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Highlights • A multi-frequency study of ultrasound (US) and nisin for microbial inactivation. • US impacts E. coli at 500 kHz only; L. innocua resists all frequencies studied. • Nisin applied before US enhances inactivation of E. coli but not when applied after. • Attributed to outer membrane destabilisation by US allowing nisin penetration. • System structure (viscosity) reduces US inactivation efficacy., Ultrasound, alone or in combination with natural antimicrobials, is a novel food processing technology of interest to replace traditional food decontamination methods, as it is milder than classical sterilisation (heat treatment) and maintains desirable sensory characteristics. However, ultrasound efficacy can be affected by food structure/composition, as well as the order in which combined treatments are applied. More specifically, treatments which target different cell components could result in enhanced inactivation if applied in the appropriate order. The microbial properties i.e. Gram positive/Gram negative can also impact the treatment efficacy. This work presents a systematic study of the combined effect of ultrasound and nisin on the inactivation of the bacteria Listeria innocua (Gram positive) and Escherichia coli (Gram negative), at a range of cavitation conditions (44, 500, 1000 kHz). The order of treatment application was varied, and the impact of system structure was also investigated by varying the concentration of Xanthan gum used to create the food model systems (0 – 0.5% w/v). Microbial inactivation kinetics were monitored, and advanced microscopy and flow cytometry techniques were utilised to quantify the impact of treatment on a cellular level. Ultrasound was shown to be effective against E. coli at 500 kHz only, with L. innocua demonstrating resistance to all frequencies studied. Enhanced inactivation of E. coli was observed for the combination of nisin and ultrasound at 500 kHz, but only when nisin was applied before ultrasound treatment. The system structure negatively impacted the inactivation efficacy. The combined effect of ultrasound and nisin on E. coli was attributed to short-lived destabilisation of the outer membrane as a result of sonication, allowing nisin to penetrate the cytoplasmic membrane and facilitate cell inactivation.

Minimal processing for microbial decontamination, such as the use of natural antimicrobials, is gaining interest in the food industry as these methods are generally milder than conventional processing, therefore better maintaining the nutritional content and sensory characteristics of food products. The aim of this study was to quantify the impact of (i) structural composition and complexity, (ii) growth location and morphology, and (iii) the natural antimicrobial nisin, on the microbial dynamics of Listeria innocua. More specifically, viscoelastic food model systems of various compositions and internal structure were developed and characterised, i.e. monophasic Xanthan gum-based and biphasic Xanthan gum/Whey protein-based viscoelastic systems. The microbial dynamics of L. innocua at 10 °C, 30 °C and 37 °C were monitored and compared for planktonic growth in liquid, or in/on (immersed or surface colony growth) the developed viscoelastic systems, with or without a sublethal concentration of nisin. Microscopy imaging was used to determine the bacterial colony size and spatial organisation in/on the viscoelastic systems. Selective growth of L. innocua on the protein phase of the developed biphasic system was observed for the first time. Additionally, significant differences were observed in the colony size and distribution in the monophasic Xanthan gum-based systems depending on (i) the type of growth (surface/immersed) and (ii) the Xanthan gum concentration. Furthermore, the system viscosity in monophasic Xanthan gum-based systems had a protective role against the effects of nisin for immersed growth, and a further inhibitory effect for surface growth at a suboptimal temperature (10 °C). These findings give a systematic quantitative insight on the impact of nisin as an environmental challenge on the growth and spatial organisation of L. innocua, in viscoelastic food model systems of various structural compositions/complexities. This study highlights the importance of accounting for system structural composition/complexity when designing minimal food processing methods with natural antimicrobials. ispartof: International Journal Of Food Microbiology vol:286 pages:15-30 ispartof: location:SPAIN, Cordoba status: Published online

A novel strategy involving Olive Leaf Extract (OLE) and Cold Atmospheric Plasma (CAP) was developed as a green antimicrobial treatment. Specifically, we reported a preliminary investigation on the combined use of OLE + CAP against three pathogens, chosen to represent medical and food industries (i.e., E. coli, S. aureus and L. innocua). The results indicated that a concentration of 100 mg/mL (total polyphenols) in OLE can exert an antimicrobial activity, but still insufficient for a total bacterial inactivation. By using plain OLE, we significantly reduced the growth of Gram positive S. aureus and L. innocua, but not Gram-negative E. coli. Instead, we demonstrated a remarkable decontamination effect of OLE + CAP in E. coli, S. aureus and L. innocua samples after 6 h. This effect was optimally maintained up to 24 h in S. aureus strain. E. coli and L. innocua grew again in 24 h. In the latter strain, OLE alone was most effective to significantly reduce bacterial growth. By further adjusting the parameters of OLE + CAP technology, e.g., OLE amount and CAP exposure, it could be possible to prolong the initial powerful decontamination over a longer time. Since OLE derives from a bio-waste and CAP is a non-thermal technology based on ionized air, we propose OLE + CAP as a potential green platform for bacterial decontamination. As a combination, OLE and CAP can lead to better antimicrobial activity than individually and may replace or complement conventional thermal procedures in food and biomedical industries.

Novel processing methods such as cold atmospheric plasma (CAP) and natural antimicrobials like nisin, are of interest to replace traditional food decontamination approaches as, due to their mild nature, they can maintain desirable food characteristics, i.e., taste, texture, and nutritional content. However, the microbial growth characteristics (planktonic growth/surface colonies) and/or the food structure itself (liquid/solid surface) can impact the inactivation efficacy of these novel processing methods. More specifically, cells grown as colonies on a solid(like) surface experience a completely different growth environment to cells grown planktonically in liquid, and thus could display a different response to novel processing treatments through stress adaptation and/or cross protection mechanisms. The order in which combined treatments are applied could also impact their efficacy, especially if the mechanisms of action are complementary. This work presents a fundamental study on the efficacy of CAP and nisin, alone and combined, as affected by food system structure. More specifically, Listeria innocua was grown planktonically (liquid broth) or on a viscoelastic Xanthan gum gel system (1.5% w/v) and treated with CAP, nisin, or a combination of the two. Both the inactivation system, i.e., liquid versus solid(like) surface and the growth characteristics, i.e., planktonic versus colony growth, were shown to impact the treatment efficacy. The combination of nisin and CAP was more effective than individual treatments, but only when nisin was applied before the CAP treatment. This study provides insight into the environmental stress response/adaptation of L. innocua grown on structured systems in response to natural antimicrobials and novel processing technologies, and is a step towards the faster delivery of these food decontamination methods from the bench to the food industry. ispartof: INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY vol:337 ispartof: location:Netherlands status: published

Natural antimicrobials are of interest to replace traditional food decontamination methods: they are milder and maintain desirable sensory characteristics. However, efficacy can be affected by food structure/composition, thus structural effects in a co-culture pathogen/microflora system are investigated. Listeria was grown planktonically (liquid broth) or on a biphasic viscoelastic system, in monoculture with/without artificial nisin, or in co-culture with L. lactis (nisin/non-nisin producing). Microbial growth kinetics were monitored and advanced microscopy techniques were utilised to quantify cellular interactions and spatial organisation. Microstructural effects are observed on the kinetics, with differences in monoculture/co- culture. Significant microscopic differences are observed in spatial organisation and colony size. We are the first to observe changing growth location for all species in monoculture/co- culture, with differences in colony size/organisation through stationary phase. This study provides insight into the environmental stress response/adaptation of Listeria grown on structured systems in response to L. lactis and natural antimicrobials.

Cold atmospheric plasma (CAP) is a minimal food processing technology of increasing interest in the food industry, as it is mild in nature compared to traditional methods (e.g. pasteurisation) and thus can maintain the food’s desirable qualities. However, due to this mild nature, the potential exists for post-treatment microbial survival and/or stress adaptation. Furthermore, biofilm inactivation by CAP is underexplored and mostly studied on specific foods or on plastic/polymer surfaces. Co-culture effects, biofilm age, and innate biofilm-associated resistance could all impact CAP efficacy, while studies on real foods are limited to the food product investigated without accounting for structural complexity. The effect of a Remote and Enclosed CAP device (Fourth State Medicine Ltd) was investigated on Escherichia coli and Listeria innocua grown as planktonic cells and as single or mixed bacterial biofilms of variable age, on a biphasic viscoelastic food model of controlled rheological and structural complexity. Post-CAP viability was assessed by plate counts, cell sublethal injury was quantified using flow cytometry, and biofilms were characterised and assessed using total protein content and microscopy techniques. A greater impact of CAP on planktonic cells was observed at higher air flow rates, where the ReCAP device operates in a mode more favourable to reactive oxygen species than reactive nitrogen species. Although planktonic E. coli was more susceptible to CAP than planktonic L. innocua, the opposite was observed in biofilm form. The efficacy of CAP was reduced with increasing biofilm age. Furthermore, E. coli produced much higher protein content in both single and mixed biofilms than L. innocua. Consequently, greater survival of L. innocua in mixed biofilms was attributed to a protective effect from E. coli. These results show that biofilm susceptibility to CAP is age and bacteria dependent, and that in mixed biofilms bacteria may become less susceptible to CAP. These findings are of significance to the food industry for the development of effective food decontamination methods using CAP.

The FDA-approved antimicrobial peptide nisin has been used to prevent contamination of food products from Gram-positive pathogens including Listeria monocytogenes (L. monocytogenes). However, some L. monocytogenes strains are tolerant or resistant to nisin treatment at a dose that would normally be lethal. Therefore, alternative or combined antimicrobial treatment strategies are required to effectively control L. monocytogenes contaminations. In this study, we investigate whether combination treatment with nisin and fatty acids can overcome the nisin resistance of L. monocytogenes by monitoring cell survival and biofilm inactivation of two strains (ATCC15313 serotype 1/2a and ATCC19115 serotype 4b). Lauric acid and N-tridecanoic acid were chosen because they exhibit antimicrobial activities. First, we observed that nisin (22.5 μg/mL) rapidly killed cells with up to a 6-log reduction after 3 h, but treatment for prolonged periods (i.e., up to 24 h) caused the cells to recover growth via the development of either inherent nisin resistance or temporary nisin tolerance. However, co-treatment of nisin and each fatty acid significantly decreased the cell survival and hindered the development of nisin resistance. In addition, the co-treatment effectively inactivated cells in biofilms. The combined antimicrobial treatment may be promising approach in the food processing environment, where Listeria contamination occurs.

The occurrence of sublethally injured cells in foods poses major public health concerns and is an essential aspect when assessing the microbial response to food preservation strategies, yet there is limited research dealing with its specific implications for mild heating. All available studies so far have been performed in broths colonized by planktonic cells, although their susceptibility to lethal agents has often been reported to be markedly different to the stress tolerance of cell colonies developed in solid foods. In this work, the effect of planktonic and colony growth, as well as the influence of colony density on sublethal injury induced by mild heating of Escherichia coli, Salmonella Typhimurium and Listeria innocua were assessed in food model systems. Detection of injured survivors relied on their inability to form visible colonies on salt-based selective media, which do not affect the growth of healthy cells. Sublethal injury (SI) increased rapidly with shorter exposure times and afterwards, decreased progressively, suggesting a mechanism of cumulative damage triggering lethal instead of SI. Cell arrangement affected the degree of SI, higher values being generally found for gelified systems, although the effect of colony density depended on the target microorganism. This information is essential for optimizing the design of food safety assurance systems.

The effect of a gelified matrix on the heat inactivation of Escherichia coli K12 and Salmonella typhimurium was investigated. Compared to liquid systems, microbial populations grow as colonies due to the gelified character of the solid environment. In addition, the effect of colony density as well as the effect of the concentration of the gelling agent on the microbial inactivation kinetics were studied. More specifically, E. coli K12 MG1655 or S. typhimurium cells were grown in a gelified medium (Brain Heart Infusion or Tryptic Soy broth supplemented with 1.5% or 2.5% (w/v) xanthan gum) at 37°C until they reached the stationary phase. Especially for E. coli, different colony densities were obtained by starting growth at 37°C from different initial cell counts, i.e., a lower inoculum level results in bigger stationary phase colonies. Hereafter, these colonies were inactivated at 54°C. Cells inactivated as colonies in the gelified environment are protected from heat stress, leading to an increased survival compared to the liquid system for both studied microorganisms. Results indicated that the density of the colonies has no significant influence on the heat resistance in the range 103-106CFU/mL, but when increasing the initial cell count to the level of 108CFU/mL less thermotolerance was observed. © 2013.

Aim The effect of spent medium, obtained after different time–temperature pre-histories, on the heat inactivation of Escherichia coli K12 MG1655 is studied. Methods and results Stationary E. coli cells were heated in BHI broth (initial pH 7.5) at different time–temperature scenarios, i.e., (1) 30 °C to 55 °C at 0.14 °C/min, (2) 30 °C to 42 °C at 0.14 °C/min and (3) 30 °C to 42 °C at 0.8 °C/min. After the heat treatment, spent medium was filter-sterilized, non-stressed cells were added and inactivation experiments took place at 54 °C and 58 °C. In all scenarios, increased resistance was observed. The main characteristics of the spent medium – compared to the unmodified BHI broth – are (1) the presence of proteins (proven via SDS-PAGE) and (2) a lower pH of approximately 6. Possibly, the increased resistance is due to these proteins and/or the lower pH. Further experiments revealed that each factor separately may lead to an increased heat resistance. Conclusions It can be concluded that this increased heat resistance resulted from both the presence of the heat shock proteins in the spent medium and the lowered pH. Experiments, which separate both effects, showed that mainly the lower pH resulted in the increased thermotolerance. Significance and impact of study This study may lead to a better understanding and control of the heat stress adaptation phenomenon as displayed by E. coli at lethal temperatures. Therefore, it contributes to an improved assessment of the effect of temperature during thermal processes in the food industry.

The aim of this work is to investigate the effect of acid treatment -before and during heat inactivation- on the heat resistance of Escherichia coli K12 MG1655 cells at lethal temperatures. E. coli cells were grown in Brain Heart Infusion broth until they reached the stationary phase (≈10(9) cfu/mL). Approximately 30 min before thermal inactivation the early stationary phase cells were added in Brain Heart Infusion broth with a specific pH value, achieved with addition of either acetic (50% (v/v)), lactic (50% (v/v)) or hydrochloric acid (30% (v/v)), and inactivation experiments took place at 54 °C and 58 °C. The inactivation dynamics are analysed using the inactivation model of Geeraerd et al. (2000). This enables to define the induced thermotolerance of E. coli as a prolongation of the shoulder and/or a reduction of the inactivation rate. Generally, addition of acids increased the heat resistance of E. coli. The induced resistance depends on the type of acid and on the quantity added, i.e. different levels of acidification lead to a different level of heat resistance. This work provides additional knowledge on the reaction of bacterial cultures to heat after acid treatment -before and during heat treatment- and, therefore, it contributes to an improved understanding of the effect of acid exposure on the bacterial heat resistance.

The objective of the present study was to validate a kinetic model for growth of spoilage bacteria in modified atmosphere packed (MAP) gilthead seabream fillets and to select a Time Temperature Integrator (TTI). The temperature and CO2 dependence of the growth of lactic acid bacteria in MAP gilthead seabream fillets was expressed by an Arrhenius-type model for the range of 0-15 °C and 20-80% CO2, which was validated at isothermal, variable and chill chain conditions. A new UV activatable photochemical TTI, was kinetically studied and the influence of the level of activation on the response of the TTI was modelled. Applying the developed models, the required charging levels were estimated so that the TTI response was tailored to monitor the shelf life of fish fillets at selected MAP conditions, during the chill chain storage. A simulation experiment of the product distribution and storage in various chill chain conditions showed the applicability of the TTIs as shelf life monitors. © 2010 Elsevier Ltd.

AIM: To quantify the influence of trimethylamine-N-oxide (TMAO) on the heat resistance of Escherichia coli K12 MG1655 cells at static temperatures. METHODS AND RESULTS: Stationary-phase E. coli cells were inactivated at 52, 54 and 58°C. The heat resistance is described as reduction in the inactivation rate, k(max) , and/or an increase in the time for one decimal reduction, D, and/or an increase in the time for the fourth decimal reduction, t(4D) . CONCLUSIONS: Resistance of E. coli changed - increased - at all temperatures under study. Generally, the addition of TMAO to the growth medium protected E. coli cells, leading to an increase in their heat resistance, i.e. reduced k(max) and increased D and t(4D) values are obtained. SIGNIFICANCE AND IMPACT OF THE STUDY: Additional knowledge on the reaction of E. coli to heat in the presence of the organic osmolyte TMAO at lethal temperatures is provided. This work contributes to an improved understanding of the level of the resistance of bacteria to heat in the presence of osmolytes.

The effect of temperature (0-15°C) and CO 2 (0-80%) on the growth of spoilage bacteria (total viable count, Pseudomonas sp. and lactic acid bacteria) in marine cultured gilthead seabream fillets was studied and kinetically modelled using an Arrhenius type equation. Pseudomonas sp. dominated the spoilage microflora of aerobically packed fillets whereas LAB defined spoilage at the MAP conditions. Modified atmosphere packaging led to a significant shelf-life extension of fillets, in terms of microbial growth and organoleptic deterioration. The preservative effect of CO 2 was greater as the CO 2 concentration in the atmosphere increased. Under this context, MAP can practically extend the shelf life of chilled gilthead seabream fillets. The growth of LAB was modelled as a function of temperature and CO 2 concentration in modified atmosphere packaging. The applicability of the models was validated at fluctuating temperature conditions. The nominal maximum CO 2 concentration (CO 2,max) for growth and the specific growth rate (k ref) at T ref (4°C) in the absence of carbon dioxide were determined to be 98.3% and 0.015 h -1, respectively. The activation energy parameter of the model, which indicates the temperature dependence of the growth rates, was 101.0 kJ/mol showing a strong dependence of LAB growth on storage temperature. Based on organoleptic acceptability the end of shelf life coincided with 10 7/g Pseudomonas sp. and 10 6/g lactobacilli for aerobically and MAP stored samples respectively. Based on the developed models this translates to 7, 8, 12 and 32 days of shelf life at 5°C and 0, 20, 50 and 80% CO 2 respectively.

The objective of the study was to establish a validated kinetic model for growth of spoilage bacteria on vacuum packed tuna slices in the temperature range of 0 to 15 degrees C and to evaluate the applicability of the TTI (Time Temperature Integrators) based SMAS (Safety Monitoring and Assurance System) system to improve tuna product quality at the time of consumption in comparison to the conventional First In First Out (FIFO) approach. The overall measurements of total flora and lactic acid bacteria (LAB) on the tuna samples used in a laboratory simulated field test were in close agreement with the predictions of the developed kinetic model. The spoilage profile of the TTI bearing products, handled with SMAS, was improved. Three out of the thirty products that were handled randomly, according to the FIFO approach, were already spoiled at the time of consumption (logN(LAB)>6.5) compared to no spoiled products when handled with the SMAS approach.