Enhanced bactericidal effect of enterocin AS-48 in combination with high-intensity pulsed-electric field treatment against Salmonella enterica in apple juice

The effect of the broad spectrum cyclic antimicrobial peptide enterocin AS-48 combination with high-intensity pulsed-electric field (HIPEF) treatment (35 kV/cm, 150 Hz, 4 micros and bipolar mode) was tested on Salmonella enterica CECT 915 in apple juice. A response surface methodology was applied to study the bactericidal effects of the combined treatment. The process variables were AS-48 concentration, temperature, and HIPEF treatment time. While treatment with enterocin AS-48 alone up to 60 microg/ml had no effect on the viability of S. enterica in apple juice, an increased bactericidal activity was observed in combination with HIPEF treatments. Survival fraction was affected by treatment time, enterocin AS48 concentration and treatment temperature. The combination of 100 micros of HIPEF treatment, 30 microg/ml of AS-48, and temperature of 20 degrees C resulted in the lowest inactivation, with only a 1.2-log reduction. The maximum inactivation of 4.5-log cycles was achieved with HIPEF treatment for 1000 micros in combination with 60 microg/ml of AS-48 and a treatment temperature of 40 degrees C. Synergism between enterocin AS-48 and HIPEF treatment depended on the sequence order application, since it was observed only when HIPEF was applied in the presence of previously-added bacteriocin. The combined treatment could improve the safety of freshly-made apple juice against S. enterica transmission.     

Inactivation of Geobacillus stearothermophilus in canned food and coconut milk samples by addition of enterocin AS-48

The cyclic bacteriocin enterocin AS-48 was tested on a cocktail of two Geobacillus stearothermophilus strains in canned food samples (corn and peas), and in coconut milk. AS-48 (7 μg/g) reduced viable cell counts below detection levels in samples from canned corn and peas stored at 45 °C for 30 days. In coconut milk, bacterial inactivation by AS-48 (1.75 μg/ml) was even faster. In all canned food and drink samples inoculated with intact G. stearothermophilus endospores, bacteriocin addition (1.75 μg per g or ml of food sample) rapidly reduced viable cell counts below detection levels and avoided regrowth during storage. After a short-time bacteriocin treatment of endospores, trypsin addition markedly increased G. stearothermophilus survival, supporting the effect of residual bacteriocin on the observed loss of viability for endospores. Results from this study support the potential of enterocin AS-48 as a biopreservative against G. stearothermophilus.     

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.     

Changes in quality attributes throughout storage of strawberry juice processed by high-intensity pulsed electric fields or heat treatments

The effects of high-intensity pulsed electric field (HIPEF) processing (35 kV/cm for 1700 μs applying 4-μs pulses at 100 Hz in bipolar mode) on color, viscosity and PME and PG activities in strawberry juice were studied and compared to those of heat treatments (90 °C for 60 s or 30 s) through 63 days of storage. L^∗ and viscosity values of the HIPEF-processed juices were higher than those found in the thermally treated. In addition, HIPEF-treated juice exhibited lower 5-(hydroxymethyl)-2-furfural (HMF) concentration and browning index than heat-treated juices throughout storage. On the other hand, HIPEF-treated juice maintained low residual pectin methylesterase (PME) activity (13.1%) for 63 days, whereas in the case of the thermally treated, 22.2 and 48.8% was retained after 60 s and 30 s, respectively. Strawberry juice treated by HIPEF achieved lower residual polygalacturonase (PG) activity (73.3%) than those of heat-processed at 90 °C for 60 s (76.2%) or 30 s (96.8%). Thus, HIPEF could be a feasible alternative to thermal processing to minimize browning and viscosity loss in strawberry juice during storage.     

Inactivation of tomato juice peroxidase by high-intensity pulsed electric fields as affected by process conditions

A response surface was used to establish the high-intensity pulsed electric fields (HIPEF) conditions in processing tomato juice to obtain the greatest peroxidase (POD) inactivation. Keeping constant the electric field strength at 35 kV/cm and the temperature below 35 °C, the treatments were set at pulse frequency from 50 to 250 Hz, pulse width from 1 to 7 μs and treatment time from 1000 to 2000 μs, using monopolar or bipolar mode. The effect of these parameters on POD inactivation was evaluated through a second order model that adequately fitted the experimental data (p = 0.0001), with a determination coefficient (R²) of 0.85. HIPEF treatment resulted to be more effective in bipolar than monopolar mode to reduce POD activity and the longer the treatment time, the greater the reduction on the enzyme activity. A pulse frequency of 200 Hz was enough to reach a minimum value of residual POD activity. The significant interaction term pulse frequency and treatment time was included in the model, showing that different combinations of both variables can lead to the same level of residual POD activity. The effect of pulse width was enhanced by using a bipolar mode, being feasible to maximize POD inactivation selecting pulse width higher than 5.5 μs in bipolar mode.     

Effectiveness of High Intensity Light Pulses (HILP) treatments for the control of Escherichia coli and Listeria innocua in apple juice, orange juice and milk

High Intensity Light Pulses (HILP) represent an emerging processing technology which uses short (100-400 μs) light pulses (200-1100 nm) for product decontamination. In this study, model and real foods of differing transparencies (maximum recovery diluent (MRD), apple and orange juices and milk) were exposed to HILP in a batch system for 0, 2, 4 or 8 s at a frequency of 3 Hz. After treatment, inactivation of Escherichia coli or Listeria innocua was evaluated in pre-inoculated samples. Sensory and other quality attributes (colour, pH, Brix, titratable acidity, non-enzymatic browning, total phenols and antioxidant capacity (TEAC)) were assessed in apple juice. Microbial kill decreased with decreasing transparency of the medium. In apple juice (the most transparent beverage) E. coli decreased by 2.65 and 4.5 after exposure times of 2 or 4 s, respectively. No cell recovery was observed after 48 h storage at 4 °C. No significant differences were observed in quality parameters, excepting TEAC and flavour score, where 8 s exposure caused a significant decrease (p < 0.05). Based on these results, HILP with short exposure times could represent a potential alternative to thermal processing to eliminate undesirable microorganisms, while maintaining product quality, in transparent fruit juices.     

Changes on flavor compounds throughout cold storage of watermelon juice processed by high-intensity pulsed electric fields or heat

The application of HIPEF processing (35 kV/cm for 1727 μs using bipolar pulses of 4-μs at 188 Hz) on watermelon juice was evaluated as an alternative to conventional heat treatments (90 °C for 30 s or 90 s) in order to achieve better preservation of watermelon aroma compounds for 56 days of storage at 4 °C. HIPEF processing not only induced a rise (roughly 20%) in the concentrations of hexanal, (E)-2-nonenal, nonanal, 6-methyl-5-hepten-2-one and geranylacetone but also achieved less reductions on the retention of volatiles than the thermal treatment at 90 °C for 60 s. In contrast, the content of (Z)-6-nonenal, 1-nonanol and (Z)-3-nonen-1-ol in the untreated and processed juices remained unchanged after processing. Despite the decrease in overall flavor compounds observed during storage irrespective of the treatment applied, HIPEF-treated juices showed better flavor retention than heat-treated samples for at least 21 days of storage. Moreover, changes in aldehydes and ketones during storage of treated watermelon juices were well fitted by a model based on the Weibull distribution function. Therefore, the application of HIPEF may be appropriate to preserve the initial volatile profile of watermelon juices during storage.     

Flavour retention and related enzyme activities during storage of strawberry juices processed by high-intensity pulsed electric fields or heat

The effects of high-intensity pulsed electric fields (HIPEF) processing (35 kV/cm for 1700 μs using pulses of 4 μs at 100 Hz in bipolar mode) and thermal treatments (90 °C for 30 s or 60 s) on lipoxygenase (LOX) and β-glucosidase (β-GLUC) activities as well as on the production of volatile compounds were assessed in strawberry juice for 56 days of storage. HIPEF-treated juice kept higher residual LOX activity than heat-treated juices during the first 28 days of storage. Moreover, β-GLUC increased its initial activity just after HIPEF processing. The concentration of DMHF in HIPEF-processed strawberry juice was above those of untreated and heat-treated juices during the first 14 days of storage. On the other hand, concentrations of ethyl butanoate and 1-butanol obtained after HIPEF processing were better maintained than after thermal processing. However, thermally-treated samples showed an increase in the amount of 1-butanol beyond day 35, causing an unpleasant flavour to the product. Thus, flavour stability in HIPEF-processed strawberry juice was greater than in thermally-treated samples during storage.     

Heat resistance of Neosartorya fischeri in various juices

Heat resistance of Neosartorya fischeri was studied in three different juices (apple, pineapple and papaya). The optimum heat activation temperature and time for the ascospores of the N. fischeri (growth for 30 days at 30 °C) was 85 °C for 10 min. Of the three juices tested, apple juice exhibited maximum 1/k values at 80, 85 and 90 °C (208.3, 30.1 and 2.0 min, respectively). The 1/k values for papaya juice (129.9, 19.0 and 1.9 min) and pineapple juice (73.5, 13.2 and 1.5 min) decreased with acidity and °Brix/acidity (ratio) level. The Z^* values for apple, papaya and pineapple juices were 5, 5.5 and 5.9 °C, respectively. The sterilization F values (4-log reduction) for apple, pineapple and papaya juices were 56.3, 38.0 and 7.2 s, respectively. Considering the thermal treatments commercially applied to pineapple (96 °C/30 s) and apple juices (95 °C/30 s), it is concluded that such treatments will not guarantee that less than 1 ascospore in each set of 10³ packs survive. Only the treatment applied to papaya juice (100 °C/30 s) will be sufficient because the F value is less than 30 s.     

Impact of high-intensity pulsed electric fields variables on vitamin C, anthocyanins and antioxidant capacity of strawberry juice

A response surface methodology was used to determine the combined effect of HIPEF critical processing conditions on vitamin C, anthocyanins and antioxidant capacity of strawberry juice. Keeping constant the electric field strength at 35 kV/cm and the treatment time at 1000 μs, the treatments were set at frequencies from 50 to 250 Hz, pulse width from 1 to 7 μs using monopolar or bipolar mode. A second order response function covering the whole range of experimental conditions was obtained for each health-related compound. Strawberry juice antioxidant potential was affected linearly by frequency, pulse width and pulse polarity. The quadratic term of frequency and the combined effect of frequency and pulse width were also significant. HIPEF treatments conducted at 232 Hz with bipolar pulses of 1 μs led to strawberry juices with the greatest presence of health-related compounds. The evaluation of the HIPEF critical parameters influence on health-related compounds can contribute to achieve optimal processing conditions to obtain strawberry juices with high antioxidant potential.     

Stability of enterocin AS-48 in fruit and vegetable juices

Enterocin AS-48 is a candidate bacteriocin for food biopreservation. Before addressing application of AS-48 to vegetable-based foods, the interaction between AS-48 and vegetable food components and the stability of AS-48 were studied. Enterocin AS-48 had variable interactions with fruit and vegetable juices, with complete, partial, or negligible loss of activity. For some juices, loss of activity was ameliorated by increasing the bacteriocin concentration, diluting the juice, or applying a heat pretreatment. In juices obtained from cabbage, cauliflower, lettuce, green beans, celery, and avocado, AS-48 was very stable for the first 24 to 48 h of storage under refrigeration, and decay of activity was markedly influenced by storage temperature. In fresh-made fruit juices (orange, apple, grapefruit, pear, pineapple, and kiwi) and juice mixtures, AS-48 was very stable for at least 15 days at 4 degrees C, and bacteriocin activity was still detectable after 30 days of storage. Gradual and variable loss of activity occurred in juices stored at 15 and 28 degrees C; inactivation was faster at higher temperatures. In commercial fruit juices (orange, apple, peach, and pineapple) stored at 4 degrees C, the bacteriocin was completely stable for up to 120 days, and over 60% of initial activity was still present in juices stored at 15 degrees C for the same period. Commercial fruit juices stored at 28 degrees C for 120 days retained between 31.5% (apple) and 67.71% (peach) of their initial bacteriocin activity. Solutions of AS-48 in sterile distilled water were stable (120 days at 4 to 28 degrees C). Limited loss of activity was observed after mixing AS-48 with some food-grade dyes and thickening agents. Enterocin AS-48 added to lettuce juice incubated at 15 degrees C reduced viable counts of Listeria monocytogenes CECT 4032 and Bacillus cereus LWL1 to below detection limits and markedly reduced viable counts of Staphylococcus aureus CECT 976.     

Combined effect of enterocin AS-48 and high hydrostatic pressure to control food-borne pathogens inoculated in low acid fermented sausages

The single and combined effects of enterocin AS-48 and high hydrostatic pressure (HHP) on Listeria monocytogenes, Salmonellaenterica, and Staphylococcus aureus was investigated in fuet (a low acid fermented sausage) during ripening and storage at 7 °C or at room temperature. AS-48 (148 AU g⁻¹) caused a drastic 5.5 log cfu g⁻¹ decrease in L. monocytogenes (P < 0.001) and a significant (P < 0.01) inhibition (1.79 logs) for Salmonella at the end of ripening (10 d). After pressurization (400 MPa) and storage Listeria counts remained below 5 cfu g⁻¹ in all fuets containing AS-48 (pressurized or not). HHP alone had no anti-Listeria effect. HHP treatment significantly reduced Salmonella counts, with lowest levels in pressurized fuets with AS-48. S. aureus showed similar growth for all treatments and storage conditions. These results indicate that AS-48 can be applied alone to control L. monocytogenes and combined with HHP treatment to control Salmonella in fuets.     

Defining treatment conditions for pulsed electric field pasteurization of apple juice

The influence of temperature and the presence of N^α-lauroyl ethylester (ethyl lauroyl arginate, LAE) on the inactivation caused by continuous pulsed electric field treatments (PEF) in Escherichia coli O157:H7 suspended in apple juice have been investigated to define treatment conditions applicable at industrial scale that promote an equivalent safety level when compared with thermal processing. In the range of experimental conditions investigated (outlet temperature: 20-40 °C, electric field strength: 20-30 kV, treatment time: 5-125 μs) at outlet temperatures equal or lower than 55 ± 1 °C, the inactivation of E. coli O157:H7 treated in apple juice ranged from 0.4 to 3.6 Log₁₀ cycles reduction and treated in apple juice supplemented with LAE (50 ppm) ranged from 0.9 to 6.7 Log₁₀ cycles reduction.An empirical mathematical model was developed to estimate the treatment time and total specific energy input to obtain 5 Log₁₀ cycles reduction in the population of E. coli O157:H7 suspended in apple juice supplemented with 50 ppm of LAE at different electric field strengths and inlet temperatures. Treatment conditions established for E. coli O157:H7 were validated with other PEF resistant Gram-positive (Listeria monocytogenes, and Staphylococcus aureus) and Gram-negative (Salmonella enterica serovar Typhimurium) strains. When the treatment was applied to the apple juice, a treatment of 25 kV/cm for 63 μs corresponding with an outlet temperature of 65 °C and input energy of 125 kJ/kg was required to achieve more than 5 Log₁₀ cycles in the four strains investigated. The addition of LAE reduced the treatment time required to obtain an equivalent inactivation (> 5 Log₁₀ cycles) in the four microorganisms to 38.4 μs, the outlet temperature to 55 °C, and the input energy to 83.2 kJ/kg.     

Combined effect of temperature and pulsed electric fields on apple juice peroxidase and polyphenoloxidase inactivation

Pulsed electric fields (PEF) were applied to freshly prepared apple juice using a laboratory scale continuous PEF system to study the feasibility of inactivating peroxidase (POD) and polyphenoloxidase (PPO). Square wave PEF using different combinations of electric field strength, pre-treatment temperature and treatment time were evaluated in this study and compared to conventional pasteurisation (72 °C; 26 s). Inactivation curves for the enzyme were plotted for each parameter and inactivation kinetics were calculated. Results showed the highest level of decrease in the enzymatic activity of 71% and 68%, for PPO and POD, respectively, were obtained by using a combination of preheating to 50 °C, and a PEF treatment time of 100 μs at 40 kV/cm. This level of inactivation was significantly higher (P < 0.05) than that recorded in juice processed by conventional mild pasteurisation where the activity of PPO and POD decreased by 46% and 48%, respectively. The kinetic data for the inactivation of both enzymes could be described using a 1st-order model (P < 0.001).     

Carotenoid and phenolic profile of tomato juices processed by high intensity pulsed electric fields compared with conventional thermal treatments

The effect of high intensity pulsed electric fields (HIPEF) processing (35 kV/cm for 1500 μs of overall treatment time with bipolar pulses of 4-μs at 100 Hz) and heat pasteurisation (90 °C for 30 s or 60 s) on carotenoids and phenolic compounds as well as on some quality attributes (pH, soluble solids and colour parameters) of tomato juice was evaluated and compared, having the untreated juice as a reference. Processing enhanced some carotenoids (lycopene, β-carotene and phytofluene) and the red colour of juices, whereas no significant changes in phenolic compounds, pH and soluble solids were observed between treated and untreated juices. A slight decrease in overall health-related compounds was observed over time, with the exception of some carotenoids (β-carotene and phytoene) and caffeic acid. However, HIPEF-processed tomato juices maintained higher content of carotenoids (lycopene, neurosporene and γ-carotene) and quercetin through the storage time than thermally and untreated juices. Hence, the application of HIPEF may be appropriate to achieve not only safe but also nutritious and fresh like tomato juice.     

Probiotic beverage from cashew apple juice fermented with Lactobacillus casei

This study optimized the conditions of Lactobacillus casei NRRL B-442 cultivation in cashew apple juice, as well as, determined the proper inoculum amount and fermentation time. Moreover, it was investigated the survivability ability of L. casei in cashew apple juice during refrigerated storage (4 °C) for 42 days. The optimum conditions for probiotic cashew apple juice production were initial pH 6.4, fermentation temperature of 30 °C, inoculation level of 7.48 Log CFU/mL (L. casei) and 16 h of fermentation process. It was observed that the L. casei grew during the refrigerated storage. Viable cell counts were higher than 8.00 Log CFU/mL throughout the storage period (42 days). The values of lightness, yellowness and total color change increased and the values of redness reduced along the fermentation and refrigerated storage periods. The fermented juice with L. casei is a good and healthy alternative functional food containing probiotics. Cashew apple juice showed to be as efficient as dairy products for L. casei growth.     

Effect of different activated coatings containing enterocin AS-48 against Listeria monocytogenes on apple cubes

Enterocin AS-48 is a circular bacteriocin with strong anti-Listeria activity. The purpose of the present study was to evaluate the effect of the bacteriocin incorporated into different coating solutions on a cocktail of five L. monocytogenes strains previously inoculated on apple cubes. Coating solutions were made with chitosan, caseinate, alginate, k-carrageenate, xanthan gum, pectin, starch, carboxymethyl cellulose or methyl cellulose. Coatings were applied singly or combined with enterocin AS-48 at 20 or 40 μg/ml. Samples were stored at 4 °C for 7 days. The single application of coatings had almost no effect (as in alginate and methyl cellulose) or had a low effect on Listeria viability (< 2.0 log cycles), with the exception of chitosan coating which showed a strong anti-Listeria activity (up to 3.7 log cycles at day 7). Coatings dosed with 20-μg/ml enterocin AS-48 reduced viable Listeria counts gradually during storage in most cases, achieving significant reductions (p < 0.05) of 1.0 to 1.9 log cycles after 7 days for k-carrageenate, xanthan gum, pectin, starch, carboxymethyl cellulose and methyl cellulose compared to the single coating. At 40 μg/ml, enterocin AS-48 significantly reduced viable counts (p < 0.05) for most coatings (by 1.4 to 3.3 log cycles, depending on the coating) compared with coatings without bacteriocin (except for chitosan). Chitosan, pectin, xanthan gum and carboxymethyl cellulose coatings, supplemented or not with 40 μg/ml AS-48 were further investigated in combination with 20 mM EDTA or with 2.0% sodium lactate. The single addition of sodium lactate showed the greatest effects at day 7, where it reduced viable counts significantly (p < 0.05) by 1.1 to 2.2 log cycles compared to the single coatings (except for chitosan), whereas the combination of sodium lactate and AS-48 reduced viable counts below detection levels also at day 7 for all coatings. The combination of EDTA and AS-48 was much more effective, reducing Listeria counts below detection levels from day 1 for most of the coatings tested. The combination of EDTA and AS-48 was also the most effective at time 0, achieving reductions of viable counts between 2.0 and 2.7 log cycles depending on the coating immediately after treatment compared with single coatings.Industrial relevanceResults from the present study suggest the potential of edible coatings containing enterocin AS-48 and EDTA for inactivation of L. monocytogenes on apple surfaces. Since edible coatings are widely used on fruit surfaces, coatings activated with enterocin AS-48 and EDTA could find application as a hurdle against L. monocytogenes in fresh-cut apple pieces.     

Comparative study on shelf life of whole milk processed by high-intensity pulsed electric field or heat treatment

The effect of high-intensity pulsed electric fields (HI-PEF) processing (35.5 kV/cm for 1,000 or 300 μ with bipolar 7-μs pulses at 111 Hz; the temperature outside the chamber was always < 40° C) on microbial shelf life and quality-related parameters of whole milk were investigated and compared with traditional heat pasteurization (75° C for 15 s), and to raw milk during storage at 4° C. A HIPEF treatment of 1,000 μ ensured the microbiological stability of whole milk stored for 5 d under refrigeration. Initial acidity values, pH, and free fatty acid content were not affected by the treatments; and no proteolysis and lipolysis were observed during 1 wk of storage in milk treated by HIPEF for 1,000 μ. The whey proteins (serum albumin, β-lactoglobulin, and α-lactalbumin) in HIPEF-treated milk were retained at 75.5, 79.9, and 60%, respectively, similar to values for milk treated by traditional heat pasteurization.     

Aseptically packaged UHPH-treated apple juice: Safety and quality parameters during storage

Ultra-high pressure homogenization (UHPH) at 300 MPa and 4 °C inlet temperature were used to preserve apple juice, and shelf-life evaluation of aseptically packaged juice was investigated. After processing Tetra Brik containers were stored at temperatures of 4, 10, 20 and 30 °C during 60 days. In this article, the effect of processing on the spoilage inactivation was evaluated after processing and during the storage trial. Non-germinated and germinated spores were found in the UHPH-treated juice, being an inactive population during storage. Patulin content was also not modified by UHPH processing, but a significant decrease was observed during storage at 30 °C (P < 0.05). Polyphenoloxidase (PPO) and pectinmethylesterase (PME) activity was not found after UHPH-processing and during storage.A kinetic study of post-processing quality loss was conducted. Vitamin C, chlorogenic acid, total polyphenols and color change were measured during storage study and were used to model the UHPH-treated apple juice shelf-life. Loss of vitamin C was correlated with the hydroxymethylfurfural (HMF) accumulation (0.59, P < 0.05). A limiting quality parameter was polyphenolic content. UHPH-treated apple juice stored at 4 °C was found to show a shelf-life for about 21 months by preserving the color characteristics of the juice with low HMF accumulation. From 15 °C changes in quality parameters were more evident.     

High-intensity pulsed electric field variables affecting Staphylococcus aureus inoculated in milk

Staphylococcus aureus is an important milk-related pathogen that is inactivated by high-intensity pulsed electric fields (HIPEF). In this study, inactivation of Staph. aureus suspended in milk by HIPEF was studied using a response surface methodology, in which electric field intensity, pulse number, pulse width, pulse polarity, and the fat content of milk were the controlled variables. It was found that the fat content of milk did not significantly affect the microbial inactivation of Staph. aureus. A maximum value of 4.5 log reductions was obtained by applying 150 bipolar pulses of 8 μs each at 35 kV/cm. Bipolar pulses were more effective than those applied in the monopolar mode. An increase in electric field intensity, pulse number, or pulse width resulted in a drop in the survival fraction of Staph. aureus. Pulse widths close to 6.7 μs lead to greater microbial death with a minimum number of applied pulses. At a constant treatment time, a greater number of shorter pulses achieved better inactivation than those treatments performed at a lower number of longer pulses. The combined action of pulse number and electric field intensity followed a similar pattern, indicating that the same fraction of microbial death can be reached with different combinations of the variables. The behavior and relationship among the electrical variables suggest that the energy input of HIPEF processing might be optimized without decreasing the microbial death.