical measures are automatically reported: standard errors of the parameter values, the Sum of Squared Errors, the Mean Sum of Squared Errors and its Root, the R(2) and the adjusted R(2). The tool can help the end-user to communicate the performance of food preservation processes in terms of the number of log cycles of reduction rather than the classical D-value and is downloadable via the KULeuven/BioTeC-homepage at the topic "Downloads" (Version 1.4, Release date April 2005).

This research deals with the development of predictive modelling approaches in the field of heat transfer and microbial inactivation. Upon making some backstage microbiological considerations, surface temperature predictions during hot dry air decontaminations are incorporated in a microbial inactivation model, in order to describe inactivation kinetics under realistic (time-varying) temperature conditions. In the present study, the following parts are presented. (i) First, a one-dimensional heat transfer model is developed taking into account exchanges by convection, radiation and evaporation. The model is subsequently validated on a laboratory setup and on a test rig, assuming no water activity changes. This test rig is developed for studying-at a later stage-surface pasteurisation treatment on food products with the use of hot dry air. (ii) Isothermal inactivation data of Escherichia coli K12 MG1655 have been collected and inactivation parameters are accurately estimated by using a primary and a secondary model in a global modelling approach. (iii) Microbiological considerations such as microbial growth effects during come-up times, initial temperature of inactivation, and heat resistance effects, based on experimental observations and on literature studies, are formulated in order to evaluate possible microbial effects arising under the dynamic temperature conditions modelled in step (i). (iv) Microbial inactivation simulations with the incorporation of surface temperature predictions are presented. (v) Finally, the level of the microbial decontamination in an example based on the design of an industrial installation is presented, outlining the importance of the combination of surface temperature and microbial inactivation modelling approaches.

This research deals with the development of a novel secondary modelling procedure within the framework of predictive microbiology. The procedure consists of three steps: (i) careful formulation of the available microbiological information, both from literature and from the experimental case study at hand, (ii) translation of these requirements in mathematical terms under the form of partial derivatives throughout the complete interpolation region of the experimental design, and (iii) determination of parameter values with suitable optimisation techniques for a flexible black box modelling approach, e.g., a polynomial model or an artificial neural network model. As a vehicle for this procedure, the description of the maximum specific growth rate of Lactobacillus sakei in modified BHI-broth as influenced by suboptimal temperature, water activity, sodium lactate and dissolved carbon dioxide concentration is under study. The procedure results in a constrained polynomial model with excellent descriptive and interpolating features in comparison with an extended Ratkowsky-type model and classical polynomial model, by combining specific properties of both model types. The developed procedure is illustrated on the description of the lag phase as well. It is stressed how the confrontation with experimental data is very important to appreciate the descriptive and interpolating capacities of new or existing models, which is nowadays not always carefully performed. Alternatively, the first two steps of the novel procedure can be used as a tool to demonstrate clearly (possible) interpolative shortcomings of an existing model with straightforward spreadsheet calculations.