Mechanistic insight into roles of α/ß-type small acid-soluble proteins, RecA, and inner membrane proteins during bacterial spore inactivation by ohmic heating.

AIM: Ohmic heating (OH) (i.e. heating by electric field) more effectively kills bacterial spores than traditional wet heating, yet its mechanism remains poorly understood. This study investigates the accelerated spore inactivation mechanism using genetically modified spores. METHODS AND RESULTS: We investigated the effects of OH and conventional heating (CH) on various genetically modified strains of Bacillus subtilis: isogenic PS533 (wild type_1), PS578 [lacking spores' α/ß-type small acid-soluble proteins (SASP)], PS2318 (lacking recA, encoding a DNA repair protein), isogenic PS4461 (wild type_2), and PS4462 (having the 2Duf protein in spores, which increases spore wet heat resistance and decreases spore inner membrane fluidity). Removal of SASP brought the inactivation profiles of OH and CH closer, suggesting the interaction of these proteins with the field. However, the reemergence of a difference between CH and OH killing for SASP-deficient spores at the highest tested field strength suggested there is also interaction of the field with another spore core component. Additionally, RecA-deficient spores yielded results like those with the wild-type spores for CH, while the OH resistance of this mutant increased at the lower tested temperatures, implying that RecA or DNA are a possible additional target for the electric field. Addition of the 2Duf protein markedly increased spore resistance both to CH and OH, although some acceleration of killing was observed with OH at 50 V/cm. CONCLUSIONS: In summary, both membrane fluidity and interaction of the spore core proteins with electric field are key factors in enhanced spore killing with electric field-heat combinations.

A microfluidic approach for studying microcolonization of Escherichia coli O157:H7 on leaf trichome-mimicking surfaces under fluid shear stress.

Escherichia coli O157:H7 have previously been associated with disease outbreaks associated with leafy green vegetables. However, the physical mechanisms that determine the spatial organization of bacteria onto leafy greens are still not clear. Microfluidics with embedded trichome-mimicking microposts were employed to investigate the role of shear flow and configuration of trichomes on E. coli O157:H7 microcolonization. We characterized the three-dimensional microcolonization of green fluorescent protein (GFP)-tagged E. coli O157:H7 using multiphoton fluorescence microscopy and compared their differences under static (no flow; incubated for 36 h at 37°C) and fluid shear conditions (750 nl/min for 36 h at 37°C). For micropatterned trichome arrays, we demonstrated that natural wax-mixed polydimethylsiloxane retains similar topographies and contact angles to the surface of trichome-bearing leafy greens. Our results showed that E. coli O157:H7 under fluid shear stress aligned their colonization parallel to the direction of flow. In a static condition, their colonization had no preferential alignment, with statistically similar angular distributions in all directions. In addition, depending on dimensions of the trichome arrays and flow conditions, different bacterial microcolonization patterns grew radially from initial attachment; they formed into filamentous structures and developed into bridges by surface hydrophobicity and flow-induced shear with a nutrient-rich medium. Collectively, these results demonstrate how the consequences of bacterial colonization in response to shear flow can affect pathogenic bacterial contamination of leafy greens and biofilm architectures.

Guidelines on reporting treatment conditions for emerging technologies in food processing.

In the last decades, different non-thermal and thermal technologies have been developed for food processing. However, in many cases, it is not clear which experimental parameters must be reported to guarantee the experiments' reproducibility and provide the food industry a straightforward way to scale-up these technologies. Since reproducibility is one of the most important science features, the current work aims to improve the reproducibility of studies on emerging technologies for food processing by providing guidelines on reporting treatment conditions of thermal and non-thermal technologies. Infrared heating, microwave heating, ohmic heating and radiofrequency heating are addressed as advanced thermal technologies and isostatic high pressure, ultra-high-pressure homogenization sterilization, high-pressure homogenization, microfluidization, irradiation, plasma technologies, power ultrasound, pressure change technology, pulsed electric fields, pulsed light and supercritical CO2 are approached as non-thermal technologies. Finally, growing points and perspectives are highlighted.

Molecular dynamics evidence for nonthermal effects of electric fields on pectin methylesterase activity.

Experimental studies relevant to the nonthermal effects of electric fields on biological systems are emerging. However, these effects are poorly understood at the molecular level. The present study investigates pectin methylesterase, a cell wall modifying enzyme in plants, exposed to various electric field strengths. Molecular dynamics (MD) of the enzyme were studied with and without (thermal-only) electric field applications. The measurements were interpreted on the basis of equivalent energy input to gain insights into the effect of electric field treatment time at a constant temperature (50 °C). Results reveal that electric fields exert nonthermal effects on both local and global protein structure. In 1 µs simulations, the results show significant (P ≤ 0.05) shrinkage of the catalytic domain and shortening of enzyme-water hydrogen bond lifetime by a 50 V cm-1 electric field. Unwinding of the helical segments, altered intra- and intermolecular hydrogen bond patterns, and increased hydration are also caused by the 50 V cm-1 electric field. This study serves to understand the electric field influence on the functional role of proteins.

Moderate Electric Field Treatment Enhances Enzymatic Hydrolysis of Cellulose at Below-Optimal Temperatures.

Saccharification of cellulosic biomass for the fermentation of transportation fuels faces several challenges. Cellulose is highly stable, and even with enzymatic assistance, decomposition of cellulose is slow. Additionally, the enzymes are expensive and sensitive to thermal and mechanical inactivation. In this work, we studied the effects of moderate electric field (MEF, in the range from 1 to 1000 V per cm) treatments on the effectiveness of enzymatic saccharification. MEF treatments were applied to determine their effects on enzyme activity. We considered the effects of field strength, frequency, application regime and temperature. It was found that the enzyme responded to alterations in the frequency of the waveform, with 50 to 60 Hz maximizing the effects of the field, although the effects of field strength and application regime were more significant. It was found that the electric field could have a positive, negative, or negligible effect depending on the field strength. Most notably, when MEF treatments were applied over a range of temperatures, it was found that MEF treatment significantly improved enzyme activity at lower temperatures, leading to the observation that MEF treatment imitates a temperature increase. Calculations simulating the electrophoretic motion of the enzymes verified that the magnitude of motion associated with the MEF treatments was qualitatively similar to the change in molecular motion associated with temperature increases.

Ohmic heating as a promising technique for extraction of herbal essential oils: Understanding mechanisms, recent findings, and associated challenges.

The applicability of ohmic heating, as a volumetric heating technique, has been explored in various sectors of the food industry. The use of ohmic heating for essential oil extraction is among its emerging applications. This chapter overviews the recent progress in this area of research, discusses the mechanisms involved in ohmic-based essential oil extraction processes, explains the effective process parameters, highlights their benefits, and explains the considerations to address the obstacles to industrial implementation. Ohmic-assisted hydrodistillation (OAHD) and ohmic-accelerated steam distillation (OASD) systems were proposed as alternatives to conventional hydrodistillation and steam distillation, respectively. These techniques have successfully extracted essential oils from several aromatic plants (e.g., thyme, peppermint, citronella, and lavender). Both OAHD and OASD possess a number of benefits, such as reducing the extraction time and energy consumption, compared to classical extraction methods. However, these techniques are in their infancy and further economic and upscaling studies are required for their industrial adaptation.

Mechanisms of enhanced bacterial endospore inactivation during sterilization by ohmic heating.

During ohmic heating, the electric field may additionally inactivate bacterial endospores. However, the exact mechanism of action is unclear. Thus, a mechanistic study was carried out, investigating the possible target of electric fields inside the spore. Bacillus subtilis spores were heated by conventional and ohmic heating in a capillary system under almost identical thermal conditions. Wild-type (PS533) spores were used, as well as isogenic mutants lacking certain components known for their contribution to spores' heat resistance: small-acid soluble proteins (SASP) protecting DNA (PS578); the coat covering the spore (PS3328); and the spore germination enzyme SleB (FB122(+)). Treatment-dependent release of the spore core's depot of dipicolinic acid (DPA) was further evaluated. Up to 2.4 log10 additional inactivation of PS533 could be achieved by ohmic heating, compared to conventional heating. The difference varied for the mutants, with a decreasing difference indicating a decreased effect of the electric field and vice versa. In particular, mutant spores lacking SASPs showed a behavior more similar to thermal inactivation alone. The combination of heat and electric field was shown to be necessary for enhanced spore inactivation. Thus, it is hypothesized that either the heat treatment makes the spore susceptible to the electric field, or vice versa.

Novel Processing Technologies as Compared to Thermal Treatment on the Bioaccessibility and Caco-2 Cell Uptake of Carotenoids from Tomato and Kale-Based Juices.

This research aimed to measure the impact of novel food processing techniques, i.e., pulsed electric field (PEF) and ohmic heating (OH), on carotenoid bioaccessibility and Caco-2 cell uptake from tomato juice and high-pressure processing (HPP) and PEF on the same attributes from kale-based juices, as compared with raw (nonprocessed) and conventional thermally treated (TT) juices. Lycopene, ß-carotene, and lutein were quantitated in juices and the micelle fraction using high-performance liquid chromatography (HPLC)-diode array detection and in Caco-2 cells using HPLC-tandem mass spectrometry. Tomato juice results were as follows: PEF increased lycopene bioaccessibility (1.5 ± 0.39%) by 150% (P = 0.01) but reduced ß-carotene bioaccessibility (28 ± 6.2%) by 44% (P = 0.02), relative to raw juice. All processing methods increased lutein uptake. Kale-based juice results were as follows: TT and PEF degraded ß-carotene and lutein in the juice. No difference in bioaccessibility or cell uptake was observed. Total delivery, i.e., the summation of bioaccessibility and cell uptake, of lycopene, ß-carotene, and lutein was independent of type of processing. Taken together, PEF and OH enhanced total lycopene and lutein delivery from tomato juice to Caco-2 cells as well as TT, and may produce a more desirable product due to other factors (i.e., conservation of heat-labile micronutrients, fresher organoleptic profile). HPP best conserved the carotenoid content and color of kale-based juice and merits further consideration.

Ohmic Heating Assisted Lye Peeling of Pears.

Currently, high concentrations (15% to 18%) of lye (sodium hydroxide) are used in peeling pears, constituting a wastewater handling and disposal problem for fruit processors. In this study, the effect of ohmic heating on lye peeling of pears was investigated. Pears were peeled using 0.5%, 1%, 2%, and 3% NaOH under different electric field strengths at two run times and their peeled yields were compared to that obtained at 2% and 18% NaOH with conventional heating. Results revealed that ohmic heating results in greater than 95% peeled yields and the best peel quality at much lower concentrations of lye (2% NaOH at 532 V/m and 3% NaOH at 426 and 479 V/m) than those obtained under conventional heating conditions. Treatment times of 30 and 60 s showed no significant differences. Within the studied range, the effects of increasing field strength yielded no significant additional benefits. These results confirm that the concentration of lye can be significantly lowered in the presence of ohmic heating to achieve high peeled yields and quality. PRACTICAL APPLICATION: Our work shows that lye concentrations can be greatly reduced while peeling pears, resulting in significant savings in use of caustic chemicals, reduced costs for effluent treatment and waste disposal.

Extraction from Food and Natural Products by Moderate Electric Field: Mechanisms, Benefits, and Potential Industrial Applications.

Several extraction techniques have been proposed recently as the food industry is seeking alternatives to tedious classical methods. Moderate electric field (MEF) extraction at low and high temperatures offers several advantages such as enhancing product quantity and quality as well as saving time and energy. This method has gained interest as a preferred extraction technique among researchers. This review is devoted to introducing the principles, mechanisms, and recent progress in the MEF extraction technique. The parameters influencing MEF treatment are also discussed and performances are compared to conventional extraction methods. Considerations for industrial development of MEF as well as its drawbacks are also discussed. MEF extraction is applicable to a wide range of food materials and offers several benefits such as reducing extraction time and energy and also enhancing the extract's quality and yield. Optimizing process parameters, such as treatment time and temperature, electric field intensity, and frequency, along with raw material selection and preparation can boost the performance of a MEF system. Further investigations on safety and sustainability of this novel technique are required prior to commercial applications.

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.

Effects of controlled-frequency moderate electric fields on pectin methylesterase and polygalacturonase activities in tomato homogenate.

The effect of controlled-frequency moderate electric field treatments on pectin methylesterase and polygalcturonase activities in tomato homogenate was investigated by subjecting identically treated control and electrically-treated samples to the same temperature history. Additionally, a model was developed for the motion of the enzyme molecules subjected to an electric field. Results show that the application of electric fields at a low field strength (0.4V/cm) constant temperature (65°C) has a statistically significant effect on pectin methylesterase activity, typically at or lower than 60 Hz. At higher frequencies, the effects are negligible. Molecular motion simulations suggest that the efficacy at low frequencies may be due to the amplitude of motion being of the order of the intermolecular distance for water. Higher frequencies result in small overall displacements due to rapid reversals in the direction of motion.

Physics of Fresh Produce Safety: Role of Diffusion and Tissue Reaction in Sanitization of Leafy Green Vegetables with Liquid and Gaseous Ozone-Based Sanitizers.

Produce safety has received much recent attention, with the emphasis being largely on discovery of how microbes invade produce. However, the sanitization operation deserves more attention than it has received. The ability of a sanitizer to reach the site of pathogens is a fundamental prerequisite for efficacy. This work addresses the transport processes of ozone (gaseous and liquid) sanitizer for decontamination of leafy greens. The liquid sanitizer was ineffective against Escherichia coli K-12 in situations where air bubbles may be trapped within cavities. A model was developed for diffusion of sanitizer into the interior of produce. The reaction rate of ozone with the surface of a lettuce leaf was determined experimentally and was used in a numerical simulation to evaluate ozone concentrations within the produce and to determine the time required to reach different locations. For aqueous ozone, the penetration depth was limited to several millimeters by ozone self-decomposition due to the significant time required for diffusion. In contrast, gaseous sanitizer was able to reach a depth of 100 mm in several minutes without depletion in the absence of reaction with surfaces. However, when the ozone gas reacted with the produce surface, gas concentration was significantly affected. Simulation data were validated experimentally by measuring ozone concentrations at the bottom of a cylinder made of lettuce leaf. The microbiological test confirmed the relationship between ozone transport, its self-decomposition, reaction with surrounding materials, and the degree of inactivation of E. coli K-12. Our study shows that decontamination of fresh produce, through direct contact with the sanitizer, is more feasible with gaseous than with aqueous sanitizers. Therefore, sanitization during a high-speed washing process is effective only for decontaminating the wash water.

Effect of the electric field frequency on ascorbic acid degradation during thermal treatment by ohmic heating.

In this work, the influence of the electric field frequency and solids content on the degradation kinetics of ascorbic acid during ohmic heating of acerola pulp and acerola serum was investigated. The degradation percentage of ascorbic acid in the pulp after 120 min of heating varied between 12 and 17%. For the serum, the degradation percentage was in the range of 13 and 18%. The results were fitted to the first-order model, and the kinetic rate constants ranged from 1.1 to 1.6×10(-3) min(-1) and from 1.1 to 1.5×10(-3) min(-1) for pulp and serum, respectively. D values ranged between 1480 and 2145 min for the pulp and between 1524 and 1951 min for the serum. A distinct behavior between the kinetic parameters of the pulp and serum in electric field frequencies ranging from 10 to 1000 Hz indicates that the presence of distinct amounts and types of solids might affect the rate of the electron transfer in electrochemical reactions. These variables may also affect the polarization process stimulated by the oscillating electric field. The non-achievement of the equilibrium of the polarization process may have an influence on oxidation reactions, affecting the predisposition to hydrogen donation from the ascorbic acid molecule.

Mathematical modeling and microbiological verification of ohmic heating of a multicomponent mixture of particles in a continuous flow ohmic heater system with electric field parallel to flow.

To accomplish continuous flow ohmic heating of a low-acid food product, sufficient heat treatment needs to be delivered to the slowest-heating particle at the outlet of the holding section. This research was aimed at developing mathematical models for sterilization of a multicomponent food in a pilot-scale ohmic heater with electric-field-oriented parallel to the flow and validating microbial inactivation by inoculated particle methods. The model involved 2 sets of simulations, one for determination of fluid temperatures, and a second for evaluating the worst-case scenario. A residence time distribution study was conducted using radio frequency identification methodology to determine the residence time of the fastest-moving particle from a sample of at least 300 particles. Thermal verification of the mathematical model showed good agreement between calculated and experimental fluid temperatures (P > 0.05) at heater and holding tube exits, with a maximum error of 0.6 °C. To achieve a specified target lethal effect at the cold spot of the slowest-heating particle, the length of holding tube required was predicted to be 22 m for a 139.6 °C process temperature with volumetric flow rate of 1.0 × 10(-4) m3/s and 0.05 m in diameter. To verify the model, a microbiological validation test was conducted using at least 299 chicken-alginate particles inoculated with Clostridium sporogenes spores per run. The inoculated pack study indicated the absence of viable microorganisms at the target treatment and its presence for a subtarget treatment, thereby verifying model predictions.

Thermal inactivation kinetics of Bacillus coagulans spores in tomato juice.

The thermal characteristics of the spores and vegetative cells of three strains of Bacillus coagulans (ATCC 8038, ATCC 7050, and 185A) in tomato juice were evaluated. B. coagulans ATCC 8038 was chosen as the target microorganism for thermal processing of tomato products due to its spores having the highest thermal resistance among the three strains. The thermal inactivation kinetics of B. coagulans ATCC 8038 spores in tomato juice between 95 and 115°C were determined independently in two different laboratories using two different heating setups. The results obtained from both laboratories were in general agreement, with z-values (z-value is defined as the change in temperature required for a 10-fold reduction of the D-value, which is defined as the time required at a certain temperature for a 1-log reduction of the target microorganisms) of 8.3 and 8.7°C, respectively. The z-value of B. coagulans 185A spores in tomato juice (pH 4.3) was found to be 10.2°C. The influence of environmental factors, including cold storage time, pH, and preconditioning, upon the thermal resistance of these bacterial spores is discussed. The results obtained showed that a storage temperature of 4°C was appropriate for maintaining the viability and thermal resistance of B. coagulans ATCC 8038 spores. Acidifying the pH of tomato juice decreased the thermal resistance of these spores. A 1-h exposure at room temperature was considered optimal for preconditioning B. coagulans ATCC 8038 spores in tomato juice.

In situ measurement of reaction volume and calculation of pH of weak acid buffer solutions under high pressure.

Direct measurements of reaction volume, so far, have been limited to atmospheric pressure. This study describes a method for in situ reaction volume measurements under pressure using a variable volume piezometer. Reaction volumes for protonic ionization of weak acid buffering agents (MES, citric acid, sulfanilic acid, and phosphoric acid) were measured in situ under pressure up to 400 MPa at 25 °C. The methodology involved initial separation of buffering agents within the piezometer using gelatin capsules. Under pressure, the volume of the reactants was measured at 25 °C, and the contents were heated to 40 °C to dissolve the gelatin and allow the reaction to occur, and cooled to 25 °C, where the volume of products was measured. Reaction volumes were used to calculate pH of the buffer solutions as a function of pressure. The results show that the measured reaction volumes as well as the calculated pH values generally quite agree with their respective theoretically predicted values up to 100 MPa. The results of this study highlight the need for a comprehensive theory to describe the pressure behavior of ionization reactions in realistic systems especially at higher pressures.

In situ measurement of pH under high pressure.

In situ measurement of pH under high pressure is complicated by the need to remain functional under extreme pressures, electrically noisy environments, and the difficulty in inferring chemical equilibrium conditions under pressure. We describe a pH sensor wherein the current flowing at a given input voltage is directly related to hydrogen ion concentration and can be operated within extreme environments. The sensor was tested at 25 °C for in situ pH measurement of several buffer solutions under high pressure up to 784.6 MPa. We found generally an increase in the acidity of the buffer solutions with increasing pressure. In all cases, pH changes were found to be less than predicted using inferred reaction volume data from the literature. Sensor response and pH changes were found to be completely reversible upon depressurization at the same temperature.

Ohmic heating of peaches in the wide range of frequencies (50 Hz to 1 MHz).

UNLABELLED: The ohmic heating (OH) rate of peaches was studied at fixed electric field strength of 60 V.cm⁻¹, square-shaped instant reversal bipolar pulses, and frequencies varying within 50 Hz to 1 MHz. Thermal damage of tissue was evaluated from electrical admittivity. It showed that the time for half disruption (τ(T)) of tissue was required more than 10 h at temperatures below 40 °C. However, cellular thermal disruption occurred almost instantly (τ(T) < 1 s) at high temperatures (> 90 °C). Electrical conductivity σ(o) and admittivity σ(o)* of tissue at T(o)= 0 °C and their temperature coefficients (m, m*) were calculated. For freeze-thawed tissues, σ and σ* as well as m and m* were nearly indifferent to the frequency. However, for the intact tissue, both σ(o), σ(o)* and m, m* were frequency dependent. For freeze-thawed product, the power factor (P) was approximately equal to 1 and indifferent to the frequency and temperature. On the other hand, strong frequency dependence was observed for intact tissue with the minimum P approximately equal to 0.68 in the range of tens of kHz. The time required to reach a target temperature t(f) was evaluated. The t(f) increased with frequency up to the middle of the range of tens of kHz and thereafter continuously decreased. Samples exposed to the low-frequency electric field demonstrated faster electro-thermal damage rates. The textural relaxation data supported more intense damage kinetics at low-frequency OH. It has been demonstrated that a combination of high-frequency OH with pasteurization at moderate temperature followed by rapid cooling minimizes texture degradation of peach tissue. PRACTICAL APPLICATION: In this study, we investigated the electric field frequency effect on the rate of OH of peaches. It was shown that the time required for reaching the target temperature is strongly dependent upon the frequency. Samples exposed to low-frequency OH demonstrated higher electro-thermal damage rates. It has been shown that the combination of high-frequency OH with pasteurization at moderate temperature followed by rapid cooling minimizes texture degradation of peach tissue. Obtained results provide new information on the impact of electric field frequency on OH, which is useful for OH process design.

Residence time distribution (RTD) of particulate foods in a continuous flow pilot-scale ohmic heater.

The residence time distribution (RTD) of a model particulate-fluid mixture (potato in starch solution) in the ohmic heater in a continuous sterilization process was measured using a radio frequency identification (RFID) methodology. The effect of solid concentration and the rotational speed of the agitators on the RTD were studied. The velocity of the fastest particle was 1.62 times the mean product velocity. In general, particle velocity was found to be greater than the product bulk average velocity. Mean particle residence time (MPRT) increased with an increase in the rotational speed of the agitators (P < 0.05), and no particular trend was observed between the MPRT and the solid concentration. The distribution curves E (theta) were skewed to the right suggesting slow moving zones in the system.