Microbial inactivation by pulsed electric fields in a batch treatment chamber: effects of some electrical parameters and food constituents

The inactivation of Escherichia coli by high voltage pulsed electric fields in a batch treatment chamber was studied in liquid, solid and semisolid foods or model systems. Treatment heterogeneity was demonstrated and found to be due to the presence of an air bubble trapped inside the chamber. Agitation of the inoculated liquid samples (16 mM sodium phosphate buffer, pH 7, ρ=460 Ωcm) during pulse processing resulted in efficient microbial inactivation (five log cycles at 33 kV/cm and 25°C after 261 μs of cumulated pulses). A slower inactivation rate was observed in inoculated solid agar gels of the same pH and resistivity, under the same pulse processing conditions. The inactivation of E. coli in inoculated dairy cream (33% fat, pH 6.8, ρ=370 Ωcm), ovalbumin solution (10% protein w/v, pH 6.7, ρ=370 Ωcm) or fish egg suspension (pH 6.8, ρ=400 Ωcm) was almost identical to that in 16 mM phosphate buffer, pH 7. Thus emulsified lipids, soluble proteins or conductive food particulates do not appear to protect against microbial inactivation by electric pulses.     

Inactivation of Yersinia enterocolitica by pulsed electric fields

The influence of growth conditions, treatment medium characteristics and PEF process parameters on the lethal effect on Yersinia enterocolitica of pulsed electric fields (PEF) treatments in batch has been investigated. Growth phase, temperature of growth, pH, conductivity of the treatment medium, pulse width and frequency of pulses did not influence the sensitivity of Y. enterocolitica to PEF. However, an A_w decrease from >0.99 to 0.93 of the treatment medium increased the PEF resistance of Y. enterocolitica with 3.5 log₁₀ cycles after a treatment of 22 kV/cm, 800 μs and 880 kJ/kg. Inactivation of Y. enterocolitica increased with the field strength, treatment time and total specific energy up to a maximum of 6 log₁₀ cycles after 28 kV/cm, 2000 μs and 3559 kJ/kg. A nonlinear relationship was found among the survival fraction and the treatment time or the specific energy that was accurately described by a mathematical model based on the Weibull distribution. The inactivation of Y. enterocolitica by PEF was characterized by maximum field strength thresholds. Above these thresholds, specific energy necessary to obtain a given level of inactivation scarcely decreased by increasing the electric field strength, and inactivation of Y. enterocolitica only depended on the specific energy applied.     

Modeling within the Bayesian framework, the inactivation of pectinesterase in gazpacho by pulsed electric fields

The inactivation of pectinesterase (PE) activity in gazpacho (a Spanish ready-to-use cold vegetable soup) under pulsed electric fields (PEFs) was studied. Samples were exposed to 4 μs monopolar or bipolar square-wave pulses at 5–35 kV cm⁻¹ electric field intensity for up to 1500 μs and 200 Hz (temperature below 40 °C). Inactivation of PE was greater when treatment time and electric field intensity increased, and bipolar pulses were more effective inactivating PE than monopolar ones. Estimation of parameters and quantities involved in the tested models were performed within the Bayesian framework. The kinetic evolution of the enzyme was explained using a 3-parameter biexponential model based on a mechanism involving two irreversible consecutive steps. Rate constant k₁ was not dependent on neither electric field intensity nor pulse polarity. Rate constant k₂ and ratio between the activities of intermediate forms and the native ones of the enzyme Λ were affected by those conditions.     

POTENTIAL USE OF 18 TESLA STATIC AND PULSED MAGNETIC FIELDS ON ESCHERICHIA COLI AND SACCHAROMYCES CEREVISIAE

Escherichia coli and Saccharomyces cerevisiae were subjected to 18T static and pulsed magnetic fields at different temperatures in either phosphate buffer, McIlvaine buffer, or peptonated water. Colony forming units were recorded after incubation in nutrient and Violet Red Bile agar for E. coli and potato dextrose agar for S. cerevisiae. No inactivation or cell injury was detected due to the influence of the magnetic field whether static or pulsed. As expected, at higher temperatures both inactivation and cell injury were increased. Although further research is needed, magnetic field technology at 18T or lower does not appear as a feasible option for processing foods.     

Influence of high-intensity pulsed electric field processing on lipoxygenase and β-glucosidase activities in strawberry juice

The influence of high-intensity pulsed electric field (HIPEF) parameters, pulse frequency, pulse width and pulse polarity in strawberry juice lipoxygenase (LOX) and β-glucosidase (β-GLUC) was studied using a response surface methodology. The studied parameters affected on both residual enzymatic activities at unchanging electric field strength of 35 kV/cm and treatment time for 1000 μs. The contour plots showed a minimum defined space where residual activity of LOX remained at 65% and 70% in monopolar and bipolar mode, respectively. Low pulse frequencies (up to 61.6 Hz) in monopolar treatments as well as pulse frequencies and widths higher than 218 Hz and 5.4 μs in bipolar treatments did not have any effect on LOX inactivation. On the other hand, the higher the pulse frequency and pulse width, the higher the β-GLUC inactivation obtained. Moreover, when the HIPEF treatment was applied in monopolar mode, an enhancement in β-GLUC activity was observed in most of the experimental range. HIPEF treatments have demonstrated adequately that can reduce activity of enzymes that are involved in the formation of desirable flavor compounds, helping processors to obtain juices that keep their fresh flavor.

Industrial relevance

High-intensity pulsed electric fields (HIPEF) have proved to be effective in the interaction of microorganisms and enzymes in juices, maintaining their quality and freshness.HIPEF juice processing has demonstrated to have some advantages with regard to conventional thermal treatment. HIPEF treatments can reduce adequately enzymes that are involved in the formation of desirable flavor or color compounds. Thus, HIPEF technology can help processors to obtain juices that keep their fresh flavor by achieving optimal inactivation of related enzymes. This would prevent the product from undesirable off-flavor formation, which in turn would result in greater acceptability by consumers.     

Predictive model of Vibrio parahaemolyticus growth and survival on salmon meat as a function of temperature

The growth and survival curves of a strain of pandemic Vibrio parahaemolyticus TGqx01 (serotype O3:K6) on salmon meat at different storage temperatures (range from 0 °C to 35 °C) were determined. In order to model the growth or inactivation kinetics of this pathogen during storage, the modified Gompertz and Weibull equations were chosen to regress growth and survival curves, respectively, and both equations produced good fit to the observed data (the average R² value equals to 0.990 for modified Gompertz and 0.920 for Weibull equation). The effect of storage temperature on the specific growth rate (μ) was modeled by square root type equation, and the relationship between μ and lag time (λ) was described by a rule of μ × λ = constant. The shape factor (n) and scale factor (b) values of the Weibull equations versus the temperature (°C) were plotted and the temperature effects on these parameters were described by two linear empirical equations. The predicted growth and survival curves from the model were compared to real enumeration results, using the correlation coefficient (R²), bias factor (B_f) and accuracy factor (A_f), to assess the performance of the established model. The results showed that the overall predictions for V. parahaemolyticus TGqx01 growth or inactivation on salmon at tested temperatures agreed well with observed plate counts, and the average R², B_f and A_f values were 0.958, 1.019 and 1.035, respectively.     

Production and stability of chlorine dioxide in organic acid solutions as affected by pH, type of acid, and concentration of sodium chlorite, and its effectiveness in inactivating Bacillus cereus spores

We studied the production and stability of chlorine dioxide (ClO₂) in organic acid solutions and its effectiveness in killing Bacillus cereus spores. Sodium chlorite (5000, 10,000, or 50,000 μg/ml) was added to 5% acetic, citric, or lactic acid solution, adjusted to pH 3.0, 4.0, 5.0, or 6.0, and held at 21 °C for up to 14 days. The amount of ClO₂ produced was higher as the concentration of sodium chlorite was increased and as the pH of the acid solutions was decreased. However, the stability in production of ClO₂ was enhanced by increasing the pH of the organic acid solutions. To evaluate the lethal activity of ClO₂ produced in various acid solutions as affected by acidulant and pH, suspensions of B. cereus spores were treated at 21 °C for 1, 3, 5, or 10 min in hydrochloric acid or organic acid solutions (pH 3.0, 4.0, 5.0, or 6.0) containing ClO₂ at concentrations of 100, 50, or 25 μg/ml. Populations of viable spores treated with ClO₂ at concentrations of 100 or 50 μg/ml in organic acid solutions decreased more rapidly than populations treated with the same concentrations of ClO₂ in HCl. Rates of inactivation tended to increase with higher pH of ClO₂ solutions. Results show that ClO₂ formed in organic acid solutions has higher stability and is more lethal to B. cereus spores than ClO₂ formed at the same concentration in HCl solution. This finding emphasizes the benefits of using organic acid solutions to prepare ClO₂ intended for use as an antimicrobial.     

Influence of pulsed electric field energy on the damage degree in alfalfa tissue

The objectives of this study were to investigate the influence of pulsed electric field (PEF) parameters on the damage degree of alfalfa mash, and to determine the relationship between the maximum damage degree and the energy used. Alfalfa mash was treated with PEF at various electric field strengths of 1.25, 1.90, and 2.50 kV/cm. The capacitance of the discharge capacitor was varied from 0.5 to 1.5 μF in steps of 0.5 μF. The pulse number was increased gradually to the point where the impedance became constant. There was no significant increase in the rate of damage beyond 0.5 kJ applied energy. The rate of change of the damage degree at 0.5 kJ was highest when the capacitance was 1.5 μF for all the voltages. Increase in the electric field strength led to decrease in energy needed to obtain the maximum damage degree. To achieve an efficient result for alfalfa juice extraction, the capacitance of the discharge capacitor should preferably be 1 μF or more. In order to minimize energy consumption for a given damage degree in alfalfa, it is desirable to have the highest energy per pulse and fewer number of pulses.     

Modelling the inactivation of Escherichia coli ATCC 25922 using pulsed electric field

Microbial tests were conducted to determine the effect of pulsed electric field treatment on microbial inactivation of gram negative Escherichia coli ATCC 25922 suspended in simulated milk ultra filtrate (SMUF). Kinetic analysis of microbial inactivation due to combined pulsed electric field (PEF) and thermal treatments of E. coli was investigated. A generalized correlation for the inactivation rate constant as a function of both electric field intensity and treatment temperature was derived. Comparison between experimental and theoretical variation of E. coli concentration with time after PEF treatment in a complete recirculation mode was conducted using the inactivation kinetics developed from the single pass measurements.

Industrial relevance

PEF technology has a tremendous potential to replace thermal pasteurisation for products which are sensitive to temperature. In this work a pulsed electric field process was mathematically modelled and a generalized correlation for the inactivation rate constant as a function of electric field intensity and treatment temperature was derived. The correlation is needed for the design of industrial PEF systems.     

Bioactivity of Ocimum gratissimum L. oil and two of its constituents against five insect pests attacking stored food products

The fumigant and repellent effects of Ocimum gratissimum L. oil and its constituents, β-(Z)-ocimene and eugenol, were evaluated against adults of Sitophilus oryzae (L.), Tribolium castaneum (Herbst), Oryzaephilus surinamensis (L.), Rhyzopertha dominica (F.) and Callosobruchus chinensis (L.). The fumigant toxicities of the oil and two of its constituents were assessed at four rates (0, 1, 5 and10 μL/L air) in space fumigation, whereas repellence of the oil and eugenol in acetone was evaluated in choice bioassays at five rates (0, 1.0, 2.0, 3.0 and 4.0 μL oil/2 g grain). Results showed that fumigant toxicity and repellence of the oil and its constituents were significantly (P<0.0001) influenced by concentration and time after treatment. At 1 μL/L air, the oil caused 98%, 99% and 100% mortality of R. dominica, O. surinamensis and C. chinensis, respectively, 24 h after treatment, whereas eugenol achieved 79%, 61% and 100% kill of the same insects. β-(Z)-ocimene produced a weaker toxicity with 8%, 11% and 59% mortality of R. dominica, O. surinamensis and C. chinensis, respectively. Except for T. castaneum which was more tolerant, LC₅₀ values for tested insects ranged from 0.20 to 14, 0.01 to 17 and 0.80 to 23 μL/L air 24 h after treatment for O. gratissimum oil, eugenol and β-(Z)-ocimene, respectively. All test insects had percentage repellence (PR) values which ranged from 37.5% to 100% and 45% to 100% for the oil and eugenol, respectively. However, C. chinensis showed a dose-dependent decrease in PR values in the eugenol bioassay with a corresponding dose-dependent mortality on treated grains. Ocimum gratissimum oil and its constituents are potential alternatives to synthetic fumigants in the treatment of durable agricultural products. Successful adoption of plant oils in the protection of food commodities promises an eco-friendly option compatible with international biosafety regulations.     

Optimising the inactivation of grape juice spoilage organisms by pulse electric fields

The effect of some pulsed electric field (PEF) processing parameters (electric field strength, pulse frequency and treatment time), on a mixture of microorganisms (Kloeckera apiculata, Saccharomyces cerevisiae, Lactobacillus plantarum, Lactobacillus hilgardii and Gluconobacter oxydans) typically present in grape juice and wine were evaluated. An experimental design based on response surface methodology (RSM) was used and results were also compared with those of a factorially designed experiment. The relationship between the levels of inactivation of microorganisms and the energy applied to the grape juice was analysed. Yeast and bacteria were inactivated by the PEF treatments, with reductions that ranged from 2.24 to 3.94 log units. All PEF parameters affected microbial inactivation. Optimal inactivation of the mixture of spoilage microorganisms was predicted by the RSM models at 35.0 kV cm^− 1 with 303 Hz pulse width for 1 ms. Inactivation was greater for yeasts than for bacteria, as was predicted by the RSM. The maximum efficacy of the PEF treatment for inactivation of microorganisms in grape juice was observed around 1500 MJ L^− 1 for all the microorganisms investigated. The RSM could be used in the fruit juice industry to optimise the inactivation of spoilage microorganisms by PEF.     

Antilisterial effects of free fatty acids and monolaurin in beef emulsions and hot dogs

This study was undertaken to screen fatty acids, conjugated isomers of linoleic acid (CLA), and monolaurin for antilisterial effects in broth, and to further test the active compounds in cooked comminuted beef and hot dogs. Capric, lauric, myristic, palmitic, stearic, oleic, linoleic, and linolenic acid, CLA and monolaurin were screened in sterile nutrient broth at concentrations of 5 to 700 μg/ml. The media were inoculated with 10³ cfu/ml of L. monocytogenes strain Scott A and incubated at 32°C for up to 8 days. Cell enumeration data showed that lauric acid was most inhibitory, followed by monolaurin, and capric acid. Tests in comminuted sterile beef stored at 5°C for 21 days showed log cfu/g of: 8.5 (control), 7.3 (500 μg/g lauric acid), and 4.7 (500 lauric acid+300 capric acid). Similar results were observed in beef hot dog emulsion to which lauricidin, lauric acid, capric acid, and the acid combination were added prior to heat treatment. At 500 μg/g, monolaurin and lauric acid caused similar delayed growth effects at 5°C, whereas the combination of the two acids showed enhanced inhibition on prolonged storage. Nonetheless, the observed 5-log increase in numbers of L. monocytogenes during 45 days of storage indicates limited control of the pathogen in refrigerated cooked meat products.     

Impact of PEF treatment inhomogeneity such as electric field distribution, flow characteristics and temperature effects on the inactivation of E. coli and milk alkaline phosphatase

High intensity pulsed electric field (PEF) treatment was investigated focusing on the alteration of electric field distribution, flow characteristics and temperature distribution due to the modification of the treatment chamber. The aim was the improvement of the effectiveness of microbial inactivation of E. coli and to reduce the PEF impact on alkaline phosphatase (ALP) activity in raw milk. Mathematical simulation of the PEF process conditions considering different treatment chamber setups was performed prior to experimental verification. Finally the impact of the treatment chamber modifications on microbial inactivation and enzyme activity was determined experimentally. Using a continuous flow-through PEF system and a co-linear treatment chamber configuration the insertion of stainless steel and polypropylene grids was performed to alter the field strength distribution, increase the turbulence kinetic energy and improve the temperature homogeneity. The Finite Element Method (FEM) analysis showed an improved electric field strength distribution with increased average electric field strength and a reduced standard deviation along the center line of the treatment zone indicating a more homogenous electric field. The velocity profile was improved resulting in an increase of turbulence kinetic energy due to the insertion of the grids. As revealed by mathematical modeling, the temperature of the liquid was decreased, and formation of temperature peaks was avoided. Measured inactivation of heat sensitive alkaline phosphatase (ALP) was reduced from 78% residual activity to 92% after PEF treatment and it could be shown that thermal effects and temperature peaks have been the main reason for enzyme inactivation due to PEF. At the same time, an increase of microbial inactivation of 0.6 log–cycles could be determined experimentally due to the modification of the treatment chamber design.

Industrial relevance

The application of pulsed electric field as a non-thermal pasteurization technology requires an accurately defined treatment intensity followed by a predictable microbial inactivation. Unavoidable thermal effects occurring during PEF treatment due to ohmic heating have to be minimized to assure the retention of heat-sensitive nutrients and bioactive compounds. The presented investigations contribute to the fulfilment of these requirements for further successful industrial implementation of the PEF technology such as the selective inactivation or retention of enzyme activity in liquid food systems.     

Water-extractable phytochemicals from Phyllanthus niruri exhibit distinct in vitro antioxidant and in vivo hepatoprotective activity against paracetamol-induced liver damage in mice

The antioxidative and hepatoprotective potential of Phyllanthus niruri, a widely used medicinal plant in Brazil, were investigated. The aqueous extracts of leaves showed inhibition against thiobarbituric acid-reactive species (TBARS), induced by different pro-oxidants (10 μM FeSO₄ and 5 μM sodium nitroprusside) in rat liver, brain and kidney homogenates. The extracts also lowered the formation of TBARS in phospholipids extracted from egg yolk. The plant exhibited strong antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl free radical (IC₅₀, 43.4 ± 1.45 μg/ml) and iron chelation assay. The hepatoprotective activity of the extracts were also demonstrated in vivo against paracetamol-induced liver damage, as evidenced by the decrease in serum glutamate oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT), and increased catalase activity in the liver in treatment groups, compared to the control. The results of the present study suggest the potential use of P. niruri in the treatment of various diseases, among which liver disease is the most important, due to its ability to act as an antioxidant. Furthermore, since the treatment of human intoxications with paracetamol is always limited to the administration of N-acetyl-cysteine, additional studies would be important to determine whether aqueous extracts of P. niruri could increase the efficacy of N-acetyl-cysteine against paracetamol acute hepatotoxicity.     

Non-nutritive functional agents in rattan-shoots, a food consumed by native people in the Philippines

The tender shoots of Calamus ornatus, one of the food items consumed by the native people, Kanawan Aytas, in the Bataan region of the Philippines, have not been studied before. A bioassay-guided investigation of its methanolic extract afforded non-nutritive functional agents (NFAs), steroidal saponins 1–3, along with its aglycone (4). The NFAs 1–4 inhibited cyclooxygenase enzymes, COX-1 and -2, by 47%, 43%, 33%, and 53% and 71%, 75%, 78%, and 73%, respectively, at 28.2, 24.2, 21.2 and 60.4 μM. Treatment of breast (MCF-7), CNS (SF-268), lung (NCI-H460), colon (HCT-116) and gastric (AGS) cancer cell lines with the extract at 100 μg/ml reduced cell proliferation. Similarly, the pure NFAs 2 and 3 reduced the cell viability of breast, CNS, lung, colon and gastric cancer cell lines by 37.5%, 22.4%, 53.3%, 58.2%, 40.3% and 29.8%, 21.3%, 45.6%, 37.1%, 25.0%, respectively, at 24.2 and 21.2 μM. The 50% reduction in cell viability (IC₅₀) concentrations of 2 and 3 against these cancer cell lines were 8.8, 6.1, 7.5, 23.8, 12.1 and 3.8, 7.1, 3.3, 14.3, 12.1 μM, respectively. This is the first report on the isolation of steroidal saponins from C. ornatus shoots and their antiinflammatory and tumor cell proliferation inhibitory activities. Therefore, our results suggest that the Kanawan Aytas may yield health benefits from rattan-shoots in their diet.     

Fumigant properties of physical preparations from mountain big sagebrush, Artemisia tridentata Nutt. ssp. vaseyana (Rydb.) beetle for stored grain insects

Vapors released from foliage of mountain big sagebrush, Artemisia tridentata Nutt. ssp. vaseyana (Rydb.) Beetle, through a patented process, were hypothesized to have an insecticidal time of action (24 h or less after time of exposure) similar to the fumigant methyl bromide. Patented preparations were more effective from plants harvested from a relatively wet site in mid to late summer (5 July to 11 September). Bioassays with the lesser grain borer, Rhyzopertha dominica (F.), 0–3 days after adult emergence indicated an LT₅₀ of 7.0±1.2 h for the volatiles generated from only 30 mg dry processed plant material (=0.56 mg active ingredients) per ml headspace. Hatching of eggs of the Indian meal moth, Plodia interpunctella (Hübner), was completely suppressed when exposed 4–20 h after oviposition to a concentration of 7 mg processed plant material per ml headspace (=0.14 mg active ingredients) in a container that allowed passive diffusion and from which the terpenes disappeared by 48 h. Adult red flour beetle, Tribolium castaneum (Herbst), had an LT₅₀ of 40.7±1.2 h when exposed to 29 mg processed plant material per ml headspace. Gas chromatography/mass spectrometry (GC/MS) analyses of the headspace above this processed plant material revealed five major peaks, all non-chlorinated and non-brominated. The two main volatiles, 1,8-cineole and camphor, occurred initially in a mean ratio of 1:3.2, gradually shifting to 1:2.4 over 24 h. The μg/ml headspace of each detectable compound in a sealed container was followed intensely (0.25, 1, 2, 12, 24, 48, and 72 h) for 72 h and at less frequent intervals for 60 days. The active compounds released by the plant material in a closed, but not airtight container, were no longer detectable after 24 h based on GC/MS analysis. Fumigative studies with the same ratio of the two main compounds generated synthetically indicated that embryos of P. interpunctella and adults of R. dominica were as sensitive to the synthetic mixture as they were to the processed plant material. Although one could apply the precise commercial terpenes in the same ratio, the plant material provides a natural formulation that is conveniently diluted (formulated) to levels safe for handling. Therefore, this preparation method and plant material shows good potential as an alternative to methyl bromide for protection of stored grain, commodity, and space fumigations. No residues are detectable in the headspace of aerated commodity, milled product, or in fumigated space.     

Inactivation of Escherichia coli in orange juice using ozone

This research investigated the efficacy of gaseous ozone for the inactivation of Escherichia coli ATCC 25922 and NCTC 12900 strains in orange juice. Orange juice inoculated with E. coli (10⁶ CFU mL^− 1) as a challenge microorganism was treated with ozone at 75–78 µg mL^− 1 for different time periods (0–18 min). The efficacy of ozone for inactivation of both strains of E. coli was evaluated as a function of different juice types: model orange juice, fresh unfiltered juice, juice without pulp, and juice filtered through 500 µm or 1 mm sieves. Fast inactivation rates for total reduction of E. coli were achieved in model orange juice (60 s) and in juice with low pulp content (6 min). However, in unfiltered juice inactivation was achieved after 15–18 min. This indicated that juice organic matter interferes with antibacterial activity of gaseous ozone. The effect of prior acid (pH 5.0) exposure of E. coli strains on the inactivation efficacy of ozone treatment was also investigated. There was a strain effect observed, where prior acid exposure resulted in higher inactivation times in some cases by comparison with the control cells. However, the overarching influence on inactivation efficacy of ozone was related to the pulp content. Generally, the applied gaseous ozone treatment of orange juice resulted in a population reduction of 5 log cycles.

Industrial relevance

To facilitate the preservation of unstable nutrients many juice processors have investigated alternatives to thermal pasteurisation, including un-pasteurised short shelf life juices with high retail value. This trend has continued within the European Union. However within the US recent regulations by the FDA have required processors to achieve a 5-log reduction in the numbers of the most resistant pathogens in their finished products. Pathogenic E. coli may survive in acid environments such as fruit juices for long periods. This study demonstrates that the use of ozone as a non-thermal technology is effective for inactivation of E. coli and acid exposed E. coli in orange juice. Information on the design of the ozone treatment for inactivation of E. coli which results into safe juice products is also among the main outputs of this work. Ozone auto-decomposition makes this technology safe for fruit juice processing.     

Essential oil composition and antibacterial activity of Thymus caramanicus at different phenological stages

Thymus species are well known as medicinal plants because of their biological and pharmacological properties. Thymus caramanicus is an endemic species grown in Iran. Variation in the quantity and quality of the essential oil of wild population of T. caramanicus at different phenological stages including vegetative, floral budding, flowering and seed set are reported. The oils of air-dried samples were obtained by hydrodistillation. The yields of oils (w/w%) at different stages were in the order of: flowering (2.5%), floral budding (2.1%), seed set (2.0%) and vegetative (1.9%). The oils were analyzed by GC and GC–MS. In total 37, 37, 29 and 35 components were identified and quantified in vegetative, floral budding, full flowering and seed set, representing 99.3, 98.6, 99.2 and 97.8% of the oil, respectively. Carvacrol was the major compound in all samples. The ranges of major constituents were as follow: carvacrol (58.9–68.9%), p-cymene (3.0–8.9%), γ-terpinene (4.3–8.0%), thymol (2.4–6.0%) and borneol (2.3–4.0%). Antibacterial activity of the oils and their main compounds were tested against seven Gram-positive and Gram-negative bacteria by disc diffusion method and determining their minimum inhibitory concentration (MIC) values. The inhibition zones (IZ) and MIC values for bacterial strains, which were sensitive to the essential oil of T. caramanicus, were in the range of 15–36 mm and 0.5–15.0 mg/ml, respectively. The oils of various phenological stages showed high activity against all tested bacteria, of which Bacillus subtilis and Pseudomonas aeruginosa were the most sensitive and resistant strains, respectively. Thus, they represent an inexpensive source of natural antibacterial substances that exhibited potential for use in pathogenic systems.     

Production and properties of crude enterotoxin of Pseudomonas aeruginosa

Pseudomonas aeruginosa CTM-3 was found to be the most potentially enteroxigenic strain out of the 12 isolates recovered from milk, as a high fluid length ratio, i.e. F/L (1.1) in rabbit gut and a strong permeability response in rabbit skin (38.5 mm² necrotic zone) was obtained with this culture. No clear-cut relationship between the two tests was observed. Six of the ethidium bromide (300 μg/ml) cured variants of this culture completely lost their ability to produce enterotoxin indicating the possible involvement of a plasmid in enterotoxin synthesis. The crude enterotoxin from P. aeruginosa CTM-3 was completely inactivated in 15 s at 72°C. However, it was fairly stable at pH values in the range 4.5–7.5. Both pepsin and trypsin inactivated the enterotoxin activity at a concentration of 40 μg/ml. Organic acids, formalin and hydrogen peroxide had no significant effect on the enterotoxin activity. The need for further investigations with purified preparations is emphasized.     

Models in a Bayesian framework for inactivation of pectinesterase in a commercial enzyme formulation by pulsed electric fields

The inactivation of pectinesterase (PE) in a commercial enzyme formulation (CEF) under pulsed electric fields (PEF) was studied. Samples of an aqueous solution of the CEF were exposed to exponential waveform pulses for up to 16 ms at electric field intensities ranging from 5 to 24 kV cm^–1. Pulses were delivered in monopolar mode. The observed inactivation of the enzyme was described by several kinetics and regarding the input of electrical energy density (Q) models using Bayesian non-linear regression. Posterior distributions of the characteristic parameters for each kinetic model (based on the Hülsheger or the Weibull equation) and each Q model (based on exponential decay or the Weibull equation) were obtained. Kinetics models based on the Weibull equation showed better goodness and accuracy than the other models.