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.     

Influence of treatment temperature on the inactivation of Listeria innocua by pulsed electric fields

The effect of treatment temperature on the bactericidal effectiveness of pulsed electric fields (PEF) applied on Listeria innocua suspended in McIllvaine buffer was investigated. Electric field intensity and number of applied pulses were applied in the ranges of 31–40 kV/cm and 5–35 pulses, respectively. Studied treatment temperatures were sustained for 10 s, and ranged between 19°C and 59°C depending on the amount of energy delivered by the PEF treatment. The application of PEF at higher temperatures proved to be more effective than either PEF at low temperatures or the applied thermal treatments by themselves. A maximum bacterial inactivation of 6-log cycles was obtained by applying either: 20 pulses of 40 kV/cm at 65°C, 25 pulses of 36 kV/cm at 61°C, or 31 pulses of 31 kV/cm at 56°C. On the other hand, a thermal treatment of 66°C sustained for 30 s reduced the bacterial population on its own by only 5-log cycles, and the application of 60 pulses of 31 kV/cm at 30°C caused only 3-log cycles of bacterial inactivation. The findings in this study suggest that PEF technology may be effectively used as an enhanced mild thermal preservation method.     

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.     

Effect of spectral range in surface inactivation of Listeria innocua using broad-spectrum pulsed light

Pulsed light (PL) treatment is an alternative to traditional thermal treatment that has the potential to achieve several log-cycle reductions in the concentration of microorganisms. One issue that is still debated is related to what specifically causes cell death after PL treatments. The main objective of this work was to elucidate which portions of the PL range are responsible for bacterial inactivation. Stainless steel coupons with controlled surface properties were inoculated with a known concentration of Listeria innocua in the stationary growth phase and treated with 1 to 12 pulses of light at a pulse rate of 3 pulses per s and a pulse width of 360 micros. The effects of the full spectrum (lambda = 180 to 1,100 nm) were compared with the effects obtained when only certain regions of UV, visible, and near-infrared light were used. The effectiveness of the treatments was determined in parallel by the standard plate count and most-probable-number techniques. At a fluence of about 6 J/cm(2), the full-spectrum PL treatment resulted in a 4.08-log reduction of L. innocua on a Mill finish surface, the removal of lambda < 200 nm diminished the reduction to only 1.64 log, and total elimination of UV light resulted in no lethal effects on L. innocua. Overwhelmingly, the portions of the PL spectrum responsible for bacterial death are the UV-B and UV-C spectral ranges (X < 300 nm), with some death taking place during exposure to UV-A radiation (300 < lambda < 400 nm) and no observable death upon exposure to visible and near-infrared light (lambda > 400 nm). This work provides additional supporting evidence that cell death in PL treatment is due to exposure to UV light. Additionally, it was shown that even a minor modification of the light path or the UV light spectrum in PL treatments can have a significant negative impact on the treatment intensity and effectiveness.     

Alkaline phosphatase and microbial inactivation by pulsed electric field in bovine milk

The effects of pulsed electric field (PEF) treatments at field intensities of 25–37 kV cm^− 1 and final PEF treatment temperatures of 15 °C and 60 °C on the inactivation of alkaline phosphatase (ALP), Total Plate Count (TPC), Pseudomonas and Enterobacteriaceae counts were determined in raw skim milk. At 15 °C, PEF treatments of 28 to 37 kV cm^− 1 resulted in 24–42% inactivation in ALP activity and < 1 log reduction in TPC and Pseudomonas count, while the Enterobacteriaceae count was reduced by at least 2.1 log units to below the detection limit of 1 CFU mL^− 1. PEF treatments of 25 to 35 kV cm^− 1 at 60 °C resulted in 29–67% inactivation in ALP activity and up to 2.4 log reduction in TPC, while the Pseudomonas and Enterobacteriaceae counts were reduced by at least 5.9 and 2.1 logs, respectively, to below the detection limit of 1 CFU mL^− 1. Kinetic studies suggested that the effect of field intensity on ALP inactivation at the final PEF treatment temperature of 60 °C was more than twice that at 15 °C. A combined effect was observed between the field intensity and temperature in the inactivation of both ALP enzyme and the natural microbial flora in raw skim milk.Industrial relevanceMilk has been pasteurised to ensure its safety and extend its shelf life. However, the need for retaining heat-sensitive nutrient and sensory properties of milk has resulted in interest in the application of alternative technologies. The results of the current study suggest that PEF as a non-thermal process can be employed for the treatment of raw milk in mild temperature to achieve adequate safety and shelf life while preserving the heat-sensitive enzymes, nutrients and bioactive compounds.     

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.     

Influence of thermosonication on Geobacillus stearothermophilus inactivation in skim milk

The influence of high intensity ultrasound coupled with thermoprocessing on the inactivation of Geobacillus stearothermophilus vegetative cells and spores in skim milk powder was explored using response surface methodology and two polynomial models were developed. Optimization of cell reduction (4.8 log) was found to be at 19.75% total solids (TS), 45 °C, and 30 s, while optimization of spore reduction (0.45 log) was found to be at 31.5% TS, 67.5 °C, and 17.5 s. Model verification experiments were performed using common milk powder processing conditions. Results showed the inactivation of cells and spores to be most effective before (9.2% TS, 75 °C, and 10 s) and after (50% TS, 60 °C, and 10 s) the evaporator during milk powder processing and may produce an additive effect in microbial reduction when the two locations are combined, resulting in a 5.8 log reduction for vegetative cells and 0.51 log reduction for spores.     

Study of butter fat content in milk on the inactivation of Listeria innocua ATCC 51742 by thermo-sonication

Ultrasound combined with heat treatment has yielded favorable results in the inactivation of microorganisms; however, the composition of food influences the rate of microbial inactivation. The objective of this research was to study the effect of butter fat content in milk on the inactivation of Listeria innocua and compositional parameters after thermo-sonication. Four butter fat contents in milk were evaluated at 63 °C for 30 min of sonication (Hielscher® UP400S, 400 W, 24 kHz, 120 μm amplitude). Results showed that inactivation of Listeria cells occurs first in fat free milk, and that the rate of inactivation decreases with increasing fat content. No degradation of protein content or color variation was observed after the treatments. The pH dropped to 6.22, and lactic acid content showed an increase of 0.015% after the treatment; solids-non-fat, density and freezing point decreased. During storage life, growth of mesophiles was retarded with sonication.Industrial relevanceUltrasound is an emerging technology that has shown positive effects in milk processing. Listeria monocytogenes represent one of the main foodborne pathogenic microorganisms in the food industry. Results of this research show that thermo-sonication is a viable technology capable of inactivating Listeria cells in milk and extending shelf-life without significant nutritional or physicochemical changes.     

The inactivation of Listeria monocytogenes by pulsed electric field (PEF) treatment in a static chamber

An experimental analysis of the effect of pulsed electric field (PEF) energy on the inactivation of Listeria monocytogenes was conducted using a custom-designed static chamber and a gel suspension medium for treatment. This allowed PEF energy to be delivered to the suspension under near isothermal conditions. The effects of variations in the number of pulses (5–50 pulses), electric field strength (15–30 kV/cm), temperature (0–60°C) and media bases (water and skim milk) on the inactivation of L. monocytogenes were examined. At temperatures less than 50°C a maximum of 1 log reduction was obtained for L. monocytogenes regardless of pulse number or electric field strength within the ranges examined. In skim milk no reduction occurred. At 50°C and 55°C synergy between PEF and thermal energy was observed. The experimental approach separated the contribution of PEF and thermal energy to total kill and thus allowed this synergy to be quantified. At 55°C the kill due to PEF energy increased to 4.5 logs with another 4.5 logs reduction attributable to thermal energy. It appears that under the conditions of this study PEF alone has a very limited effect on the reduction of L. monocytogenes. However, the addition of thermal energy not only contributed to the kill, but also increased the susceptibility of L. monocytogenes to PEF energy.     

Pulsed electric fields inactivation of attached and free-living Escherichia coli and Listeria innocua under several conditions

The use of pulsed electric fields (PEF) is considered as a mild process in the inactivation of microorganisms present in liquid food products. PEF treatments of Escherichia coli and Listeria innocua suspended in milk and phosphate buffer, with same pH and same conductivities, yielded to similar inactivation. Reduction rates obtained in distilled water indicated that conductivity of the food product is a main parameter in bacterial inactivation. Bacteria attached to polystyrene beads were inactivated by PEF at a greater (E. coli) or equal rate (L. innocua) than free-living bacteria. Base on the use of selective and non-selective enumeration media, no clear indications were obtained for sublethal damage of microorganisms surviving the PEF treatment. E. coli cells subjected to 60 pulses at 41 kV/cm were examined by transmission and scanning electron microscopy. Changes in the cytoplasm were observed and the cell surface appeared rough. The cells outer membranes were partially destroyed allowing leaking of cell cytoplasm.     

Effect of the pressurizing ramp on the inactivation of Listeria innocua considering thermofluiddynamical processes

The effect of the pressurization ramp on the inactivation kinetics of Listeria innocua suspended in Tris buffer has been studied experimentally in an optical pressure cell. Additionally, the thermofluiddynamical processes occurring at the different pressure ramps have been considered. The results obtained at room temperature show that the inactivation effect of high pressure remains the same in case of a fast pressurization ramp (500 MPa/min) and a slow depressurization ramp (100 MPa/min) in comparison to a slow pressurization ramp (100 MPa/min) and a fast depressurization ramp (500 MPa/min). At the chosen test conditions, the fluiddynamical and thermal processes of the two variations differ only slightly from each other. This has been ascertained by measuring the velocity and temperature distributions occurring at the pressure processes. The conclusion is that the pressurization and depressurization rate, respectively, do not effect per se the inactivation kinetics of Listeria innocua.     

Kinetic models for pulsed electric field and thermal inactivation of Escherichia coli and Pseudomonas fluorescens in whole milk

Inactivation of Escherichia coli ATCC 11775 and Pseudomonas fluorescens ATCC 948 in UHT whole (4% fat) milk during thermal processing at 56–62 °C and pulsed electric field (PEF) processing at 30 or 35 kV cm⁻¹ at approximately 30, 40 or 50 °C was investigated. E. coli ATCC 11775 was more heat-resistant than P. fluorescens ATCC 948, but more susceptible to PEF processing. All inactivation kinetics showed strong deviations from log-linearity. Thus, a simplified logistic (log-decay) regression model was used to accurately predict thermal and PEF inactivation of E. coli ATCC 11775 and P. fluorescens ATCC 948 under various treatment conditions. This is a useful tool for identifying processing conditions to inactivate pathogenic and spoilage microorganisms in whole milk at sub-pasteurisation temperatures.     

Combined effect of temperature and pulsed electric fields on soya milk lipoxygenase inactivation

Pulsed electric fields (PEF) were applied to freshly prepared soya milk using a laboratory scale continuous PEF system to study the feasibility of inactivating lipoxygenase (LOX). Square wave PEF using different combinations of pre-treatment temperature, electric field strength and treatment time were evaluated in this study. Inactivation curves for the enzyme were plotted for each parameter and inactivation kinetics were calculated and modelled. Results showed the highest level of inactivation (84.5%) was obtained using a combination of preheating to 50 °C, and a PEF treatment time of 100 μs at 40 kV/cm. Inactivation of LOX activity as a function of treatment time could be described using a first order kinetic model. Calculated D values following pre-heating to 50 °C were 172.9, 141.6 and 126.1 μs at 20, 30 and 40 kV/cm, respectively.     

Evaluation of pulsed electric field and minimal heat treatments for inactivation of pseudomonads and enhancement of milk shelf-life

Pulsed Electric Field (PEF) treatment of milk provides the opportunity to increase the shelf-life of fresh milk for distribution to distant markets. PEF treatments were evaluated in sterile (UHT) milk to determine the inactivation of added spoilage Pseudomonas isolates and the subsequent gains in microbial shelf-life (time taken to reach 10⁷ CFU mL^− 1). Little inactivation of Pseudomonas was achieved at 15 or 40 °C compared with 50 or 55 °C. The greatest inactivation (> 5 logs) was achieved by processing at 55 °C with 31 kV cm^− 1 (139.4 kJ L^− 1). Heat treatment at the application temperature without PEF treatment caused minimal inactivation of Pseudomonas (only 0.2 logs), demonstrating that the inactivation of the Pseudomonas was due to the PEF treatment rather than the heat applied to the milk. At added Pseudomonas levels of 10³ and 10⁵ CFU mL^− 1, the microbial shelf-life of PEF-treated milk was extended by at least 8 days at 4 °C compared with untreated milk. The total microbial shelf-life of the PEF-treated milk was 13 and 11 days for inoculation levels of 10³ and 10⁵ CFU mL^− 1 respectively. The results indicate that PEF treatment is useful for the reduction of pseudomonads, the major spoilage bacteria of milk.Industrial relevancePseudomonads are the major psychrotrophic spoilage microflora of refrigerated, stored HTST pasteurised milk. Long-life (UHT) products are an important component of milk sales in South-East Asia, but in recent years there has been an increasing demand for less processed milk products with extended shelf-life. The recent practice of shipping fresh bulk milk from Australia to South-East Asian countries has necessitated additional heat treatment prior to export and on arrival, to achieve the required shelf-life. Pulsed electric field treatment of HTST milk, applied alone or in combination with mild heat under optimised conditions, offers the opportunity of shelf-life extension, while limiting the reduction in quality attributes of milk associated with more severe additional heat treatments.     

Effect of a post-packaging pasteurization process on inactivation of a Listeria innocua surrogate in meat products

Effects of surface pasteurization on inactivation of Listeria innocua were investigated. Surface temperature, monitored during post-packaging pasteurization, was used to predict the lethality of L. monocytogenes. Temperatures reached 70°C for lean and fat sausages within 9 min of treatment. An inoculation study validated the efficacy of post-processing and the thermal lethality of L. monocytogenes. Pre-cooked sausage and ham, inoculated with approximately 10⁷ CFU/cm² of L. innocua, were heated to a surface temperature of 70°C. Numbers of L. innocua were reduced by 7 log on surface-inoculated sausage. Guidelines for safe, ready-to-eat meat products are provided for small scale meat processors.     

The effect of homogenization of whole milk, skim milk and milk fat on nisin activity against Listeria innocua

Whole milk, skim milk and an emulsion of milk fat in water, inoculated with approx. 10(5) cfu/ml of Listeria innocua, were treated at 30 degrees C with 100 IU/ml of nisin, homogenization at 200 bar or both procedures. Nisin activity and survival of L. innocua after treatments were determined. Recovery of nisin activity from non-homogenized whole milk treated with 100 IU/ml of nisin was complete, whereas a loss of 18 to 28% of activity was detected in non-homogenized fat-in-water emulsion. Loss in nisin activity due to homogenization represented up to 64% in whole milk and 62% in fat-in-water emulsion. Nisin addition by itself achieved a reduction in L. innocua counts of 3.7-3.8 log units in whole milk and 3.6 log units in fat-in-water emulsion compared to numbers in untreated samples. When nisin-containing whole milk and fat-in-water emulsion were homogenized, L. innocua counts were only reduced by 2.6-2.9 log units and 2.5 log units, respectively, compared to numbers in untreated samples. Homogenization of nisin-containing skim milk resulted in a loss of nisin activity of 20% but achieved a reduction of 3.0 log units in L. innocua counts.     

Inactivation of Escherichia coli, Listeria innocua and Saccharomyces cerevisiae in relation to membrane permeabilization and subsequent leakage of intracellular compounds due to pulsed electric field processing

Membrane permeabilization, caused by pulsed electric field (PEF) processing of microbial cells, was investigated by measurement of propidium iodide (PI) uptake with flow cytometry. Inactivation of Escherichia coli, Listeria innocua and Saccharomyces cerevisiae was determined by viable counts, and leakage of intracellular compounds, such as ATP and UV-absorbing substances, was measured in the extracellular environment. Electrical field strength and pulse duration influenced membrane permeabilization of all three tested organisms of which S. cerevisiae was the most PEF sensitive, followed by E. coli and L. innocua. It was shown by viable counts, PI uptake and leakage of intracellular compounds that L. innocua was the most resistant. Increased inactivation corresponded to greater numbers of permeabilized cells, which were reflected by increased PI uptake and larger amounts of intracellular compounds leaking from cells. For E. coli and L. innocua, a linear relationship was observed between the number of inactivated cells (determined as CFU) and cells with permeated membranes (determined by PI uptake), with higher number of inactivated cells than permeated cells. Increased leakage of intracellular compounds with increasing treatment severity provided further evidence that cells were permeabilized. For S. cerevisiae, there was higher PI uptake after PEF treatments, although very little or no inactivation was observed. Results suggest that E. coli and L. innocua cells, which took up PI, lost their ability to multiply, whereas cells of S. cerevisiae, which also took up PI, were not necessarily lethally permeabilized.     

Modelling inactivation of Listeria monocytogenes by pulsed electric fields in media of different pH

A study of the effect of square-wave pulsed electric fields (PEF) on the inactivation of Listeria monocytogenes in McIlvaine buffer of different pH (3.5-7.0) was conducted. L. monocytoges was more PEF sensitive at higher electric field strengths (E) and in media of low pH. A treatment at 28 kV/cm for 400 mus that inactivated 1.5, 2.3 and 3.0 Log10 cycles at pH 7.0, 6.5 and 5.0 respectively destroyed almost 6.0 Log10 cycles at pH 3.5. The general shape of survival curves of L. monocytogenes PEF treated at different pH was convex/concave upwards. A mathematical model based on the Weibull distribution accurately described these survival curves. At each pH, the shape parameter (n value) did not depend on E. The relationship between n value of the Weibull model and the pH of the treatment medium was described by the Gompertz equation. A multiple linear regression model using three predictor variables (E, E2, pH2) related the Log10 of the scale paramenter (b value) of the Weibull model with E and pH of the treatment medium. A tertiary model developed using McIlvaine buffer as treatment medium predicted satisfactorily the inactivation of L. monocytogenes in apple juice.