Published Article / The development of tolerance to low pH levels and the existence of cross-resistance may promote survival of bacteria in acidic foodstuff and in acidic environments such as the human stomach, in so doing escalating the probability of food poisoning. Similar to antimicrobial resistance developing, there is growing concern that effectiveness of organic acids may decrease as a result of the emergence of acid-tolerant food-borne pathogens. The objectives of this study were to determine the development of acid tolerance in selected food-borne pathogenic bacteria and to explore the activity of organic acids against acid tolerant pathogens. Bacterial strains were screened for acid-tolerance and susceptible strains were induced through exposure to increasing concentrations of an inorganic acid, as well as acidic foodstuffs. Susceptibility to six organic acids at various pH levels was assessed in order to evaluate the possible relationship between altered antimicrobial activity and acid tolerance. Salmonella enterica sv. Enteritidis ATCC 13076 and Escherichia coli ATCC 25922 were found to rapidly develop acid tolerance, while intrinsic acid tolerance was noted in Salmonella enterica sv. Typhimurium ATCC 14028. Pseudomonas aeruginosa ATCC 27853 demonstrated intermediate intrinsic acid tolerance. As expected, pH played a significant role in inhibitory activity of the organic acids as these compounds exhibit optimum antimicrobial activity at a lower pH (pH ≤5). It is, however, necessary to further elucidate the two-way role of pH in foodstuff concomitant to the addition of an organic acid.

Increasing consumer demand for new products with high nutritional qualities has spurred a search for new alternatives to food preservation. Pulsed electric field (PEF) is an emerging technology for non thermal food pasteurisation. Using this technology, enzymes, pathogenic and spoilage microorganisms can be inactivated without affecting the colour, flavour, and nutrients of the food. PEF treatment may be provided by applying pulsed electric field to a food product in a treatment zone between two electrodes at ambient , or slightly above ambient temperature. Exposure of microbial cells to the electric field induces a transmembrane potential in the cell membrane, which results in electroporation (the permeabilization of the membranes of cells and organelles) and/or electrofusion (the connection of two separate membranes into one) of the cells. An innovative pulsed electric field (PEF) unit was designed and constructed in the University of Auckland using modern IGBT technology. The system consists of main equipments, the high voltage pulse generator and the treatment chambers. The main focus of this work was to design an innovative PEF treatment chamber that provide uniform distribution of electric field, minimum increase in liquid temperature, minimum fouling of electrodes and an energy efficient system. Four multi pass treatment chambers were designed consisting of two stainless steel mesh electrodes in each chamber, with the treated fluid flowing through the openings of the mesh electrodes. The two electrodes are electrically isolated from each other by an insulator element designed to form a small orifice where most of the electric field is concentrated. Dielectric breakdown inside the chambers was prevented by removing the electrodes far from the narrow gap. The effect of the chambers different geometries on the PEF process in terms of electric parameters and microbial inactivation were investigated. Electric field intensity in the range of (17-43 kV/cm) was applied with square bipolar pulses of 1.7 µs duration. The effect of PEF treatment on the inactivation of gram-negative Escherichia coli ATCC 25922 suspended in simulated milk ultra-filtrate (SMUF) of 100%, 66.67% and 50% concentration was investigated. Treatments with the same electrical power input but higher electric field strengths provided larger degree of killing. The inactivation rate of E coli was significantly increased with increasing the electric field strength, treatment time and processing temperature. Morphological changes on E coli as a result of PEF treatment were studied under transmission electron microscopy (TEM). Significant morphological changes on E coli after PEF treatment were observed. The TEM studies suggested that the microbial inactivation was a consequence of electroporation and electrofusion mechanisms. Kinetic analysis of microbial inactivation due to PEF and thermal treatment of E coli suspended in SUMF were also studied. Comparison between measured (experimental) and predicted (theoretical) variation of E coli concentration with time following the PEF treatment was discussed, taking into consideration the recirculation mode of the PEF treatment. The treated liquid was circulated more than once through the treatment chamber to provide higher microbial inactivation. Arrhenius constants and activation energies of E coli inactivation using combined PEF and thermal treatment were calculated and generalized correlation for the inactivation rate constant as a function of electric field intensity and treatment temperature was developed. / Fonterra Research Institute (NZ) and the Foundation for Research Science and Technology (NZ)

Increasing consumer demand for new products with high nutritional qualities has spurred a search for new alternatives to food preservation. Pulsed electric field (PEF) is an emerging technology for non thermal food pasteurisation. Using this technology, enzymes, pathogenic and spoilage microorganisms can be inactivated without affecting the colour, flavour, and nutrients of the food. PEF treatment may be provided by applying pulsed electric field to a food product in a treatment zone between two electrodes at ambient , or slightly above ambient temperature. Exposure of microbial cells to the electric field induces a transmembrane potential in the cell membrane, which results in electroporation (the permeabilization of the membranes of cells and organelles) and/or electrofusion (the connection of two separate membranes into one) of the cells. An innovative pulsed electric field (PEF) unit was designed and constructed in the University of Auckland using modern IGBT technology. The system consists of main equipments, the high voltage pulse generator and the treatment chambers. The main focus of this work was to design an innovative PEF treatment chamber that provide uniform distribution of electric field, minimum increase in liquid temperature, minimum fouling of electrodes and an energy efficient system. Four multi pass treatment chambers were designed consisting of two stainless steel mesh electrodes in each chamber, with the treated fluid flowing through the openings of the mesh electrodes. The two electrodes are electrically isolated from each other by an insulator element designed to form a small orifice where most of the electric field is concentrated. Dielectric breakdown inside the chambers was prevented by removing the electrodes far from the narrow gap. The effect of the chambers different geometries on the PEF process in terms of electric parameters and microbial inactivation were investigated. Electric field intensity in the range of (17-43 kV/cm) was applied with square bipolar pulses of 1.7 µs duration. The effect of PEF treatment on the inactivation of gram-negative Escherichia coli ATCC 25922 suspended in simulated milk ultra-filtrate (SMUF) of 100%, 66.67% and 50% concentration was investigated. Treatments with the same electrical power input but higher electric field strengths provided larger degree of killing. The inactivation rate of E coli was significantly increased with increasing the electric field strength, treatment time and processing temperature. Morphological changes on E coli as a result of PEF treatment were studied under transmission electron microscopy (TEM). Significant morphological changes on E coli after PEF treatment were observed. The TEM studies suggested that the microbial inactivation was a consequence of electroporation and electrofusion mechanisms. Kinetic analysis of microbial inactivation due to PEF and thermal treatment of E coli suspended in SUMF were also studied. Comparison between measured (experimental) and predicted (theoretical) variation of E coli concentration with time following the PEF treatment was discussed, taking into consideration the recirculation mode of the PEF treatment. The treated liquid was circulated more than once through the treatment chamber to provide higher microbial inactivation. Arrhenius constants and activation energies of E coli inactivation using combined PEF and thermal treatment were calculated and generalized correlation for the inactivation rate constant as a function of electric field intensity and treatment temperature was developed. / Fonterra Research Institute (NZ) and the Foundation for Research Science and Technology (NZ)

Increasing consumer demand for new products with high nutritional qualities has spurred a search for new alternatives to food preservation. Pulsed electric field (PEF) is an emerging technology for non thermal food pasteurisation. Using this technology, enzymes, pathogenic and spoilage microorganisms can be inactivated without affecting the colour, flavour, and nutrients of the food. PEF treatment may be provided by applying pulsed electric field to a food product in a treatment zone between two electrodes at ambient , or slightly above ambient temperature. Exposure of microbial cells to the electric field induces a transmembrane potential in the cell membrane, which results in electroporation (the permeabilization of the membranes of cells and organelles) and/or electrofusion (the connection of two separate membranes into one) of the cells. An innovative pulsed electric field (PEF) unit was designed and constructed in the University of Auckland using modern IGBT technology. The system consists of main equipments, the high voltage pulse generator and the treatment chambers. The main focus of this work was to design an innovative PEF treatment chamber that provide uniform distribution of electric field, minimum increase in liquid temperature, minimum fouling of electrodes and an energy efficient system. Four multi pass treatment chambers were designed consisting of two stainless steel mesh electrodes in each chamber, with the treated fluid flowing through the openings of the mesh electrodes. The two electrodes are electrically isolated from each other by an insulator element designed to form a small orifice where most of the electric field is concentrated. Dielectric breakdown inside the chambers was prevented by removing the electrodes far from the narrow gap. The effect of the chambers different geometries on the PEF process in terms of electric parameters and microbial inactivation were investigated. Electric field intensity in the range of (17-43 kV/cm) was applied with square bipolar pulses of 1.7 µs duration. The effect of PEF treatment on the inactivation of gram-negative Escherichia coli ATCC 25922 suspended in simulated milk ultra-filtrate (SMUF) of 100%, 66.67% and 50% concentration was investigated. Treatments with the same electrical power input but higher electric field strengths provided larger degree of killing. The inactivation rate of E coli was significantly increased with increasing the electric field strength, treatment time and processing temperature. Morphological changes on E coli as a result of PEF treatment were studied under transmission electron microscopy (TEM). Significant morphological changes on E coli after PEF treatment were observed. The TEM studies suggested that the microbial inactivation was a consequence of electroporation and electrofusion mechanisms. Kinetic analysis of microbial inactivation due to PEF and thermal treatment of E coli suspended in SUMF were also studied. Comparison between measured (experimental) and predicted (theoretical) variation of E coli concentration with time following the PEF treatment was discussed, taking into consideration the recirculation mode of the PEF treatment. The treated liquid was circulated more than once through the treatment chamber to provide higher microbial inactivation. Arrhenius constants and activation energies of E coli inactivation using combined PEF and thermal treatment were calculated and generalized correlation for the inactivation rate constant as a function of electric field intensity and treatment temperature was developed. / Fonterra Research Institute (NZ) and the Foundation for Research Science and Technology (NZ)

Increasing consumer demand for new products with high nutritional qualities has spurred a search for new alternatives to food preservation. Pulsed electric field (PEF) is an emerging technology for non thermal food pasteurisation. Using this technology, enzymes, pathogenic and spoilage microorganisms can be inactivated without affecting the colour, flavour, and nutrients of the food. PEF treatment may be provided by applying pulsed electric field to a food product in a treatment zone between two electrodes at ambient , or slightly above ambient temperature. Exposure of microbial cells to the electric field induces a transmembrane potential in the cell membrane, which results in electroporation (the permeabilization of the membranes of cells and organelles) and/or electrofusion (the connection of two separate membranes into one) of the cells. An innovative pulsed electric field (PEF) unit was designed and constructed in the University of Auckland using modern IGBT technology. The system consists of main equipments, the high voltage pulse generator and the treatment chambers. The main focus of this work was to design an innovative PEF treatment chamber that provide uniform distribution of electric field, minimum increase in liquid temperature, minimum fouling of electrodes and an energy efficient system. Four multi pass treatment chambers were designed consisting of two stainless steel mesh electrodes in each chamber, with the treated fluid flowing through the openings of the mesh electrodes. The two electrodes are electrically isolated from each other by an insulator element designed to form a small orifice where most of the electric field is concentrated. Dielectric breakdown inside the chambers was prevented by removing the electrodes far from the narrow gap. The effect of the chambers different geometries on the PEF process in terms of electric parameters and microbial inactivation were investigated. Electric field intensity in the range of (17-43 kV/cm) was applied with square bipolar pulses of 1.7 µs duration. The effect of PEF treatment on the inactivation of gram-negative Escherichia coli ATCC 25922 suspended in simulated milk ultra-filtrate (SMUF) of 100%, 66.67% and 50% concentration was investigated. Treatments with the same electrical power input but higher electric field strengths provided larger degree of killing. The inactivation rate of E coli was significantly increased with increasing the electric field strength, treatment time and processing temperature. Morphological changes on E coli as a result of PEF treatment were studied under transmission electron microscopy (TEM). Significant morphological changes on E coli after PEF treatment were observed. The TEM studies suggested that the microbial inactivation was a consequence of electroporation and electrofusion mechanisms. Kinetic analysis of microbial inactivation due to PEF and thermal treatment of E coli suspended in SUMF were also studied. Comparison between measured (experimental) and predicted (theoretical) variation of E coli concentration with time following the PEF treatment was discussed, taking into consideration the recirculation mode of the PEF treatment. The treated liquid was circulated more than once through the treatment chamber to provide higher microbial inactivation. Arrhenius constants and activation energies of E coli inactivation using combined PEF and thermal treatment were calculated and generalized correlation for the inactivation rate constant as a function of electric field intensity and treatment temperature was developed. / Fonterra Research Institute (NZ) and the Foundation for Research Science and Technology (NZ)

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Este trabalho teve como objetivo estudar a utilizaÃÃo de glicerol oriundo da indÃstria do biodiesel (glicerol bruto) como substrato em ensaios fermentativos para a produÃÃo de etanol. Os ensaios foram realizados em mesa agitadora com velocidade de 200 rpm, nas temperaturas de 30 e 37 ÂC, respectivamente para os microrganismos Saccharomyces sp. 1238 e Escherichia coli 224 ATCC 25922. Nos experimentos realizados com a levedura Saccharomyces sp., variaram-se as concentraÃÃes de glicerol adicionado ao meio fermentativo em 0,5; 1,0 e 5,0 % v/v e fixou-se o volume de inÃculo em 1 % m/v. Observou-se que o microrganismo Saccharomyces sp. nÃo utilizou glicerol como fonte de carbono para produÃÃo de etanol, porÃm em ensaios teste com glicose P.A., observou-se que este substrato foi rapidamente consumido pelo microrganismo apresentando uma produÃÃo de etanol de 5,5 g/L. Nas fermentaÃÃes com a bactÃria Escherichia coli, variou-se a concentraÃÃo de glicerol adicionado ao meio fermentativo em: 1, 10, 15 e 20 g/L. Pela avaliaÃÃo da influÃncia da concentraÃÃo de substrato no meio atravÃs dos resultados obtidos, pode-se concluir que a melhor condiÃÃo para a produÃÃo de etanol com esse microrganismo foi a concentraÃÃo inicial de 10 g/L de glicerol. O consumo de glicerol pela bactÃria Escherichia coli foi afetado pela variaÃÃo deste substrato. Observou-se que o etanol foi produzido a partir de 8 h de cultivo nas fermentaÃÃes tanto com glicerol bruto quanto P.A. nas concentraÃÃes de 10, 15 e 20 g/L adicionado ao meio de cultivo. Observou-se tambÃm a formaÃÃo de Ãcido acÃtico nas primeiras horas da fermentaÃÃo. A produÃÃo de Ãcido acÃtico foi baixa, atingindo a concentraÃÃo de 0,15 g/L na fermentaÃÃo utilizando 10 g/L de glicerol bruto. Analisando os dois microrganismos estudados, verificou-se que apenas a bactÃria Escherichia coli 224 ATCC 25922 mostrou-se adequada ao objetivo desta pesquisa, jà que com a levedura nÃo foi produzido etanol em quantidade significativa. / The aim of this work was to investigate the use of glycerol from biodiesel industry (raw glycerol) as a substrate in fermentation assays for production of ethanol. The assays were performed in shaker with agitation of 200 rpm, at temperatures of 30 and 37 ÂC, respectively for the microorganisms Saccharomyces sp. 1238 and 224 Escherichia coli ATCC 25922. In experiments with yeast Saccharomyces sp., it was varied concentrations of glycerol from fermentation medium at 0.5, 1.0 and 5.0 % v/v and the inoculum was set to 1% w/v. It was observed that the microorganism Saccharomyces sp. could not use glycerol as carbon source for ethanol production, but in assays using glucose, this substrate was rapidly consumed by the microorganism achieving an ethanol production of 5.5 g/L. It was varied the concentration of glycerol added to the fermentation medium: 1, 10, 15 and 20 g/L when Escherichia coli was used. By analyzing the influence of substrate concentration in fermentations, it can be concluded that the best condition for ethanol production, with this microorganism, was initial concentration of glycerol of 10 g/L. The consumption of glycerol by Escherichia coli was affected by the change of substrate concentration. It was observed that ethanol was produced after 8 h of fermentation with both raw and PA glycerol at 10, 15 and 20 g/L. It also observed the formation of acetic acid in the first hours of fermentation. The production of acetic acid was low, reaching a concentration of 0.15 g/L in fermentation using 10 g/L of raw glycerol. Analyzing the two microorganisms studied, it was found that only 224 bacteria Escherichia coli ATCC 25922 was adequate to the aim of this research, since the yeast was not produced ethanol in significant amounts.