Combining nonthermal technologies to control foodborne microorganisms

Novel nonthermal processes, such as high hydrostatic pressure (HHP), pulsed electric fields (PEFs), ionizing radiation and ultrasonication, are able to inactivate microorganisms at ambient or sublethal temperatures. Many of these processes require very high treatment intensities, however, to achieve adequate microbial destruction in low-acid foods. Combining nonthermal processes with conventional preservation methods enhances their antimicrobial effect so that lower process intensities can be used. Combining two or more nonthermal processes can also enhance microbial inactivation and allow the use of lower individual treatment intensities. For conventional preservation treatments, optimal microbial control is achieved through the hurdle concept, with synergistic effects resulting from different components of the microbial cell being targeted simultaneously. The mechanisms of inactivation by nonthermal processes are still unclear; thus, the bases of synergistic combinations remain speculative. This paper reviews literature on the antimicrobial efficiencies of nonthermal processes combined with conventional and novel nonthermal technologies. Where possible, the proposed mechanisms of synergy is mentioned.     

Inactivation of Listeria innocua in liquid whole egg by pulsed electric fields and nisin

Consumer demand for fresh-like products with little or no degradation of nutritional and organoleptic properties has led to the study of new technologies in food preservation. Pulsed electric fields (PEF) is a nonthermal preservation method used to inactivate microorganisms mainly in liquid foods. Microorganisms in the presence of PEF suffer cell membrane damage. Nisin is a natural antimicrobial known to disrupt cell membrane integrity. Thus the combination of PEF and nisin represents a hurdle for the survival of Listeria innocua in liquid whole egg (LWE). L. innocua suspended in LWE was subjected to two different treatments: PEF and PEF followed by exposure to nisin. The selected frequency and pulse duration for PEF was 3.5 Hz and 2 micros, respectively. Electric field intensities of 30, 40 and 50 kV/cm were used. The number of pulses applied to the LWE was 10.6, 21.3 and 32. The highest extent of microbial inactivation with PEF was 3.5 log cycles (U) for an electric field intensity of 50 kV/cm and 32 pulses. Treatment of LWE by PEF was conducted at low temperatures, 36 degrees C being the highest. Exposure of L. innocua to nisin following the PEF treatment exhibited an additive effect on the inactivation of the microorganism. Moreover, a synergistic effect was observed as the electric field intensity, number of pulses and nisin concentration increased. L. innocua exposed to 10 IU nisin/ml after PEF exhibited a decrease in population of 4.1 U for an electric field intensity of 50 kV/cm and 32 pulses. Exposure of L. innocua to 100 IU nisin/ml following PEF resulted in 5.5 U for an electric field intensity of 50 kV/cm and 32 pulses. The model developed for the inactivation of L. innocua by PEF and followed by exposure to nisin proved to be accurate (p = 0.05) when used to model the inactivation of the microorganism by PEF in LWE with 1.2 or 37 IU nisin/ml. The presence of 37 IU nisin/ml in LWE during the PEF treatment for an electric field intensity of 50 kV/cm and 32 pulses resulted in a decrease in the population of L. innocua of 4.4 U.     

Enhancing the lethal effect of high-intensity pulsed electric field in milk by antimicrobial compounds as combined hurdles

High-intensity pulsed electric field (HIPEF) is a nonthermal treatment studied for its wide antimicrobial spectrum on liquid food, including milk and dairy products. Moreover, the antimicrobial effect of HIPEF may be enhanced by combining HIPEF with other treatments as hurdles. Nisin and lysozyme are natural antimicrobial compounds that could be used in combination with HIPEF. Therefore, the purpose of this study was to determine the effect of combining HIPEF with the addition of nisin and lysozyme to milk inoculated with Staphylococcus aureus with regard to different process variables. The individual addition of nisin and lysozyme did not produce any reduction in cell population within the proposed range of concentrations, whereas their combination resulted in a pH-dependent microbial death of Staph. aureus. The addition of nisin and lysozyme to milk combined with HIPEF treatment resulted in a synergistic effect. Applying a 1,200-μs HIPEF treatment time to milk at pH 6.8 containing 1 IU/mL of nisin and 300 IU/mL of lysozyme resulted in a reduction of more than 6.2 log units of Staph. aureus. Final counts resulting from the addition of nisin and lysozyme and applying HIPEF strongly depended on both the sequence of application and the milk pH. Thus, more research is needed to elucidate the mode of action of synergism as well as the role of different process variables, although the use of HIPEF in combination with antimicrobial compounds such as nisin and lysozyme is shown to be potentially useful in processing milk and dairy products.     

Nonthermal preservation of foods using combined processing techniques

In the last 2 decades, consumer demand for fresher, higher quality, and safer food has promoted research on nonthermal methods of food preservation for the inactivation of microorganisms and enzymes as an alternative to thermal processes. However, the high resistance of certain enzymes and microorganisms to nonthermal processes, especially bacterial spores, limit their application. To expand the use of nonthermal processes in the food industry, combinations of these technologies with traditional or emerging food preservation techniques are being studied. The use of nonthermal processes in combination with other preservation technologies presents a number of potential benefits to food preservation. The purpose of this article is to review some successful combinations of different nonthermal technologies, such as high hydrostatic pressure, ultrasound, pulsed electric fields, and irradiation, with traditional or emerging food preservation technologies.     

The effect of adding antimicrobial peptides to milk inoculated with Staphylococcus aureus and processed by high-intensity pulsed-electric field

The use of high-intensity pulsed-electric field (HIPEF) and antimicrobial substances of natural origin, such as enterocin AS-48 (AS-48), nisin, and lysozyme, are among the most important nonthermal preservation methods. Thus, the purpose of this study was to evaluate the combined effect on milk inoculated with Staphylococcus aureus of the addition of AS-48 with nisin or lysozyme, or both, together with the use of HIPEF. Synergy was observed in the reduction of Staph. aureus counts with the following combination methods: i) addition of AS-48 and nisin, ii) addition of AS-48 plus use of HIPEF, and iii) addition of AS-48 and nisin plus use of HIPEF. Specifically, when 28 arbitrary units/mL of AS-48 and 20 IU/mL of nisin were added to the milk, and it was treated with HIPEF for 800 μs, over 6 log reductions were observed in the microorganism. In general, Staph. aureus inactivation was dependent on HIPEF treatment time, antimicrobial doses, and medium pH. During storage of the treated milk, survivor population was related to peptide concentration and temperature. Final cell viability was influenced by the sequence in which the treatments were applied: the addition of AS-48 or AS-48 and nisin was more effective before than after HIPEF treatment. The results obtained indicate that the combination of HIPEF and antimicrobials could be of great interest to the dairy industry, although it is necessary to study further the way in which the combined treatments act.     

Transmission electron microscopy of Listeria innocua treated by pulsed electric fields and nisin in skimmed milk

Pulsed electric fields (PEF) is a nonthermal food preservation process where organoleptic and nutritional properties of the food are maintained. PEF is known to inactivate microorganisms by causing dielectric breakdown of the cell membrane, thus altering the functionality of the membrane as a semipermeable barrier. The extent of damage of the cell membrane, whether visible in the form of a pore or as loss of membrane functionality leads to the inactivation of the microorganism. The objective of this study was to investigate under transmission electron microscopy (TEM) the morphological changes on Listerit innocua as a result of PEF treatment in skimmed milk containing nisin. L. innocua was subjected to PEF at selected electric field intensities of 30, 40, and 50 kV/cm. L. innocua was treated by PEF in both skimmed milk with and without 37 IU nisin/ml. L. innocua treated by PEF in skimmed milk exhibited an increase in the cell wall roughness. cytoplasmic clumping, leakage of cellular material, and rupture of the cell walls and cell membranes. L. innocua subjected to PEF in skimmed milk containing 37 IU nisin/ml exhibited an increased cell wall width. At the highest electric field intensity, 50 kV/cm, elongation of the cell length was observed. There were no morphological differences between cells treated by PEF in skimmed milk with or without nisin. The combination of PEF and nisin exhibit an additive effect in the morphological damage observed on L. innocua. Pore formation was observed on L. innocua for an electric field intensity of 40 kV/cm. The inactivation of L. innocua was a consequence of rupture of the cell membrane and loss of cell membrane functionality.     

Review: Pulsed Electric Fields Processing of Protein-Based Foods

Pulsed electric fields (PEF) processing is a promising nonthermal food preservation technology, which is ongoing from laboratory and pilot plant scale level to the industrial level. Application of PEF processing may be a good alternative treatment to thermal methods in protein-based foods. A large number of literatures have fully demonstrated that small molecule compounds in plant-based foods, mainly aroma compounds and health-related phytochemicals, were not significantly affected by PEF. However, there was a lack of knowledge on the effects of PEF on proteins and qualities of protein-based foods. This review focuses on effects of PEF processing on endogenous enzymes, safety, and quality of protein-based foods. Finally, the ways to achieve food quality assurance and food safety in PEF processing of protein-based foods are proposed.     

Nonthermal Food Processing Alternatives and Their Effects on Taste and Flavor Compounds of Beverages

Food drinks are normally processed to increase their shelf-life and facilitate distribution before consumption. Thermal pasteurization is quite efficient in preventing microbial spoilage of many types of beverages, but the applied heat may also cause undesirable biochemical and nutritious changes that may affect sensory attributes of the final product. Alternative methods of pasteurization that do not include direct heat have been investigated in order to obtain products safe for consumption, but with sensory attributes maintained as unchanged as possible. Food scientists interested in nonthermal food preservation technologies have claimed that such methods of preserving foods are equally efficient in microbial inactivation as compared with conventional thermal means of food processing. Researchers in the nonthermal food preservation area also affirm that alternative preservation technologies will not affect, as much as thermal processes, nutritional and sensory attributes of processed foods. This article reviews research in nonthermal food preservation, focusing on effects of processing of food drinks such as fruit juices and dairy products. Analytical techniques used to identify volatile flavor-aroma compounds will be reviewed and comparative effects for both thermal and nonthermal preservation technologies will be discussed.     

Inactivation of Listeria innocua in skim milk by pulsed electric fields and nisin

Pulsed electric fields (PEF) is an emerging nonthermal processing technology used to inactivate microorganisms in liquid foods such as milk. PEF results in loss of cell membrane functionality that leads to inactivation of the microorganism. There are many processes that aid in the stability and safety of foods. The combination of different preservation factors, such as nisin and PEF, to control microorganisms should be explored. The objective of this research was to study the inactivation of Listeria innocua suspended in skim milk by PEF as well as the sensitization of PEF treated L. innocua to nisin. The selected electric field intensity was 30, 40 and 50 kV/cm and the number of pulses applied was 10.6, 21.3 and 32. The sensitization exhibited by PEF treated L. innocua to nisin was assessed for 10 or 100 IU nisin/ml. A progressive decrease in the population of L. innocua was observed for the selected field intensities, with the greatest reduction being 2 1/2 log cycles (U). The exposure of L. innocua to nisin after PEF had an additive effect on the inactivation of the microorganism as that exhibited by the PEF alone. As the electric field, number of pulses and nisin concentration increased, synergism was observed in the inactivation of L. innocua as a result of exposure to nisin after PEF. The reduction of L. innocua accomplished by exposure to 10 IU nisin/ml after 32 pulsed electric fields was 2, 2.7, and 3.4 U for an electric field intensity of 30, 40, and 50 kV/cm, respectively. Population of L. innocua subjected to 100 IU nisin/ml after PEF was 2.8-3.8 U for the selected electric field intensities and 32 pulses. The designed model for the inactivation of L. innocua as a result of the PEF followed by exposure to nisin proved to be accurate in the prediction of the inactivation of L. innocua in skim milk containing 1.2 or 37 IU nisin/ml. Inactivation of L. innocua in skim milk containing 37 IU nisin/ml resulted in a decrease in population of 3.7 U.     

Inactivation of Salmonella Typhimurium in orange juice containing antimicrobial agents by pulsed electric field

Combinations of different hurdles, including moderately high temperatures (<60 degrees C), antimicrobial compounds, and pulsed electric field (PEF) treatment, to reduce Salmonella in pasteurized and freshly squeezed orange juices (with and without pulp) were explored. Populations of Salmonella Typhimurium were found to decrease with an increase in pulse number and treatment temperature. At a field strength of 90 kV/cm, a pulse number of 20, and a temperature of 45 degrees C, PEF treatment did not have a notable effect on cell viability or injury. At and above 46 degrees C, however, cell death and injury were greatly increased. Salmonella numbers were reduced by 5.9 log cycles in freshly squeezed orange juice (without pulp) treated at 90 kV/cm, 50 pulses, and 55 degrees C. When PEF treatment was carried out in the presence of nisin (100 U/ml of orange juice), lysozyme (2,400 U/ml), or a mixture of nisin (27.5 U/ml) and lysozyme (690 U/ml), cell viability loss was increased by an additional 0.04 to 2.75 log cycles. The combination of nisin and lysozyme had a more pronounced bactericidal effect than did either nisin or lysozyme alone. An additional Salmonella count reduction of at least 1.37 log cycles was achieved when the two antimicrobial agents were used in combination. No significant difference (P > 0.05) in cell death was attained by lowering the pH value; only cell injury increased. Inactivation by PEF was significantly more extensive (P < 0.05) in pasteurized orange juice than in freshly squeezed orange juice under the same treatment conditions. This increase might be due to the effect of the chemical composition of the juices.     

Pulsed electric field‐assisted extraction of valuable compounds from microorganisms

Microorganisms (bacteria, yeast, and microalgae) are a promising resource for products of high value such as nutrients, pigments, and enzymes. The majority of these compounds of interest remain inside the cell, thus making it necessary to extract and purify them before use. This review presents the challenges and opportunities in the production of these compounds, the microbial structure and the location of target compounds in the cells, the different procedures proposed for improving extraction of these compounds, and pulsed electric field (PEF)‐assisted extraction as alternative to these procedures. PEF is a nonthermal technology that produces a precise action on the cytoplasmic membrane improving the selective release of intracellular compounds while avoiding undesirable consequences of heating on the characteristics and purity of the extracts. PEF pretreatment with low energetic requirements allows for high extraction yields. However, PEF parameters should be tailored to each microbial cell, according to their structure, size, and other factors affecting efficiency. Furthermore, the recent discovery of the triggering effect of enzymatic activity during cell incubation after electroporation opens up the possibility of new implementations of PEF for the recovery of compounds that are bounded or assembled in structures. Similarly, PEF parameters and suspension storage conditions need to be optimized to reach the desired effect. PEF can be applied in continuous flow and is adaptable to industrial equipment, making it feasible for scale‐up to large processing capacities.     

A comparative study on the structure of Saccharomyces cerevisiae under nonthermal technologies: high hydrostatic pressure, pulsed electric fields and thermo-sonication

Nonthermal technologies are becoming more popular in food processing; however, little detailed research has been conducted on the study of the lethal effect of these technologies on certain microorganisms. Saccharomyces cerevisiae is a yeast related to spoilage of fruit products such as juices; novel technologies have been explored to inactivate this yeast. Three nonthermal technologies, high hydrostatic pressure (HHP), pulsed electric fields (PEF) and thermo-sonication (TS), were used to evaluate and to compare the structural damage of yeast cells after processing. Processing conditions were chosen based on previous experiments to ensure the death of cells; HHP was conducted at 600 MPa for 7 min (room temperature, 21 °C); for PEF, 30.76 kV/cm at 40 °C and 21 pulses (2 μs each), and finally for TS the conditions were 120 μm, 60 °C and 30 min in continuous and pulsed modes; all treatments were applied in apple juice. Cells were prepared for electron microscopy using an innovative and short microwave assisted dehydration technique. Scanning electron microscopy showed the degree of damage to the cells after processing and illustrated the important and particular characteristics of each technology. Cells treated with high hydrostatic pressure showed a total disruption of the cell membrane, perforation, and release of the cell wall; scars were also observed on the surface of the pressurized cells. PEF treated cells showed less superficial damage, with the main changes being the deformation of the cells, apparent fusion of cells, the formation of pores, and the breakdown of the cell wall in some cells. Finally, the thermo-sonicated cells showed a similar degree of cellular damage to their structure regardless of whether the TS was applied continuously or pulsed. The main characteristics of cellular death for this technology were the erosion and disruption of the cellular membrane, formation of orifices on the surface, lysis of cells causing the release of intracellular contents, roughness of the cell membrane, and displacement of cell debris to the surface of other cells. This study confirms some theories about cell inactivation and presents new and detailed results about nonthermal technologies, but also shows that after using the above mentioned conditions, recovery of cells, specifically those that are pressurized and thermo-sonicated, it is not possible to do it following the high extent of damage observed in the entire population. Furthermore, a faster methodology that was used in sample preparation for electron microscopy provided high quality resolution images, allowing closer study of the detail of structural lethal effects on treated cells.     

Biological Aspects in Food Preservation by Ultraviolet Light: a Review

The potential to commercialize nonthermal ultraviolet (UV) light technologies as new methods for preserving food products has caught the attention of a food industry that wishes to fulfill consumers' demands for fresh products. Numerous investigations have demonstrated UV light's ability to inactivate a wide range of microorganisms. However, the lack of UV sensitivity data from pathogenic and spoilage bacteria is evident. In addition, the main factors associated with UV light in terms of microbial lethality remain unclear. This review surveys critical factors (process, microbial, and environmental parameters) that determine UV microbial resistance and assess the effects of such factors on the inactivation mechanism and repair pathway efficiency. The effects of some of these factors, such as prior sublethal stresses and post-recovery conditions of UV treatments, may extensively improve the damage repair capacity and thus microbial survivability. Further research is needed to establish adequate control measures pre- and post-UV treatments. Furthermore, the possibility of combining UV light with conventional preservatives and other nonthermal technologies was assessed. The combination of UV light with mild heating or oxidant compounds could offer promising treatments to enhance the safety and stability of minimally processed foods.     

Enhancing phenolic content in carrots by pulsed electric fields during post-treatment time: Effects on cell viability and quality attributes

The impact of pulsed electric fields (PEF) and post-treatment time on the phenolic content and quality attributes of carrots was studied. Additionally, their influence on cellular permeability and viability was analyzed. Carrots were subjected to different electric field strengths (0.8, 2 and 3.5 kV·cm⁻¹) and number of pulses (5, 12 and 30). The largest increases in phenolic content were produced 24 h after applying 30 pulses of 0.8 kV·cm⁻¹ (40.1%) and 5 pulses of 3.5 kV·cm⁻¹ (39.5%). At such conditions, the colour was not affected but softening occurred after applying the highest electric field strength. Moreover, the increase in the specific energy input correlated with the decrease in cell viability. Carrot weight loss over time, media conductivity increase and cell viability decrease are related to the destabilization of cell membranes, which would entail a physiological response to stress, leading to a higher content in phenolic compounds.Industrial relevanceDetermining the response of plant tissues to processing technologies is of great interest from an industrial point of view. Pulsed electric fields (PEF), as well as other processing technologies, may trigger metabolic responses that are directly related to the quality of final products. This paper studies the impact of PEF treatments on the phenolic content and quality attributes of carrots. Results show that PEF application allows improving the polyphenol content in carrots. However, it must be considered that firmness, weight, and colour may suffer modifications if PEF parameters are not properly selected. The information provided could be beneficial to develop processed foods with enhanced health-related compounds content. Furthermore, in order to optimize treatments, it is critical to study structural changes as affected by PEF processing. In this study, tetrazolium staining and conductivity measurements were used to visualize and determine the cell damage on carrot tissues. Industry can apply PEF to achieve other aims such as enhancing intracellular metabolite extraction or improving the drying efficiency; therefore, PEF treatment suitability could be evaluated based on this approach. Results show that PEF could be a promising pre-treatment to enhance the phenolic content of carrots and obtain derived products with improved nutritional value.     

高压脉冲电场对绿茶饮料杀菌的研究

研究了高压脉冲电场对中性的绿茶饮料的杀菌效果，结果表明，当电场强度达到40kV／cm，处理时间达到120μs时，绿茶饮料中接种的大肠杆菌数量可降低5个对数级以上。此条件下生产的绿茶饮料样品，其茶多酚含量及色泽都无明显变化。     

Nonthermal Processes for Shelf‐Life Extension of Seafoods: A Revisit

For the past two decades, consumer demand for minimally processed seafoods with good sensory acceptability and nutritive properties has been increasing. Nonthermal food processing and preservation technologies have drawn the attention of food scientists and manufacturers because nutritional and sensory properties of such treated foods are minimally affected. More importantly, shelf‐life is extended as nonthermal treatments are capable of inhibiting or killing both spoilage and pathogenic organisms. They are also considered to be more energy‐efficient and to yield better quality when compared with conventional thermal processes. This review provides insight into the nonthermal processing technologies currently used for shelf‐life extension of seafoods. Both pretreatments such as acidic electrolyte water and ozonification and processing technologies, including high hydrostatic pressurization, ionizing radiation, cold plasma, ultraviolet light, and pulsed electric fields, as well as packaging technology, particularly modified atmosphere packaging, have been implemented to lower the microbial load in seafood. Thus, those technologies may be the ideal approach for the seafood industry, in which prime quality is maintained and safety is assured for consumers.     

Effect of thermal and non‐thermal pasteurisation on the microbial inactivation and phenolic degradation in fruit juice: a mini‐review

Fruit juice has been traditionally preserved by thermal pasteurisation. However, the applied heat can cause detrimental effects on health‐promoting components such as phenolic compounds. Several non‐thermal technologies such as membrane filtration, pulsed electric field (PEF) and ultraviolet (UV) exposure are promising methods developed for liquid food preservation. In particular, the combination of UV and PEF has proven to be more effective for microbial inactivation and maintaining nutritional quality of fruit juice compared with individual applications. © 2012 Society of Chemical Industry     

The antimicrobial activity of hydrophobic essential oil constituents acting alone or in combined processes of food preservation

This work evaluates the antimicrobial activity of widespread hydrophobic essential oil (EO) constituents, 3 hydrocarbon monoterpenes (α-pinene, β-pinene, and p-cymene) and 8 oxygenated monoterpenes (thymol, carvacrol, borneol, linalool, terpineol-4-ol, 1,8-cineole, α-terpinyl acetate, and camphor), as a function of the treatment medium pH, and possible synergistic effects in combination with mild heat or pulsed electric fields (PEF) treatments. Results obtained using the disk diffusion technique highlight phenols and alcohols as the best growth inhibitors and discount hydrocarbons due to their poorer activity. However, the evaluation of the bactericidal effect at pH 4.0 shows that most compounds assayed, including some hydrocarbons, were very effective against Escherichia coli and Listeria monocytogenes. Most EO constituents caused membrane permeabilization and sublethal injuries within survivors. Outstanding synergistic lethal effects were shown using mild heat (54 °C/10 min) or PEF (30 kV/cm/25 pulses) combined with 0.2 μl/ml of some antimicrobials, achieving 5 log₁₀ cycles of cell inactivation as a function of the treatment conditions. In most cases, combined treatments were more effective in apple than in orange juice.

Industrial relevance

The efficacy of EO constituents improves when combining with mild heat or PEF treatments, which allows us to propose very low doses of antimicrobials. The valuable synergistic effects observed offer the potential to improve traditional heat treatments by reducing treatment intensity and consequently adverse effects on food quality, and to enhance novel PEF treatments by achieving a higher degree of microbial inactivation.     

Toward a Philosophy and Theory of Volumetric Nonthermal Processing

Nonthermal processes for food preservation have been under intensive investigation for about the past quarter century, with varying degrees of success. We focus this discussion on two volumetrically acting nonthermal processes, high pressure processing (HPP) and pulsed electric fields (PEF), with emphasis on scientific understanding of each, and the research questions that need to be addressed for each to be more successful in the future. We discuss the character or “philosophy” of food preservation, with a question about the nature of the kill step(s), and the sensing challenges that need to be addressed. For HPP, key questions and needs center around whether its nonthermal effectiveness can be increased by increased pressures or pulsing, the theoretical treatment of rates of reaction as influenced by pressure, the assumption of uniform pressure distribution, and the need for (and difficulties involved in) in‐situ measurement. For PEF, the questions include the rationale for pulsing, difficulties involved in continuous flow treatment chambers, the difference between electroporation theory and experimental observations, and the difficulties involved in in‐situ measurement and monitoring of electric field distribution.     

Pulsed electric field treatment of red wine: Inactivation of Brettanomyces and potential hazard caused by metal ion dissolution

Pulsed electric field (PEF) technology is an attractive alternative method of wine preservation by inactivating Brettanomyces bruxellensis, a major spoilage concern affecting wines worldwide. Currently, wine preservation using SO₂ can have negative effects on consumers including headaches and allergic reactions. Therefore, the objectives of this study were to investigate the effect of PEF processing conditions, B. bruxellensis yeast strain and alcohol concentration on B. bruxellensis inactivation in red wine, as well as whether PEF treatment could have a harmful effect on wine through the release of metal ions. Electric field intensity was found to have a greater impact on inactivation than specific energy, with 31, 40 and 50 kV/cm treatments resulting in B. bruxellensis D values of 181.8, 36.1 and 13.0 μs, respectively. At 50 kV/cm, a temperature rise of almost 10 °C, doubled inactivation to 3.0 log reductions (cfu/mL). Yeast strain and alcohol concentration were also shown to influence inactivation, even though cell size comparisons of the three yeasts tested proved inconclusive. Overall, PEF treatment of wine was shown to be a possible preservation alternative for the wine industry. After PEF treatment, the wine produced remained safe for human consumption, with Fe, Cr and Ni ions contents well below dangerous levels.