Temperature and presence of ethanol affect accumulation of intracellular trehalose in Lactobacillus plantarum WCFS1 upon pulsed electric field treatment.

Pulsed electric field (PEF) treatment can be used to increase intracellular small molecule concentrations in bacteria, which can lead to enhanced robustness of these cells during further processing. In this study we investigated the effects of the PEF treatment temperature and the presence of 8% (v/v) ethanol in the PEF medium on cell survival, membrane fluidity and intracellular trehalose concentrations of Lactobacillus plantarum WCFS1. A moderate PEF treatment temperature of 21 °C resulted in a high cell survival combined with higher intracellular trehalose concentrations compared to a treatment at 10 and 35 °C. Interestingly, highest intracellular trehalose concentrations were observed upon supplementing the PEF medium with 8% ethanol, which resulted in more than a doubling in intracellular trehalose concentrations, while culture survival was retained. Overall, this study shows that treatment temperature and PEF medium optimization are important directions for improving molecule uptake upon PEF processing.

Reversibility of membrane permeabilization upon pulsed electric field treatment in Lactobacillus plantarum WCFS1.

Pulsed electric field (PEF) treatment, or electroporation, can be used to load molecules into cells. The permeabilizing effect of the PEF treatment on the cellular membrane can be either reversible or irreversible depending on the severity of the PEF treatment conditions. The influence of PEF on the reversibility of membrane permeabilization in Lactobacillus plantarum WCFS1 by two different fluorescent staining methods was investigated in this study. Whereas staining with propidium iodide (PI) before and after PEF treatment indicated small reversible permeabilized fractions of maximum 14%, the use of a double staining method with PI and SYTOX Green suggested larger reversible permeabilized fractions up to 40% of the population. This difference shows that the choice for a fluorescent staining method affects the conclusions drawn regarding reversibility of membrane permeabilization. Additionally, the effect of PEF treatment conditions on membrane integrity was compared, indicating a relation between critical electric field strength, cell size and membrane permeabilization. Overall this study showed the possibilities and limitations of fluorescent membrane integrity staining methods for PEF studies.

Moderate intensity Pulsed Electric Fields (PEF) as alternative mild preservation technology for fruit juice.

Moderate intensity Pulsed Electric Fields (PEF) was studied for microbial inactivation as an alternative to high intensity PEF or to classical thermal pasteurization. The process is characterized by the application of electric pulses, allowing an increase of the product temperature by the ohmic heat generated by the pulses. A systematic evaluation of the effect of parameters electric field strength (E) and pulse width (τ) on the inactivation of Escherichia coli, Listeria monocytogenes, Lactobacillus plantarum, Salmonella Senftenberg and Saccharomyces cerevisiae in orange juice was carried out in a continuous flow system. A wide range of conditions was evaluated, and both E and τ were shown to be important in the efficacy to inactivate micro-organisms. Remarkably, PEF conditions at E = 2.7 kV/cm and τ = 15-1000 µs showed to be more effective in microbial inactivation than at E = 10 kV/cm and τ = 2 µs. Inactivation kinetics of the tested PEF conditions were compared to an equivalent thermal process to disentangle non-thermal effects (electroporation) from thermal effects responsible for the microbial inactivation. At standard high intensity PEF treatment a non-thermal inactivation at E = 20 kV/cm and τ = 2 µs pulses was observed and attributed to electroporation. Non-thermal effects could also be resolved with moderate intensity PEF at E = 2.7 kV/cm and pulse width between τ = 15-1000 µs. Microbial inactivation at these moderate intensity PEF conditions was studied in more detail at different pH and medium conductivity for E. coli and L. monocytogenes in watermelon juice and coconut water. Under moderate intensity PEF conditions the effectiveness of treatment was independent of pH for all evaluated matrices in the pH range of 3.8-6.0, whereas under high intensity PEF conditions the pH of the product is a critical factor for microbial inactivation. This suggests that the inactivation proceeds through a different mechanism at moderate intensity PEF, and speculations for this mechanism are presented. In conclusion, moderate intensity PEF conditions at E = 2.7 kV/cm and pulse width of 15-1000 µs has potential for industrial processing for the preservation of fruit juices and pH neutral liquid food products.

Evaluation of the Gauss-Eyring model to predict thermal inactivation of micro-organisms at short holding times.

Application of mild (non)-thermal processing technologies have received considerable interest as alternative to thermal pasteurisation, because of its shorter holding time and lower temperature aiming for an improved product quality. To understand and develop these alternative technologies, like pulsed electric fields, a proper comparison between the conventional thermal and alternative process is necessary. Up to recent, no suitable models were available to predict the inactivation of micro-organisms by a thermal process at a chosen short holding time, due to non-linearity. The recently developed Gauss-Eyring model with two variables temperature and time has the properties to be a suitable model to apply for short holding times, and was tested for this purpose. Therefore, this study aims to validate if the Gauss-Eyring model can be used to describe non-linear isothermal (a fixed temperature with varying holding time) and isotime (a fixed holding time with varying temperature) thermal inactivation data, and if it is a suitable model to predict the thermal inactivation as a function of temperature for short holding times. Inactivation data of Escherichia coli, Listeria monocytogenes, Lactobacillus plantarum, Salmonella Senftenberg and Saccharomyces cerevisiae in orange juice were collected via isothermal and isotime inactivation kinetics. Survival of the tested micro-organisms was modelled with the Gauss-Eyring model, which contains three parameters σ, Tr and Z. The transition of 'no inactivation' to 'inactivation' (i.e. the 'shoulder' in inactivation curves) can be characterised as the temperature-time (T,t) combination where T=Tr-Z·log10(t), with Tr as the reference temperature defined for 1s treatment, Z as the temperature needed for a 10-fold increase of decrease of the holding time t, and σ as the temperature width of the distribution. The Gauss-Eyring model fitted well to the experimental data, and revealed different sensitivity for the tested micro-organisms. Based on the parameter estimations, survival curves for the desired short holding times were predicted.

The Gauss-Eyring model: A new thermodynamic model for biochemical and microbial inactivation kinetics.

A new primary model has been developed, using Gaussian distributed populations and Eyrings rate constant for the transition state, to describe inactivation kinetics of enzymes and micro-organisms subjected to heat and chemical treatment. The inactivation of both enzymes and micro-organisms could be associated with the irreversible transition to an inactivated state, as suggested by the Lumry-Eyring model for protein denaturation and enzyme inactivation. The characteristic inactivation model parameters, standard activation enthalpy and entropy, are directly related to the reference temperature and Z-value commonly used for kinetic analysis in food microbiology. An essential feature of the kinetic model is that its parameters, and hence the transition temperature, are treated as stochastic variables. The characteristic line shape of the primary model is the log-normal distribution. The performance of the model was validated, using literature data for enzyme and microbial inactivation over a wide range of temperature and pH.

Effect of electrical field strength applied by PEF processing and storage temperature on the outgrowth of yeasts and moulds naturally present in a fresh fruit smoothie.

Pulsed electrical field (PEF) technology offers an alternative to thermal pasteurisation of high-acid fruit juices, by extending the shelf life of food products, while retaining its fresh taste and nutritional value. Substantial research has been performed on the effect of electrical field strength on the inactivation kinetics of spoilage and pathogenic micro-organisms and on the outgrowth of spoilage micro-organisms during shelf life. However, studies on the effect of electrical field strength on the inactivation and outgrowth of surviving populations during shelf life are missing. In this study, we assessed the influence of electrical field strength applied by PEF processing and storage temperature on the outgrowth of surviving yeast and mould populations naturally present in fresh fruit smoothie in time. Therefore, an apple-strawberry-banana smoothie was treated in a continuous-flow PEF system (130L/h), using similar inlet and outlet conditions (preheating temperature 41°C, maximum temperature 58°C) to assure that the amount of energy across the different conditions was kept constant. Smoothies treated with variable electrical field strengths (13.5, 17.0, 20.0 and 24.0kV/cm) were compared to smoothies without treatment for outgrowth of yeasts and moulds. Outgrowth of yeasts and moulds stored at 4°C and 7°C was analysed by plating and visual observation and yeast growth was modelled using the modified logistic growth model (Zwietering model). Results showed that the intensity of the electrical field strength had an influence on the degree of inactivation of yeast cells, resulting in a faster outgrowth over time at lower electrical field strength. Outgrowth of moulds over time was not affected by the intensity of the electrical field strength used. Application of PEF introduces a trade-off between type of spoilage: in untreated smoothie yeasts lead to spoilage after 8days when stored at 4 or 7°C, whereas in PEF treated smoothie yeasts were (partly) inactivated and provided outgrowth opportunities for moulds, which led to spoilage by moulds after 14days (7°C) or 18days (4°C).

Pulsed electric field processing of different fruit juices: impact of pH and temperature on inactivation of spoilage and pathogenic micro-organisms.

Pulsed electrical field (PEF) technology can be used for the inactivation of micro-organisms and therefore for preservation of food products. It is a mild technology compared to thermal pasteurization because a lower temperature is used during processing, leading to a better retention of the quality. In this study, pathogenic and spoilage micro-organisms relevant in refrigerated fruit juices were studied to determine the impact of process parameters and juice composition on the effectiveness of the PEF process to inactivate the micro-organisms. Experiments were performed using a continuous-flow PEF system at an electrical field strength of 20 kV/cm with variable frequencies to evaluate the inactivation of Salmonella Panama, Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae in apple, orange and watermelon juices. Kinetic data showed that under the same conditions, S. cerevisiae was the most sensitive micro-organism, followed by S. Panama and E. coli, which displayed comparable inactivation kinetics. L. monocytogenes was the most resistant micro-organism towards the treatment conditions tested. A synergistic effect between temperature and electric pulses was observed at inlet temperatures above 35 °C, hence less energy for inactivation was required at higher temperatures. Different juice matrices resulted in a different degree of inactivation, predominantly determined by pH. The survival curves were nonlinear and could satisfactorily be modeled with the Weibull model.