This study aimed to identify the principal mechanisms of action by which Listeria monocytogenes EGD-e responds to pulsed electric field (PEF) treatments at pH 7.0, given its recognition as a robust target microorganism and strain. Microbiologically challenged buffer samples (pH 7.0) were subjected to pulses with an electric field strength of 20 kV/cm and their transcriptional response was assessed using RNA sequencing. Our analysis revealed 119 differentially expressed genes, 51 of which were upregulated and 68 downregulated. From the 51 upregulated genes, 4 were transcription regulators (lmo1974, glnR, lmo806 and lmo0371) with the potential to influence the resistance of L. monocytogenes EGD-e. Additionally, assessment of 11 isogenic mutants at a PEF treatment (20 kV/cm, 184 kJ/kg) relative to the wild type identified the Delta yneA and Delta clpB deletion mutants as more resistant and more sensitive (p<0.05). Finally, the isogenic mutant Delta clpB was assessed against the wild type at 25 kV/cm at different total specific energies (54, 113, 135 and 160 kJ/kg) resulting in statistical difference(p<0.05) only under the highest parameter. In conclusion, transcriptomic analysis revealed that the primary mechanistic pathways of L. monocytogenes in response to PEF involve the preservation of homeostasis, energy availability, and quorum sensing. Additionally, the increased sensitivity of the Delta clpB mutant highlights a supplementary mechanism related to protein disaggregation and refolding under high-energy. These findings suggest that L. monocytogenes mounts a complex and multifaceted response to PEF treatments. These results can provide insights and support PEF treatment decontamination alone or as pretreatment in combination with other hurdles.

Pulsed Electric Fields (PEF) have proven effective in inactivating Anisakis in fish fillets. One of the parameters conditioning the lethal efficacy of PEF is the electrical conductivity of the treatment medium in which the fish is immersed. However, the underlying cause of this increased efficacy remains unknown. It was hypothesized that the difference in electrical conductivity between fish cells and Anisakis larvae could influence the electric field applied to each cell type. As measuring cells' electric fields is difficult, the study adopted an alternative dual approach. First, based on the experimental data, a mathematical equation was developed to predict Anisakis inactivation in hake belly fillets within the study's parameter ranges (1-5 kV/cm, 10-40 kJ/kg, 7-30 mu s pulse width, and 0.4-10 mS/cm). Additionally, an increase in electrical conductivity under the same PEF treatment was experimentally observed to result in a greater degree of parasite inactivation. To investigate this phenomenon, the numerical simulation strategy was employed to estimate the electric field strength affecting the elements in the treatment chamber. The results showed that as the electrical conductivity of the medium increased, the electric field strength in the parasite also increased, thus explaining the greater inactivation observed.

Toxoplasma gondii is an intracellular protozoan transmitted via environmentally resistant oocysts present in food and water, as well as through the consumption of meat containing infective bradyzoites. This study evaluated the inactivation of T. gondii oocysts and bradyzoites (ME-49 strain) by Pulsed Electric Field technology (PEF). Treatment efficacy was determined by mouse bioassay combining brain qPCR and indirect immunofluorescence (IFA), with complementary qPCR in Hs27 cells. The infectious dose (ID50) of T. gondii was estimated at 34.6 oocysts. PEF-treated oocysts (15 kV/cm; 50 kJ/kg; 225 µs) showed a significant reduction in infectivity compared with untreated controls; accordingly, the dose required to establish infection increased to 85.3 oocysts after PEF treatment. Brain qPCR and IFA were highly correlated, whereas heart tissue was less sensitive. Bradyzoites recovered from PEF-treated meat (3.3 kV/cm; 27 kJ/kg; 1600 µs) showed a 50% infectivity reduction compared with untreated samples. In vitro assays confirmed an in vivo reduction in infectivity, indicating that cell cultures can serve as an ethical and efficient tool for preliminary viability assessment. This is the first evidence of T. gondii inactivation by PEF, highlighting its potential as a non-thermal strategy. Further studies are needed to optimize treatment parameters.

Featured Application The findings of this study have direct practical relevance for the design and optimization of UV-C-based surface decontamination protocols in food processing environments, particularly for ready-to-eat products where pathogen control is critical. The results demonstrate that UV-C treatment is most effective when applied at early stages of contamination, before Listeria monocytogenes colonies reach the diameter range where UV-C-induced growth cessation becomes less reliable. In this work, bootstrap analysis of the neural-network predictions placed this reduced-efficacy region around 0.8-0.9 mm, with the minimum mean predicted growth-cessation probability occurring at approximately 0.862 mm. On the other hand, the neural network model developed in this work provides a preliminary framework for predicting treatment outcomes based on colony size. These findings suggest that such data-driven approaches could support quality control frameworks for assessing decontamination efficacy under varying contamination conditions, ultimately contributing to improved food safety management and reduced risk of listeriosis-associated foodborne outbreaks.Abstract UV-C light is a promising non-thermal technology for microbial inactivation on food surfaces; however, its efficacy may be compromised by the spatial structure of microbial colonies. The present work investigated the influence of Listeria monocytogenes colony size on UV-C treatment effectiveness using agar-based model systems. Petri dishes were inoculated at defined concentrations and incubated to generate colonies of varying sizes, which were subsequently exposed to a UV-C dose of 0.12 J/cm2. Colony growth was monitored over 48 h using an image-based analysis workflow implemented in MATLAB, combined with individual colony tracking. A neural network model was developed to predict the probability of growth cessation based on colony diameter, and quantitative PCR combined with bead-beating was used to estimate cell counts per colony. UV-C treatment applied immediately after inoculation achieved high inactivation efficacy, consistent with minimal cell aggregation. As colony size increased, treatment effectiveness declined markedly. Bootstrap analysis of the neural-network predictions identified a minimum mean growth cessation probability at a colony diameter of approximately 0.862 mm. At this diameter, the predicted probability was 56.8%, with a pointwise 95% bootstrap interval of 50.3-62.8%, corresponding to approximately 106.14 (viable + non-viable) cells per colony. These findings demonstrate that colony spatial structure substantially limits UV-C efficacy and underscore the importance of early-stage intervention in food surface UV-C decontamination protocols.

Pulsed Electric Fields (PEFs) are a technology increasingly used in the food industry for various purposes. However, their potential benefits as a pretreatment prior to the culinary preparation of a product have rarely been investigated. No previous study has investigated the use of PEFs in obtaining gazpacho, a typical Spanish dish. We aimed to evaluate the possibility of applying this technology in pretreating the vegetables used in gazpacho; furthermore, we evaluated its impact on the final product by comparing results with control samples. Applied at several different intensities (0.5–1.5 kV/cm and 4–40 kJ/kg), PEFs softened and decreased the vegetables’ water-holding capacity. In addition, this technique beneficially affected the organoleptic characteristics of gazpacho, increasing its consistency, improving its color (which became more reddish and intense), and enhancing its flavor. Moreover, the use of PEFs allowed us to reduce the amount of water in the mix, thus saving natural resources, concentrating nutrients, and decreasing energy consumption. Although further studies are required, PEFs can be considered a technology of interest in this productive sector.

The aim of this study is to determine the role that sigma(B) factor plays in the development of growth phase- and temperature-dependent thermoresistance in Staphylococcus aureus cells and its possible relationship with membrane fluidity. Cells of S. aureus strain Newman and its isogenic Delta sigB mutant (strain IK184) were grown at different temperatures to exponential and stationary growth phases, and the relationship between sigma(B) activity (estimated by RT-qPCR), heat resistance, and membrane fluidity (fatty acid profiles and DPH fluorescence anisotropy) was assessed. sigma(B) was required for the development of the growth phase- and temperature-dependent increases in thermoresistance and membrane rigidity (up to 37 degrees C), although a direct correlation between sigma(B) activity and heat resistance or membrane fluidity in S. aureus could not be established. Results obtained also revealed the existence of sigma(B)-independent growth phase and temperature (>37 degrees C) heat adaptation mechanisms. Moreover, it was demonstrated that sigma(B) does not control the fatty acid synthesis in S. aureus. Results obtained in this work contribute to a deeper knowledge of S. aureus physiology and to the development and design of more effective processes used by the food industry for microbial inactivation.

Larvae of the nematode Trichinella are capable of causing parasitic infections in humans after the consumption of uncooked meat or meat products, leading to severe symptoms and even death. Meat inspection is costly and tends to progressively be derogated in holdings applying controlled housing conditions in certain regions. Our study focuses on the evaluation of the efficacy of Pulsed Electric Fields (PEF) in the inactivation of Trichinella spp., including excysted larvae isolated by artificial digestion and encapsulated larvae found in meat from naturally infected wild boars. Microscopic examination of unstained and propidium-iodide-stained larvae suspensions showed that most of the excysted larvae were inactivated ten minutes after an intermediate PEF treatment (1 kV/cm, 0.41 kJ/kg). Treating excysted larvae with the mildest PEF treatment (0.5 kV/cm, 0.05 kJ/kg) combined with a 3% NaCl incubation resulted in synergistic inactivation. The application of 3 kV/cm (20 kJ/kg) to wild boar meat resulted in the inactivation of over 90% of encapsulated Trichinella larvae. The viability of Trichinella in meat was inversely correlated to the field strength applied (1–6 kV/cm) for equal energy input (20 kJ/kg). These findings demonstrate that PEF technology can potentially serve as a novel strategy for the inactivation of Trichinella larvae in meat.

Salmonella is the most frequently reported cause of foodborne outbreaks with known origin in Europe, with eggs and egg products standing out as the most frequent food source (when it was known). The growth and survival of Salmonella in eggs and egg products have been extensively studied and, recently, it has been reported that factors such as the initial concentration and thermal history of the egg product can also influence its growth capability. Therefore, the objective of this study was to define the boundary zones of the growth/no growth domain of Salmonella Enteritidis (4 strains) as a function of temperature (low temperature boundary) and the initial concentration in different egg products. A series of polynomial logistic regression equations were successfully adjusted, allowing the study of these factors and their interaction on the probability of growth of S. Enteritidis in these products. Results obtained indicate that the minimum growth temperatures of Salmonella Enteritidis are higher in egg white (9.5-18.3 degrees C) than in egg yolk (7.1-7.8 degrees C) or liquid whole egg (7.2-7.9 degrees C). Results also demonstrate that in raw liquid whole egg and raw and pasteurized egg white, the minimum growth temperature of Salmonella Enteritidis does depend on the initial concentration. Similarly, the previous thermal history of the egg product only influenced the minimum growth temperature in some of them. On the other hand, large differences in the minimum growth temperatures among strains were observed in some products (up to approx. 6 degrees C in egg white). Finally, it should be noted that none of the strains grew at 5 degrees C under any of the conditions assayed. Therefore, storage of egg products (particularly whole liquid egg and egg yolk) below this temperature might be regarded/proposed as a good management approach. Our experimental approach has allowed us to provide a more accurate prediction of S. Enteritidis minimum growth temperatures in egg products by taking into account additional factors (initial concentration and thermal history) while also providing a quantification of the intra-specie variability. This would be of high relevance for improving the safety of egg products.

Ohmic cooking is considered a fast and homogeneous process. However, achieving heating uniformity depends on several process parameters and intrinsic product characteristics. Furthermore, reference indicators for evaluating the ohmic process and generating reliable comparisons with conventional cooking are still lacking. The objective of this study was to investigate the reliability of the use of power input and cooking value as process indicators. The results showed that the specific ohmic power did affect only the heating rate but not the heating uniformity and the tissue softening rate. Therefore, the power input as process acceleration tool is not sufficient as stand-alone process indicator because other critical parameters (i.e., electrical conductivity) need to be taken into account to display the complex product-process-interactions. The cooking value was proven to be not valid as indicator for ohmic heating, as it does not take into account additional effects not attributable to only thermal exposure.

Anisakis is a zoonotic parasite found in the stomach of marine mammals. Its eggs are released into the sea and ingested by fish and cephalopods. Humans accidently become hosts when they consume raw or undercooked fish, or cephalopods, leading them to suffer from intestinal syndromes and allergic reactions. In Europe, the officially prescribed methods for inactivation of Anisakis are heat treatment or freezing, both of which can affect fish quality. Several studies have demonstrated the effectiveness of PEF for the inactivation of Anisakis; however, none of them have featured naturally infected samples. This study focuses on 1) the inactivation of Anisakis by PEF in naturally infected hake belly fillets (as hake is one of the most parasitized species in Europe) and 2) the evaluation of the quality of fish samples during their shelf life after PEF treatment. Results showed that it was necessary to apply higher PEF intensities of up to 5 kV/cm to inactivate Anisakis when it is naturally parasitized in comparison to artificial scenarios or when the parasite is present in water. The degree of inactivation increased over time when the samples were stored after PEF treatments in a modified atmosphere containing 50% CO2. After PEF treatments, quality analyses during shelf-life indicated that fish microbiota evolved similarly to untreated samples; however, the modified atmosphere limited the growth of the microbiota. In PEF-treated samples, quality parameters (drip loss, moisture, water holding capacity, and cooking loss) were closer to those of fresh hake and superior, in terms of quality, to the values obtained in frozen/thawed samples during their entire shelf-life.

Ohmic heating (OH) of food has been investigated for many years as an alternative to conventional heating because it allows fast and homogeneous heating. The processing parameters that influence the most uniformity of the heating in OH are the electric field strength and the frequency. Therefore, recent trends have focused on studying the application of frequencies in the order of kHz and electric fields higher than 100 V/cm. In this regard, and considering only the applied field strength in a way to easily differentiate them, three ohmic systems could be distinguished: OH (< 100 V/cm), moderated electric fields (MEF) (100–1000 V/cm), and ohmic-pulsed electric fields (ohmic-PEF) (> 1000 V/cm). The advantages of applying higher electric fields (MEF and ohmic-PEF) over OH are, on the one hand, their much higher heating rate and, on the other hand, their capability to electroporate cells, causing the release of intracellular ionic compounds, and therefore, uniformizing the electrical conductivity of the product. This strategy is especially interesting for large solid foods where conventional heating applications lead to large temperature gradients and quality losses due to surface overtreatment. Therefore, the aim of this work is to review the state of the art of OH technologies, focusing on MEF and ohmic-PEF. The advantages and disadvantages of MEF and ohmic-PEF compared to OH and their potential for improving processes in the food industry are also discussed.

This study aimed to acquire a deeper knowledge of the mechanisms of PEF resistance development after the exposure of Staphylococcus aureus to sublethal alkaline and heat shocks, with a particular focus on the modifications of cell envelope properties and their impact on electroporation and its reversion. Both shocks significantly (p < 0.05) increased the surface negative charge but they barely affected surface hydrophobicity or membrane fluidity. This resulted in an increased electroporation threshold (approximate to 2 kV/cm) for alkaline-shocked but not for heat-shocked cells. Heat and alkaline shock-dependent development of PEF resistance did not require de novo RNA, protein, or lipid synthesis. Addition of nisin (100 UI/mL) to the treatment medium not only counteracted the protective effect of sublethal shocks against PEF, but even increased the lethality of PEF treatments (up to 8.9-fold increase in Log cycles of inactivation) against heat-shocked and alkaline-shocked cells. Industrial relevance: This work contributed to a deeper understanding of the mechanisms leading to the development of PEF resistance, which is essential for PEF process optimization and for the design of PEF-based combined processes for food decontamination or pasteurization.

The safety of novel proteins is routinely evaluated in various regulated areas of the food and feed chain, including genetically modified (GM) crops and novel foods (NFs). This project aimed to map the food and feed products containing protein from the main GM crops, relevant food categories falling under the NF Regulation, and unconventional feed, together with their production processes and to discuss the effect of the mapped processes on the safety of the corresponding novel proteins. A scoping literature review (1,325 documents included), an open online survey and a stakeholder workshop were the basis to build up the mappings for products and processes, also including operational conditions for each processing step. In the case of crops, the information gathered also helped identify more than 40 products, and the corresponding production processes, not included in the OECD consensus documents for compositional considerations of GM crops. Moreover, a systematic literature review (154 documents included), carried out within the project, assisted in the identification of the available evidence on the impact of processing on protein safety. Overall, certain processes, such as thermal treatments, fermentation, or enzymatic hydrolysis, significantly enhanced protein digestibility across various food/feed matrices. Similarly, fermentation, ensiling, and extraction processes have been shown to improve nutritional properties in various products. The data collected seemed to indicate that heating can effectively reduce the activity of NEPs from GM crops and that heating and enzymatic hydrolysis can reduce IgE reactivity for certain proteins and operational conditions. However, exceptions to these trends were also reported in the literature, and in certain cases (e.g., impact on gut microbiota), the evidence gathered was insufficient to draw substantiated conclusions. This project also contributed to identify existing knowledge gaps and research needs towards regulatory risk assessment of food and feed products containing protein.

Pulsed electric field (PEF) is a food processing technology based on the phenomenon of electroporation for the inactivation of microorganisms with main advantage the minimal effect on the quality (nutritional, functional, and sensorial) characteristics of the food products. Despite the plethora of research literature on PEF-processed food safety, PEF's industrial application as an alternative of classical pasteurization is limited and mainly at industrial level is focused on high acid-liquid food products. Thus, the thorough assessment of the antimicrobial efficiency of PEF, coupled with the meticulous identification of key microbial resistance mechanisms is scientifically imperative. These efforts are essential for refining the process and exploring potential enhancements through synergistic integration and combination with other methods or/and hurdles. On this basis this manuscript aims to critically review and summarise: a) the antimicrobial mechanism of action, b) the microbial inactivation efficiency, and c) the effect of PEF at a microbial genomic/transcriptomic level.Industrial applicationEvaluating the effectiveness of inactivation and understanding the underlying resistance mechanisms can help on strategies to optimize PEF-mediated decontamination practices, and thereby enhancing the overall process efficiency.

In this study, resistant variants of Salmonella enterica serovar Typhimurium SL1344 to different stressors were selected. In addition, a genetic and phenotypic study was performed to explore the mechanisms underlying the acquisition of resistance. We isolated 4 variants with increased stable resistance to acid, osmotic stress, high hydrostatic pressure (HHP) and Ultraviolet-C light (UV-C) after repeated rounds of exposure to these agents and outgrowth of survivors. A PEF-resistant variant (SL1344-RS), previously isolated by Sagarzazu et al. (2013), was also included in the analysis. The results indicated that the isolated variants showed resistance to at least one other agent. This increased resistance, in general terms, had a fitness cost in growth, and exerted a variable impact on virulence (mainly in cell adhesion capacity), increased antibiotic resistance but did not influence in biofilm formation capacity. Whole Genome Sequencing (WGS) analysis allowed us to identify the genetic changes responsible for these phenotypic differences, and revealed that in 3 out of the 5 variants (including SL1344-RS) a mutation was found in hnr gene, an anti-sigma factor that promotes RpoS proteolysis. Hence the expression of several rpoS-regulated genes was quantified and found higher in these variants. This increase in RpoS activity would explain the lower growth rates observed in these 3 variants, as it would lead to increased transcription of genes involved in growth arrest and resistance to various types of stress. However, further analysis of a set of 22 additional Salmonella strains obtained from different culture collections indicated that a direct relationship between RpoS activity and stress resistance might not exist within Salmonella.

Pulsed Electric Fields (PEF) technology is regarded as one of the most interesting alternatives to current food preservation methods, due to its capability to inactivate vegetative microorganisms while leaving the product's organoleptic and nutritional properties mostly unchanged. However, many aspects regarding the mechanisms of bacterial inactivation by PEF are still not fully understood. The aim of this study was to obtain further insight into the mechanisms responsible for the increased resistance to PEF of a Salmonella Typhimurium SL1344 variant (SL1344-RS, Sagarzazu et al., 2013), and to quantify the impact that the acquisition of PEF resistance has on other aspects of S. enterica physiology, such as growth fitness, biofilm formation ability, virulence and antibiotic resistance. WGS, RNAseq and qRT-PCR assays indicated that the increased PEF resistance of the SL1344-RS variant is due to a higher RpoS activity caused by a mutation in the hnr gene. This increased RpoS activity also results in higher resistance to multiple stresses (acidic, osmotic, oxidative, ethanol and UV-C, but not to heat and HHP), decreased growth rate in M9-Gluconate (but not in TSB-YE or LB-DPY), increased ability to adhere to Caco-2 cells (but no significant change in invasiveness) and enhanced antibiotic resistance (to six out of eight agents). This study significantly contributes to the understanding of the mechanisms of the development of stress resistance in Salmonellae and underscores the crucial role played by RpoS in this process. Further studies are needed to determine whether this PEF-resistant variant would represent a higher, equal or lower associated hazard than the parental strain.

Background: Parasites are concerning food-borne pathogens. Some of them are currently not being routinely controlled in food, probably because their burden on public health is underestimated and the relative importance of different transmission routes is not completely known. Parasitic incidences could be avoided if preventive technologies were applied during food processing. Effective inactivation treatments are currently based on heat or freezing, but their side effects collide head-on with current consumer trends and new culinary habits.Scope and approach: This review describes the potential application of Pulsed Electric Field (PEF) technology in the control of food-borne parasites, with the aim of reducing the viability and infectivity of parasite transmission stages without affecting food quality. Results of published studies performed on different media are critically analyzed and factors affecting the outcomes are examined. Key findings and conclusions: Recent studies on the topic demonstrate the feasibility of PEF as an alternative to traditional freezing processes for the inactivation of Anisakis in fish. The development of new PEF equipment is advancing at a rapid pace, allowing for food treatment at a scale that would have been unimaginable some years ago. A review of more basic-science studies carried out on buffer media would contribute to progress in addressing the underlying drawbacks that remain to be solved. Thoroughly different fields (parasitology, physics, food engineering, water sanitation, etc.) should converge to achieve the industrial implementation of PEF for the inactivation of food-borne parasites.

The efficacy of applying ultrasound (US) as a system to homogenize emulsions has been widely demonstrated. However, research has not yet shown whether the effect achieved by homogenizing milk with US is modified by subsequent pasteurization treatments that use new processing technologies such as pulsed electric fields (PEF), microwaves (MW), and high hydrostatic pressure (HPP). The aim of this study was, therefore, to optimize the application of US for milk homogenization and to evaluate the effect of PEF, HPP, and MW pasteurization treatments on the sensorial, rheological, and microbiological properties of milk throughout its shelf life. To homogenize whole milk, a continuous US system (20 kHz, 0.204 kJ/mL, 100%, 40 °C) was used, and different ultrasonic intensities (0.25, 0.5, and 1.0 kJ/mL) were evaluated. The optimal ultrasonic treatment was selected on the basis of fat globule size distribution and pasteurization treatments by MW (5800 W, 1.8 L/min), PEF (120 kJ/kg, 20 kV/cm) and HPP (600 MPa, 2 min, 10 °C) was applied. The ultrasound intensity that achieved the highest reduction in fat globule size (0.22 ± 0.02 µm) and the most homogeneous distribution was 1.0 kJ/mL. Fat globule size was smaller than in commercial milk (82% of volume < 0.5 µm for US milk versus 97% of volume < 1.2 µm for commercial milk). That size was maintained after the application of the different pasteurization treatments, and the resulting milk had better emulsion stability than commercial milk. After 28 days of storage, no differences in viscosity (4.4–4.9 mPa s) were observed. HPP pasteurization had the greatest impact on color, leading to higher yellowness values than commercial milk. Microbial counts did not vary significantly after 28 days of storage, with counts below 102 CFU/mL for samples incubated at 15 °C and at 37 °C. In summary, the homogenization of milk obtained by US was not affected by subsequent pasteurization processes, regardless of the technology applied (MW, PEF, or HPP). Further research is needed to evaluate these procedures’ effect on milk’s nutritional and functional properties.

Larvae of the nematode family Anisakidae are capable of causing parasitic infections in humans associated with the consumption of fishery products, leading to intestinal syndromes and allergic reactions. Anisakidae larvae are widely distributed geographically, with rates of parasitism close to 100% in certain fish species. Methods need to be established for their inactivation and elimination, especially in fishery products that are to be consumed raw, pickled, or salted, or which have been insufficiently treated to kill the parasite. Many strategies are currently available (such as freezing and heat treatment), but further ones, such as pulsed electric fields (PEF), have hardly been investigated until now. This study focuses on the experimental evaluation of the efficacy of PEF in the inactivation of Anisakis spp. larvae in terms of electric field strength, specific energy, and pulse width, as well as on the evaluation of the quality of fish samples after PEF treatment. Results show that viability of Anisakis was highly dependent on field strength and specific energy. Pulse width exerted a considerable influence at the lowest field strengths tested (1 kV/cm). Central composite design helped to define a PEF treatment of 3 kV/cm and 50 kJ/kg as the one capable of inactivating almost 100% of Anisakis present in pieces of hake, while affecting the investigated quality parameters (moisture, water holding capacity, and cooking loss) to a lesser extent than freezing and thawing. These results show that PEF could serve as an alternative to traditional freezing processes for the inactivation of Anisakis in fish.