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The antibacterial and antibiofilm activity of essential oil of clove against Listeria monocytogenes and Salmonella Enteritidis were investigated. The chemical composition of the oil was characterized by gas chromatography–mass spectrometry. Stock solution of the essential oil of clove was prepared in 95% (v/v) ethanol (EOC). The antibacterial assays were performed by disk diffusion assay and minimal inhibitory concentration (MIC). The biomass of adhered cells and preformed biofilms after incubation with different concentrations of EOC was assessed by crystal violet. Eugenol was the major bioactive compound of clove essential oil, accounting for 78.85% of the total composition. The MIC values for L. monocytogenes and S. Enteritidis were 0.05 mg/ml and 0.1 mg/ml, respectively. The initial cell adhesion at MIC was inhibited by 61.8% for L. monocytogenes and 49.8% for S. Enteritidis. However, the effect of EOC was less marked on biofilm eradication than on cell adhesion. At MIC and within 1 hour of incubation with the EOC, the preformed biofilms were reduced by 30.2% and 20.3% for L. monocytogenes and S. Enteritidis, respectively. These results suggest that sanitizers based on clove essential oil could be a potential strategy to control biofilms in food-related environments.

Consumers’ demands for ready-to-eat, fresh-like products are on the rise during the last years. This type of products have minimal processing conditions that can enable the survival and replication of pathogenic microorganisms. Among them, Listeria monocytogenes is of special concern, due to its relatively high mortality rate and its ability to replicate under refrigeration conditions. Previous research works have shown that nanoemulsified essential oils in combination with thermal treatments are effective for inactivating L. monocytogenes. However, previous research works were limited to isothermal conditions, whereas actual processing conditions in industry are dynamic. Under dynamic conditions, microorganism can respond unexpectedly to the thermal stress (e.g. adaptation, acclimation or increased sensitivity). In this work, we assess the combination of nanoemulsified D-limonene with thermal treatments under isothermal and dynamic conditions. The nanoemulsion was prepared following an innovative methodology using soya lecithin, a natural compound as well as the essential oil. Under isothermal heating conditions, the addition of the antimicrobial enables a reduction of the treatment time by a factor of 25. For time-varying treatments, dynamic effects were relevant. Treatments with a high heating rate (20 °C/min) are more effective than those with a slow heating rate (1 °C/min). This investigation demonstrates that the addition of nanoemulsified D-limonene can greatly reduce the intensity of the thermal treatments currently applied in the food industry. Hence, it can improve the product quality without impacting its safety.

The antibacterial and antibiofilm activity of essential oil of clove against

The heat resistance of the bacterial spores of Moorella thermoacetica, Clostridium sporogenes, Geobacillus stearothermophilus and Bacillus coagulans was determined over a wide range of temperatures using the capillary method and thermoresistometer Mastia. The results showed that the two experimental methods gave similar heat resistance values excepted for Geobacillus stearothermophilus. The effect of temperature on thermal resistance was evaluated using the Arrhenius and Bigelow models. The fit of the heat sensitivity parameters of the Arrhenius and Bigelow models on the heat resistance parameter values obtained over a wide temperature range was equally good. Despite the apparent mathematical incompatibility of the two equations, it is recognized that they yield the same goodness of fit. This paper finds a mathematical reason for this convergence and explains why inside a temperature range of at least 100 °C, no significant difference in the quality of fit between these two models can be found.

This contribution presents a mathematical model to describe non-isothermal microbial inactivation processes taking into account the acclimation of the microbial cell to thermal stress. The model extends the log-linear inactivation model including a variable and model parameters quantifying the induced thermal resistance. The model has been tested on cells of Escherichia coli against two families of non-isothermal profiles with different constant heating rates. One of the families was composed of monophasic profiles, consisting of a non-isothermal heating stage from 35 to 70 °C; the other family was composed of biphasic profiles, consisting of a non-isothermal heating stage followed by a holding period at constant temperature of 57.5 °C. Lower heating rates resulted in a higher thermal resistance of the bacterial population. This was reflected in a higher D-value. The parameter estimation was performed in two steps. Firstly, the D and z-values were estimated from the isothermal experiments. Next, the parameters describing the acclimation were estimated using one of the biphasic profiles. This set of parameters was able to describe the remaining experimental data. Finally, a methodology for the construction of diagrams illustrating the magnitude of the induced thermal resistance is presented. The methodology has been illustrated by building it for a biphasic temperature profile with a linear heating phase and a holding phase. This diagram provides a visualization of how the shape of the temperature profile (heating rate and holding temperature) affects the acclimation of the cell to the thermal stress. This diagram can be used for the design of inactivation treatments by industry taking into account the acclimation of the cell to the thermal stress.

Heterogeneity in the response of microbial cells to environmental conditions is inherent to every biological system and can be very relevant for food safety, potentially being as important as intrinsic and extrinsic factors. However, previous studies analyzing variability in the microbial response to thermal treatments were limited to data obtained under isothermal conditions, whereas in the reality, environmental conditions are dynamic. In this article we analyse both empirically and through mathematical modelling the variability in the microbial response to thermal treatments under isothermal and dynamic conditions. Heat resistance was studied for four strains of Listeria monocytogenes (Scott A, CECT 4031, CECT 4032 and 12MOB052), in three different matrices (buffered peptone water, pH 7 Mcllvaine buffer and semi-skimmed milk). Under isothermal conditions, between-strain and between-media variability had no impact in the heat resistance, whereas it was very relevant for dynamic conditions. Therefore, the differences observed under dynamic conditions can be attributed to the variability in the ability for developing stress acclimation. The highest acclimation was observed in strain CECT 4031 (10-fold increase of the D-value), while the lowest acclimation was observed in strain CECT 4032 (50% increase of the D-value). Concerning the different media, acclimation was higher in buffered peptone water and semi-skimmed milk than in Mcllvaine buffer of pH 7.0. To the knowledge of the authors, this is the first research work that specifically analyses the variability of microbial adaptation processes that take place under dynamic conditions. It highlights that microbial heat resistance under dynamic conditions are sometimes determined by mechanisms that cannot be observed when cells are treated in isothermal conditions (e.g. acclimation) and can also be affected by variability. Consequently, empirical evidence on variability gathered under isothermal conditions should be extrapolated with care for dynamic conditions.

The estimation of the concentration of microorganisms in a sample is crucial for food microbiology. For instance, it is essential for prevalence studies, challenge tests (growth and/or inactivation studies) or microbial risk assessment. The application of serial dilutions followed by viable counts in Petri dishes is probably the most extended experimental methodology for this purpose. However, this enumeration technique is also a source of uncertainty. In this article, the uncertainty of the serial dilution and viable count methodology related to the sampling error is analyzed, as well as the approximation of the microbial concentration by the number of colonies in a Petri dish. We analyze from a theoretical point of view (statistical analysis) the application of the binomial and Poisson models, demonstrating that the Poisson distribution increases the variance when used to model individual serial dilutions. On the other hand, the binomial model produces unbiased results. Therefore, the Poisson distribution is only applicable when it is a good approximation of the binomial distribution, so the use of the latter is recommended. The relevance of this uncertainty is demonstrated by Monte Carlo simulations of a generic microbial inactivation experiment, where the only source of uncertainty/variability considered is the one generated by serial plating and viable cell enumeration. Due to both the uncertainty of the methodology and the omission of zero-count plates because of the log-transformation, the simulated survival curve can have a tail. Therefore, this phenomenon, which is usually attributed to biological variability, can be to some extent an artefact of the experimental design and/or methodology.

Enabling temperature dependent experiments in Atomic Force Microscopy is of great interest to study materials and surface properties at the nanoscale. By studying Curie temperature of multiferroic materials, temperature dependent phase transitions on crystalline structures or resistive switching phenomena are only a few examples of applications. We present an equipment capable of cooling samples using a thermoelectric cooling stage down to −61.4°C in a 15 × 15 mm2 sample plate. The equipment uses a four-unit thermoelectric stack to achieve maximum temperature range, with low electrical and mechanical noise. The equipment is installed into a Keysight 5500LS Atomic Force Microscopy maintaining its compatibility with all Electrical and Mechanical modes of operation. We study the contribution of the liquid cooling pump vibration into the cantilever static deflection noise and the temperature dependence of the cantilever deflection. A La0.7Sr0.3MnO3-y thin film sample is used to demonstrate the performance of the equipment and its usability by analyzing the resistive switching phenomena associated with this oxide perovskite., This research was supported by Consolider NANOSELECT (CSD 2007-00041). ICMAB acknowledges financial support from the Spanish Ministry of Economy and Competitiveness, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV- 2015-0496). The authors thank ICMAB Scientific and Technical Services.

Decisions regarding microbial risk assessment usually have to be carried out with incomplete information. This is due to the large number of possible scenarios and the lack of specific data for the problem considered. Consequently, risk assessment studies are based on the information obtained with a small number of bacterial cells which are considered the most heat resistant and/or more capable of multiplying during storage. The identification of the most resistant strains is usually based on D and z-values, normally estimated from isothermal experiments. This procedure omits the potential effect that the shape of the dynamic thermal profile applied in industry has on the microbial inactivation. One example of such effects is stress acclimation, which is related to a physiological response of the cells during sub-lethal treatments that increases their resistance. In this article, we use a recently published mathematical model to compare the development of thermal resistance for Escherichia coli K12 MG1655 and E. coli CECT 515 using inactivation data already published for these strains. Based only on the isothermal experiments, E. coli K12 MG1655 would be identified as more resistant to the thermal treatment than the CECT 515 strain in the 50-65oC temperature range. However, we conclude that stress acclimation is strain (and/or media)-dependent; the CECT 515 strain has a higher capacity for developing a stress acclimation than K12 MG1655 (300% increase of the D-value for CECT 515, 50% for K12 MG1655). It, thus, has the potential to be more resistant to the thermal treatment than the K12 MG1655 strain for some conditions allowing acclimation. A methodology is proposed to identify for which conditions this may be the case. After calibrating the model parameters representing acclimation using real experimental data, the applicability of the proposed approach is demonstrated using numerical simulations, showing how the CECT 515 strain can be more resistant for some heating profiles. Consequently, the most resistant bacterial strain to a dynamic heating profile should not be identified based only on isothermal experiments (D- and z-value). The relevance of stress acclimation for the treatment studied should also be evaluated.

The application of oily antimicrobials in form of nanoemulsion has been proved to solve the problem of their immiscibility in aqueous media, still preventing microbial growth and even improving the antimicrobial effect observed when applied directly. At present, only a few documented studies have evaluated the combined effect of nanoemulsions with other factors of stress for the microorganism. The present research shows very promising results on the combination of nanoemulsified d -limonene with thermal treatments on the inactivation of Listeria monocytogenes . The thermal resistance of L . monocytogenes was reduced two to five times when 0.5 mM d -limonene was added directly to the heating medium. However, when the same concentration of d -limonene was present in the heating medium in form of nanoemulsion, the heat resistance was reduced by one hundred times at all heating temperatures tested. The addition of nanoemulsified antimicrobials would allow to reduce greatly the intensity of the thermal treatments currently applied in the food processing industry.

Heat resistance determinations, necessary for the design of safe thermal treatments, are usually performed in batch systems, but those on continuous systems are more realistic. In continuous systems, samples and temperatures are only taken from the inlet and outlet. Therefore, microbial inactivation kinetics and heat-labile compound degradation on these systems remain a black box. A pilot-scale heat exchanger, with several temperature sensors and sampling tubes along the system, was built. The aims of this research were to determine the residence time distribution, the flow type, and the inactivation kinetics of Salmonella Senftenberg and Staphylococcus aureus on the heat exchanger. Microbial inactivation data were obtained both under isothermal and non-isothermal conditions and compared to those obtained in a batch system. Flow was estimated as turbulent. Under isothermal conditions, similar inactivation levels were reached for S. aureus in both equipments (batch or continuous). Under non-isothermal conditions, for both microorganisms, higher inactivation of the population was observed in the last step of the treatment in the heat exchanger than in the batch system. These differences could be attributed to faster heating rates in the heat exchanger. The heat exchanger used in this investigation is a suitable tool to understand microbial inactivation kinetics in continuous systems.

The inactivation of Escherichia coli, S. Enteritidis and Listeria monocytogenes after UV-C radiation with 0, 2.5, 5, 7.5, 10 and 15 kJ UV-C m−2 on fresh-cut kailan-hybrid broccoli was explored. Inactivation did not follow linear kinetics. Hence, it was modelled by using the Weibull distribution function, obtaining adjusted R2 values higher than 94%, indicative of the accuracy of the model to the experimental data. The UV-C doses needed to reduce 1 log cycle the E. coli, S. Enteritidis and L. monocytogenes counts were 1.07, 0.02 and 9.26 kJ m−2, respectively, being S. Enteritidis the most sensitive microorganism to UV-C radiation while L. monocytogenes was the most resistant. According to experimental data, UV-C doses higher than 2.5 kJ m−2 did not achieve great microbial reductions. No differences in the growth behaviour of these microorganisms was observed in the treated samples stored under air conditions at 5, 10 and 15 °C, compared to the control. Conclusively, low UV-C doses are effective to reduce E. coli, S. Enteritidis and L. monocytogenes populations in fresh-cut kailan-hybrid broccoli keeping such counts stable during shelf life at 5–10 °C. The current study provides inactivation models for these foodborne pathogens that can be used in microbial risk assessment.

The present work evaluated the antibacterial effect of nanoemulsions from natural compounds d-limonene and nisin against Listeria monocytogenes in tryptic soy broth growth medium, chicken broth, and vegetable cream. Experiments were performed by means of optical density growth curves and studies of viability in culture media and foods. Stability of nanoemulsions was evaluated by a Mastersizer 2000 equipment. Results showed greater effectiveness when applying d-limonene in form of nanoemulsion than when applying it directly, and when using both compounds together, both directly and in the form of nanoemulsion. Concentration of L. monocytogenes decreased at least in three log cycles in all the culture media and foods used within the first 90 min after the addition of the natural antimicrobials combined in form of nanoemulsion. Moreover, the growth of L. monocytogenes was inhibited with the combination of antimicrobials in the four weeks that the experiment lasted. Nanoemulsion technology would solve present problems of solubility and stability of oily antimicrobials in the food industry.

Cronobacter sakazakii is an opportunistic pathogen in neonates which can cause meningitis, septicaemia and enterocolitis related to the consumption of contaminated Powdered Infant Formula (PIF). C. sakazakii has an unusual ability to survive under dry conditions and it could be among the most thermotolerant members of the Enterobacteriaceae. Little is known about how Cronobacter species respond to heat stress and the mechanisms involved in the process. In the current study we determined the heat resistance of a particularly stress tolerant C. sakazakii strain, C. sakazakii DPC 6529, and monitored the behavior of a lux-tagged derivative under different reconstitution and handling scenarios in a commercial brand of PIF. Some of the molecular mechanisms involved in the heat stress response were investigated using a transposon mutagenesis approach. Survival curves of C. sakazakii DPC 6529 in Luria-Bertani (LB) broth and PIF at various temperatures (58, 60, 62 and 64 °C) displayed an upward concavity and were fitted to the non-linear Weibull model. While at the highest treatment temperatures heat resistance was lower in PIF than in LB broth, at lower temperatures no significant differences in heat resistance were observed. Experiments in real time with artificially inoculated PIF reconstituted at different water temperatures (50, 55, 60, 65, 70 °C) and cooled at different rates confirmed that C. sakazakii can survive for long time periods in powdered formula, and is capable of proliferating after reconstitution. The use of water at temperatures between 50 and 65 °C for reconstitution did not provide a significant inactivation of C. sakazakii cells. Reconstitution at 70 °C reduced the bacterium to levels below the detection limit, although survivors were able to proliferate and reached dangerous levels when the reconstituted product was stored for a long time at room temperature. The cooling rate had an important impact on survival and subsequent growth of C. sakazakii, which makes it advisable to avoid rapid cooling of baby formula. Transposon mutagenesis allowed the identification of some of the molecular mechanisms involved in the response of C. sakazakii DPC6529 to heat stress. Genes identified included the Ribosome Maturation Protein RimP and Outer Membrane Porin L (OmpL). Results suggest that de novo protein synthesis, and the uptake of cysteine for the formation of disulfide bonds for protein stabilization, are key processes.

The present work studies the effect of acid shock on cell viability of Bacillus cereus and Bacillus weihenstephanensis. Experiments were performed by means of flow cytometry (FCM) combined with fluorescent labelling. Propidium iodide, for membrane evaluation, and carboxyfluorescein diacetate, for esterase activity were used for differential staining of both strains. pH values of 3.4, 3.8 and 4.2 were selected on the basis of previous screening tests. FCM analysis showed to clearly discriminate between different populations, viable and damage cells, leading to successful assessment of acid shock effect on B. cereus and B. weihenstephanensis vegetative cell viability. Cellular viability decreased at lower pH values and longer exposure times, being B. weihenstephanensis more acid resistant than B. cereus. Reliability of FCM analysis for detection of B. cereus and B. weihenstephanensis vegetative cells was compared with classical viable plate count techniques (VPC). Although FCM and VPC data were not directly correlated and classical analytical methods show higher reliability, FCM analysis provides high-speed information at real time on damage at single cell level, whereas VPC only gives an indication of cells able to grow at a certain time. These data could help to establish more accurately the potential risk associated to foods or processing conditions where pH is involved, taking damage into consideration.

Yeasts are tolerant to acid pH values, are able to grow in anaerobic media and have minimum nutrition requirements. These capabilities enable them to survive and even grow in foods prepared from acid fruits or vegetables. Among yeasts, Candida is one of the genus most frequently isolated from fruit juices. Bacteriocins and essential oils from spices and aromatic herbs are an alternative to preservatives and other technological treatments and have the advantage that their natural origins do not lead to consumer rejection. However, before the food industry uses them on a large scale, it is necessary to know their effects on microorganisms. The objective of this research was to study the effect of different concentrations of nisin, thymol, carvacrol and cymene on the growth of Candida lusitaniae in pH 5 broth at 25 ℃, and their potential uses as food preservatives. The addition of nisin at the concentrations tested (up to 3 µmol L−1) did not affect the yeast growth. Thymol, carvacrol and cymene completely inhibited the yeast growth at concentrations over 1 mM for at least 21 days at 25 ℃. Below this concentration, inhibitions on yeast growth were observed at increasing concentrations. The effect of thymol was also proved in tomato juice. This study indicates the potential use of essential oils for preservation of minimally processed foods.

The aim of this study was to evaluate the combined effect of a previous mild heat treatment (15 min at 55 ℃) with the use of antimicrobials, nisin and carvacrol, on the growth of Salmonella enteritidis and Salmonella senftenberg. Natural antimicrobials, alone or combined with a previous mild heat treatment, affected the growth of these two serovars in Tryptone Soy Broth at 37 ℃. Increasing concentrations of carvacrol had a significant effect on both growth rate and lag phase duration of both strains. The time to reach stationary phase was almost doubled in the case of S. enteritidis when a concentration of 0.77 mM in carvacrol was added. For S. senftenberg the effect was smaller. The effect of nisin and of heat, applied individually, was lower for both microorganisms. A combination of 1.2 µM nisin with 0.77 mM carvacrol significantly delayed the growth of heat treated cells, compared to the control without antimicrobials, showing additive effects.

The critical control points of the refrigeration and air conditioning systems serving the food processing and packaging plants are analyzed from the hygienic viewpoint. They identified the problems of hygienic design of the various components of refrigeration systems and air conditioning installations, and several solutions are described. The lack of hygienic design of air conditioning and refrigeration systems, and their inadequate cleaning and disinfection procedures, can cause significant economic losses and epidemic diseases in people, employees, or visitors, or in the final food consumers. So, the present manuscript analyses: (1) the microbial control in refrigeration systems, including the control of Legionella, and the limitations of using biocides embedded in plastic materials (for cooling tower filling and air ducts in air conditioning systems); (2) the hygienic design of evaporative coolers (evaporative condensers and cooling towers) as a new way for controlling Legionella contamination; and (3) the microbial contamination and hygienic design of air conditioning systems for food processing and packaging rooms, including the microbial control in the air by means of UV lamps, essential oil aerosolization, and filtration. The interest of the clean rooms and microbiologically controlled environments for food processing and packaging is explained through a case study of slicing and packaging of ready-to-eat meat products.

The aim of the present study was to characterize the thermal inactivation of Alicyclobacillus acidoterrestris spores under isothermal and non-isothermal conditions simulating industrial heating processes applied to tangerine vesicles. A microbiological time temperature integrator (TTI) suitable for estimating the severity of thermal processes applied to acid foods was also developed. The heat resistance of A. acidoterrestris was characterized by D105 °C = 0.63 min and z = 10.8 °C in tangerine juice, showing linear survival curves, without shoulders and tails. Under non-isothermal conditions, the use of isothermal data allowed for an accurate prediction of the inactivation. The spores were included in alginate TTIs and they were used to estimate the severity of thermal treatments applied both in a thermoresistometer Mastia and in a food pilot plant scale system, which allows fast heating of the product to 93 °C and then a short holding time (2 min). In the thermoresistometer, tangerine juice was used as heating medium. In the food pilot plant scale system, thermal treatments were applied in batch to unpackaged tangerine vesicles. In both equipments, the TTIs accurately predicted the lethality of the thermal treatments applied. The percent coefficients of variation for survivor counting in TTIs showed that distribution of heat is homogeneous both in the thermoresistometer and in the reactor, being lower than 10% in all cases. The logistic and normal distributions were found to be the best for fitting the different survivor datasets.

PurposeMost of the preservation processes are based on temperature control; they are economical, safe and well established. However, for certain foods these processes modify the valuable nutritional content and organoleptic properties. In recent years, a number of emerging food preservation processes have been developed to fill a market niche in which the consumer prefers to pay more to obtain processed foods with their natural properties. The paper seeks to address this issue.Design/methodology/approachIn this study a review of the available scientific data in relation to high hydrostatic pressure (HHP) and pulsed electric fields (PEF) are presented, highlighting the quality advantages, industrial application and safety risks in their use.FindingsNowadays, more than 100 industrial applications are marketed and several companies design process equipments. Damage to cell membranes, enzymes or DNA is the most commonly cause of microorganisms' death by these technologies. In addition, within a population of microorganisms, some bacteria are killed, others survive, and a proportion is damaged. The latter is the concern population as the damage may be repaired and the microorganism maybe viable during the product shelf life. Acquisition of new or modified characteristics such as higher treatment resistance could also occur. Another potential risk has to do with the death pattern of microorganisms showing deviations from the traditional log‐linear kinetic model (shoulders, tails and sigmoidal shape). Therefore the developing of simple mathematical models, which can adequately interpret this behaviour, is necessary.Originality/valueIn summary, PEF and HHP technologies need to be evaluated by industries and regulatory authorities and more affordable equipments should be marketed to expand their use at industrial level.

Spore-forming bacteria with high heat resistance increasingly challenge industrial sterilisation processes in foods. To ensure stability of manufactured foods, generally worst case scenarios are applied often leading to unwanted over processing of foods. This means bigger requirements of energy and larger emissions of CO2 into the atmosphere. A heat-resistant spore-forming bacterium, Bacillus sporothermodurans, was tested for its ability to produce consistently highly heat-resistant spore crops to be used to more precisely determine the minimal thermal process that is compatible with microbial stability, thus avoiding the indicated overprocessing. Isothermal survival curves of B. sporothermodurans IC4 spores were determined in distilled water at temperatures up to 125 °C using the capillary tube technique and a thermoresistometer Mastia. B. sporothermodurans consistently produced crops of heat-resistant spores with kmax values at 121 °C up to 0.46 min−1. After consecutive sporulation cycles, the measured heat resistance of B. sporothermodurans was not reduced. Survival curves showed shoulders that were characterized by means of existing models (Geeraerd, Weibull) that were compared with the classical, log-linear one. Shoulders are a common phenomenon in heat-resistant spore-forming bacteria and may be related to the need of more stringent processing conditions. B. sporothermodurans spores were considered adequate to optimize sterilisation processes of low acid foods under a wide variety of conditions. Establishing precise heat treatments can be a way to guarantee food safety and stability while reducing the use of energy and contaminating emissions.

In general, food engineers are trained to solve engineering problems in the food industry. More specifically, the food engineer must specify the functional requirements, design, and testing of food products, and finally, the evaluation of products to check for overall efficiency, cost, reliability, and most importantly, safety. Food safety must be considered foremost as the overall engineering problem encountered in the food supply chain, and it must be solved from a food safety engineering perspective. This article will show that the food safety engineering perspective is needed in order to produce high quality food products (minimally processed) that are both safe and secure. This multi-disciplinary approach will involve certain engineering components: (i) predictive microbiology as a tool to evaluate and improve food safety in traditional and new processing technologies, (ii) advanced food contaminants detection methods, (iii) advanced processing technologies, (iv) advanced systems for re-contamination control, (v) advanced systems for active and intelligent packaging.

The antimicrobial properties of oregano, rosemary and laurel extracts obtained by supercritical fluid extraction were investigated by examining their influence on the growth and viability of Listeria monocytogenes in laboratory medium and broccoli juice at 30 and 8 °C. Important decreases in the L. monocytogenes population were shown in presence of all the extracts obtained from rosemary and one oregano extract. The counts were reduced below the level of detection after 4 h of exposure at 30 °C in laboratory medium. A bactericidal effect was observed also when L. monocytogenes was exposed to rosemary at 30 °C and 8 °C in broccoli juice. Significant reductions in growth rate and an increase in lag phase of L. monocytogenes were observed in presence of some of the laurel and oregano extracts at both temperatures. Flow cytometry was used as a rapid method to determine the antibacterial effect of supercritical extracts and the physiological state of L. monocytogenes. Bacterial viability performed by dual staining of L. monocytogenes with SYTO 9 and propidium iodide revealed three different cell populations, specifically, living, dead and compromised cells. Live cell percentage decreased with the time of exposure, whereas the percentage of compromised cells remained constant and the dead cells increased in the same period.

The aim of this research was to evaluate the effect of thermal treatments (isothermal or nonisothermal) combined with nisin, a natural antimicrobial, on the survival and recovery of Clostridium sporogenes spores. The addition of nisin to the heating medium at concentrations up to 0.1 mg liter(-1) did not reduce the heat resistance of C. sporogenes. Without a thermal treatment, nisin added at concentrations up to 0.1 mg liter(-1) did not reduce the viable counts of C. sporogenes when added to the recovery medium, but inactivation of more than 4 log cycles was achieved after only 3 s at 100°C. At 100°C, the time needed to reduce viable counts by more than 3 log cycles was nine times shorter when 0.01 mg liter(-1) nisin was added to the recovery medium than without it. The heat resistance values calculated under isothermal conditions were used to predict the survival in the nonisothermal experiments, and the predicted values accurately fit the experimental data. The combination of nisin with a thermal treatment can help control C. sporogenes.

The microbial safety and stability of minimally processed foods are based on the application of combined preservative factors. Since microorganisms are able to develop adaptive networks to survive under conditions of stress, food safety may be affected, and therefore understanding of stress adaptive mechanisms plays a key role in designing safe food processing conditions. In the present study, the viability and the sublethal injury ofListeria monocytogenesexposed to moderate heat (55°C) and/or essential oil compounds (carvacrol and thymol, 0.3 mM) treatments were studied. Synergistic effects were obtained when combining mild heat (55°C) with one or both essential oil compounds, leading to inactivation kinetics values three to four times lower than when using heat alone. All the treatments applied caused some injury in the population. The injury levels ranged from around 20% of the surviving population under the mildest conditions to more than 99.99% under the most stringent conditions. Protein extracts of cells exposed to these treatments were analysed by two-dimensional gel electrophoresis. The results obtained revealed that stressed cells exhibited differential protein expression to control cells. The proteins upregulated under these stressing conditions were implicated, among other functions, in stress response, metabolism, and protein refolding.

A model for prediction of bacterial spore inactivation was developed. The influence of temperature, pH and NaCl on the heat resistance of Bacillus stearothermophilus spores was described using low-complexity, black box models based on artificial neural networks. Literature data were used to build and train the neural network, and new experimental data were used to evaluate it. The neural network models gave better predictions than the classical quadratic response surface model in all the experiments tried. When the neural networks were evaluated using new experimental data, also good predictions were obtained, providing fail-safe predictions of D values in all cases. The weights and biases values of neurons of the neural network that gave the best results are presented, so the reader can use the model for their own purposes. The use of this non-linear modelling technique makes it possible to describe more accurately interacting effects of environmental factors when compared with classical predictive microbial models.

The antibacterial action of carvacrol and cymene on two Listeria monocytogenes strains (STCC4031 and NCTN4032) was studied. Carvacrol or cymene showed inhibitory effect on the growth of L. monocytogenes during lag and exponential growth phases and was more evident with increasing concentrations in brain heart infusion broth at 30 degrees C. Carvacrol or cymene also decreased the survival of mid-exponential-growth-phase L. monocytogenes STCC4031 cells in potassium-N-2-hydroxy-ethylpiperazine-N-ethanesulfo nic acid, at 30 degrees C. The combination of carvacrol and cymene resulted in an increased antibacterial effect on the growth and a synergistic effect on the viability of L. monocytogenes compared with the natural compounds applied separately. The analysis of survival curves by the Weibull frequency distribution function allowed an accurate prediction of the level of inactivation achieved. Interestingly, an important bactericidal effect (4.7-log reduction) of low concentrations of both antimicrobials combined (0.75 mM) was observed on L. monocytogenes in carrot juice. This study indicates the potential use of carvacrol and cymene applied simultaneously for preservation of minimally processed foods.

The bactericidal action of thymol and cymene on two Bacillus cereus strains (INRA-AVTZ415 and INRA-AVZ421) was studied. Increasing concentrations of thymol (0.2–1.0 mmol l−1) or cymene (0.2–2.0 mmol l−1) showed higher bactericidal effect on exponential growth phase B. cereus cells suspended in HEPES buffer (pH 7), at 30°C. The two strains tested presented different sensitivity to these natural antimicrobials. When thymol and cymene were combined, it resulted in a greater bactericidal effect on B. cereus cells than when these compounds in the essential oil fraction of aromatic plants were applied separately. There was a synergistic effect of both natural antimicrobials on the viability of exponential growth phase B. cereus cells in pH 7 HEPES buffer, at 30°C. Survivor curves did not follow a first-order kinetics, which makes it difficult to establish processing conditions. Experimental data were modeled using a frequency distribution function (Weibull). This model guaranteed a good description of the experimental results and allowed predictions of time to a specific decrease in bacterial populations. This study indicates the potential use of thymol and cymene applied separately or simultaneously for preservation of minimally processed foods, as well as the validity of the Weibull distribution to describe the resulting nonlinear data and establish effective treatment conditions with these compounds.

The antibacterial action of thymol and cymene on two Bacillus cereus strains (INRA-AVTZ415 and INRA-AVZ421) was studied. Thymol and cymene alone showed higher antibacterial effect on the growth of B. cereus in BHI at 30 °C, both in lag and in exponential growth phases, with increasing added concentrations. However, the two strains tested showed different sensitivity to these natural compounds. When thymol and cymene were applied simultaneously, it resulted in a greater antibacterial effect than when the compounds were applied separately. The synergistic behaviour of both compounds was also shown for lag phase B. cereus INRA-AVZ421 cells in food substrates (carrot juice, chicken soup and pumpkin cream) at 30 °C, though a clear food-related effect was observed. This study indicates the potential use of thymol and cymene applied simultaneously for preservation of minimally processed foods.

Different inactivation kinetics data have been used to predict the number of survivors exposed to a heat treatment and, in consequence, to design thermal processes for the food industry. In this work, spores of an acidophilic strain of Bacillus subtilis were heated under isothermal and non-isothermal conditions. Experimental results obtained after isothermal treatments were analysed using the classical two-step linear regression procedure and a one-step non-linear regression method. Data obtained after non-isothermal treatments were analysed using a one-step, non-linear procedure. Kinetic parameters obtained from isothermal heating were close, either using the two-step linear regression (D 100=6.5 min) or the one-step non-linear regression (D 100=6.3 min), although the second method gave smaller 95% confidence intervals. The z values derived from non-isothermal heating were higher than those obtained in isothermal conditions (z=9.3 °C for non-isothermal heating at 1 °C/min versus z=7.7 °C for isothermal heating one step non-linear regression). Results were validated with experimental data obtained after different heat treatments, consisting of a phase of temperature increase at a fixed rate, followed by a holding phase. Non-isothermal methods predicted accurately the number of survivors after the heating ramp, while isothermal methods were more accurate for the holding phase of the treatment. When a temperature profile of a typical heat treatment process applied in the food industry was simulated, all predictions were on the safe side.

The selection of a primary model to describe microbial growth in predictive food microbiology often appears to be subjective. The objective of this research was to check the performance of different mathematical models in predicting growth parameters, both by absorbance and plate count methods. For this purpose, growth curves of three different microorganisms (Bacillus cereus, Listeria monocytogenes, and Escherichia coli) grown under the same conditions, but with different initial concentrations each, were analysed. When measuring the microbial growth of each microorganism by optical density, almost all models provided quite high goodness of fit (r(2) > 0.93) for all growth curves. The growth rate remained approximately constant for all growth curves of each microorganism, when considering one growth model, but differences were found among models. Three-phase linear model provided the lowest variation for growth rate values for all three microorganisms. Baranyimodel gave a variation marginally higher, despite a much better overall fitting. When measuring the microbial growth by plate count, similar results were obtained. These results provide insight into predictive microbiology and will help food microbiologists and researchers to choose the proper primary growth predictive model., This research was financially supported by the Ministerio de Economia y Competitividad of the Spanish Government and Fondo Europeo de Desarrollo Regional (FEDER) through Projects AGL2010-19775 and AGL2013-48993-C2-1-R.

The effect of irradiation with accelerated electrons on Bacillus cereus and Bacillus subtilis spore counts and spore heat resistance was investigated. Spore counts reduced approximately two log cycles for B. cereus and up to five log cycles for B. subtilis after a radiation dose of 7.6 kGy, with D10 values ranging from 1.5 to 3.8 kGy. Radiation had an important heat-sensitising effect on bacterial spores. After a 3.3 kGy treatment DT values were reduced more than three times for both B. cereus and B. subtilis spores. This study indicates the potential use of accelerated electrons, alone or followed by a heat treatment, for food preservation.

Highly heat-resistant spore-forming Bacillus cause nonsterility problems in canned food and reduce the shelf life of many processed foods. The aim of this research was to evaluate the thermal inactivation of Bacillus sporothermodurans IIC65 , Bacillus subtilis IC9, and Geobacillus stearothermophilus T26 under isothermal and nonisothermal conditions. The data obtained showed that B. sporothermodurans and B. subtilis were more heat resistant than G. stearothermophilus. The survival curves of B. sporothermodurans and B. subtilis showed shoulders, while the survival curves of G. stearothermophilus showed tails. Under nonisothermal treatment, at heating rates of 1 and 20 ℃/min, time needed to completely inactivate G. stearothermophilus was shorter than that required for B. sporothermodurans and B. subtilis. In complex heat treatments (heating–holding–cooling), the survival curves of B. sporothermodurans and B. subtilis showed the same activation shoulders than those obtained under isothermal treatments and the activation shoulders were again absent in the case of G. stearothermophilus. Predictions fitted quite well the data obtained for B. sporothermodurans. In contrast, the data for B. subtilis showed half a log cycle more survival than expected and in the case of G. stearothermophilus, the survival curve obtained showed much higher inactivation than expected.

The influence of mild heat pretreatment on the bactericidal action of nisin, carvacrol and thymol on stationary and exponential phase cells of two Bacillus cereus strains was studied. Carvacrol or thymol concentrations of 0.3 mmol/L had no bactericidal effect on unheated, and a minor effect on heated B. cereus cells either in stationary or exponential growth phases. Nisin (0.15 mg/mL) alone decreased the viable counts in all cases (unheated and heated cells in stationary and exponential phase) although the two strains tested showed different sensitivity to this natural antimicrobial between them. When carvacrol or thymol were combined with nisin, it resulted in a greater loss of viability of heated cells than when nisin was applied alone. There was a synergistic effect of nisin and both essential oils with a mild heat pretreatment on the viability of B. cereus cells. This study pointed out the potential use of nisin alone and in combination with carvacrol or thymol applied after a mild heat pretreatment for preservation of minimally processed foods.

The effect of the pH or the composition of the heating medium and of the sporulation temperature on the heat resistance of spores of a thermoacidophilic spore-forming microorganism isolated from a dairy beverage containing orange fruit concentrate was investigated. The species was identified as Alicyclobacillus acidocaldarius. The spores showed the same heat resistance in citrate-phosphate buffers of pH 4 and 7, in distilled water, and in orange juice at any of the temperatures tested (D120 degrees C = 0.1 min and z = 7 degrees C). A raise in 20 degrees C in the sporulation temperature (from 45 to 65 degrees C) increased the heat resistance eightfold (from D110 degrees C = 0.48 min when sporulated at 45 degrees C to 3.9 min when sporulated at 65 degrees C). The z-values remained constant for all sporulation temperatures. The spores of this strain of A. acidocaldarius were very heat resistant and could easily survive any heat treatment currently applied to pasteurize fruit juices.

The influence of growth phase, cell concentration, pH and conductivity of treatment medium on the inactivation of Salmonella senftenberg by high electric field pulses (HELP) was studied. Cells were more resistant to HELP treatments at the beginning of the logarithmic phase and at the stationary phase. Microbial inactivation was not a function of the initial cell concentration. At constant input voltage, electric field strength obtained in the treatment chamber depended on medium conductivity. At the same electric field strength, conductivity did not influence S. senftenberg inactivation. At the same conductivity, inactivation of S. senftenberg was bigger at neutral than acidic pH.

Sporulation temperature is one of the most important factors that determine the heat resistance of spores. Bacterial spores are usually more heat resistant when they are formed at higher temperatures. Sporulation temperature also influences the effect of pH of heating menstruum and the effect of different acids on spore heat resistance. Heat resistance is maximum at neutral pH and decreases with acidification, but when spores are sporulated at high temperatures the effect of acidic pH on heat resistance is lower at high heat treatment temperatures. Lactic and acetic acid decrease spore heat resistance more than other acidulants, but only when spores had been sporulated at high temperatures. These effects should be considered by canning factories of acid/acidified foods, especially in hot climate areas, where these high temperatures can be easily reached. The level of mineralization of spores obtained at different Sporulation temperatures seems to be the most important mechanism responsible for their heat resistance.

The influence of pH of heating menstruum (McIlvaine buffer) on the heat resistance of Bacillus coagulans spores has been investigated and compared with the heat resistance in homogenized tomato and asparagus at pH 7 and 4 at a wide range of temperatures. Spores were less heat resistant in all menstrua at acid pH. The magnitude of this effect was greatest at the lowest heating temperatures tested. z values in buffer increased from 8.9 degrees C at pH 7 to 10.5 degrees C at pH 4. pH of menstrua was the main influencing factor, but media composition also influenced heat resistance: at pH 7 heat resistance was similar in all menstrua (D111 degrees C = 1.6 min) but at pH 4 the heat resistance in homogenized foods (D111 degrees C = 0.26 min in tomato and D111 degrees C = 0.28 min in asparagus) was lower than in buffer (D111 degrees C = 0.49 min). The reduced influence of the acidification of media on the heat resistance of B. coagulans at higher temperatures should be taken into account when a rise in the temperature of treatment for canned vegetables is considered to shorten duration of heat processes.