Tesis por compendio / [ES] Generalmente, se utilizan tratamientos térmicos para la esterilización de emulsiones. Sin embargo, el calentamiento ha demostrado inducir la hidrólisis de lípidos y lecitina. En este sentido, las tecnologías no térmicas están surgiendo en la industria para alcanzar la estabilidad microbiana evitando la pérdida de calidad relacionada con el calor. El CO2 supercrítico (SC-CO2) y los campos eléctricos pulsados (PEF) son tecnologías no térmicas para la inactivación microbiana. Sin embargo, estas técnicas en ocasiones requieren altas intensidades o tiempos de tratamiento largos para garantizar la seguridad del producto. La literatura ha demostrado la capacidad de los ultrasonidos de alta potencia (HPU) para intensificar fenómenos de transferencia de masa y calor. Por lo tanto, su aplicación a tecnologías no térmicas podría ser un enfoque interesante para mejorar la efectividad de la inactivación microbiana. En este contexto, el objetivo fue evaluar el efecto de los tratamientos SC-CO2, PEF y HPU, aplicados de forma individual y combinada, sobre la inactivación de diferentes microorganismos en emulsiones. Para ello, por un lado, se estudió el efecto de la aplicación de HPU a los tratamientos SC-CO2 sobre diferentes tipos de microorganismos y sobre medios con diferente contenido en aceite. Por otro lado, se evaluó el efecto de los tratamientos PEF y HPU individuales y combinados sobre diferentes microorganismos Los resultados mostraron que, en general, la aplicación de HPU intensificó la capacidad de inactivación de SC-CO2. Los HPU probablemente facilitaron la solubilidad del CO2 en el medio y provocaron daños en las células. En este sentido, el análisis microscópico de las células inactivadas reveló importantes cambios morfológicos, incluyendo paredes celulares dañadas y pérdida del contenido citoplasmático. En cambio, los HPU no mejoraron la inactivación de SC-CO2 de las esporas de A. niger en emulsión. El aumento de la presión llevó a una mayor inactivación, a excepción de E. coli en agua, donde no se encontró efecto de la presión. Sin embargo, las presiones por encima de 350 bar no parecen ejercer ninguna inactivación adicional. El aumento de temperatura tuvo un efecto significativo para todos los tratamientos y microorganismos. En cuanto al efecto del medio, se sabe que la presencia de aceite protege a los microorganismos, como se observó en la inactivación de bacterias SC-CO2 en agua y en emulsiones con diferente contenido en aceite. Sin embargo, la aplicación de HPU enmascaró el efecto protector que ejerce el aceite en las emulsiones. En cambio, para las esporas de A. niger no se encontró efecto del medio sobre la efectividad de los tratamientos. En relación al efecto de los tratamientos de SC-CO2 + HPU sobre la calidad de las emulsiones, se encontró un efecto leve de las condiciones del proceso y mediante la selección de condiciones adecuadas de SC-CO2 + HPU, se pudieron obtener cambios mínimos en la calidad de las emulsiones y una inactivación satisfactoria de todos los microorganismos, excepto para las esporas de G. stearothermophilus. Con respecto a los tratamientos de PEF y HPU, no se logró la inactivación completa de las emulsiones con los tratamientos individuales. Sin embargo, cuando el PEF (152,3-176,3 kJ / kg) fue seguido de HPU (3 min), se obtuvieron niveles de inactivación de 8,2, 6,6 y 1,0 ciclos-log para E. coli, A. niger y B. pumilus. Además, la inactivación lograda por el tratamiento con PEF-HPU fue mayor que la de la suma de los tratamientos individuales para todos los microorganismos. Por el contrario, la inactivación lograda por el tratamiento HPU-PEF fue menor que la de la suma de los tratamientos individuales. Por lo tanto, la secuencia más eficaz fue aquella en la que el PEF fue seguido de los HPU. Se puede concluir que, la combinación de HPU con SC-CO2 o PEF generalmente mejoró la inactivación microbiana. En consecuencia, se podrían utili / [CA] Generalment, s'utilitzen tractaments tèrmics per a l'esterilització d'emulsions. No obstant això, el calfament ha demostrat induir la hidròlisi de lípids i lecitina. En aquest sentit, les tecnologies no tèrmiques estan sorgint en la indústria per a aconseguir l'estabilitat microbiana evitant la pèrdua de qualitat relacionada amb la calor. El CO¿ supercrític (SC-CO¿) i els camps elèctrics premuts (PEF) són tecnologies no tèrmiques per a la inactivació microbiana. No obstant això, aquestes tècniques a vegades requereixen altes intensitats o temps de tractament llargs per a garantir la seguretat del producte. La literatura ha demostrat la capacitat dels ultrasons d'alta potència (HPU) per a intensificar fenòmens de transferència de massa i calor. Per tant, la seua aplicació a tecnologies no tèrmiques podria ser un enfocament interessant per a millorar l'efectivitat de la inactivació microbiana. En aquest context, l'objectiu va ser avaluar l'efecte dels tractaments SC-CO¿, PEF i HPU, aplicats de manera individual i combinada, sobre la inactivació de diferents microorganismes en emulsions. Per a això, d'una banda, es va estudiar l'efecte de l'aplicació de HPU als tractaments SC-CO¿ sobre diferents tipus de microorganismes i sobre mitjans amb diferent contingut en oli. D'altra banda, es va avaluar l'efecte dels tractaments PEF i HPU individuals i combinats sobre diferents microorganismes Els resultats van mostrar que, en general, l'aplicació de HPU va intensificar la capacitat d'inactivació de SC-CO2. Els HPU probablement van facilitar la solubilitat del CO¿ en el mitjà i van provocar danys en les cèl·lules. En aquest sentit, l'anàlisi microscòpica de les cèl·lules inactivades va revelar importants canvis morfològics, incloent parets cel·lulars danyades i pèrdua del contingut citoplasmàtic. En canvi, els HPU no van millorar la inactivació de SC-CO2 de les espores de A. niger en emulsió. L'augment de la pressió va portar a una major inactivació, a excepció d'E. coli en aigua, on no es va trobar efecte de la pressió. No obstant això, les pressions per damunt de 350 bar no semblen exercir cap inactivació addicional. L'augment de temperatura va tindre un efecte significatiu per a tots els tractaments i microorganismes. Quant a l'efecte del medi, se sap que la presència d'oli protegeix els microorganismes, com es va observar en la inactivació de bacteris SC-CO¿ en aigua i en emulsions amb diferent contingut en oli. No obstant això, l'aplicació de HPU va emmascarar l'efecte protector que exerceix l'oli en les emulsions. En canvi, per a les espores de A. niger no es va trobar efecte del medi sobre l'efectivitat dels tractaments. En relació a aquest efecte dels tractaments de SC-CO2 + HPU sobre la qualitat de les emulsions, es va trobar un efecte lleu de les condicions del procés i mitjançant la selecció de condicions adequades de SC-CO2 + HPU, es van poder obtindre canvis mínims en la qualitat de les emulsions i una inactivació satisfactòria de tots els microorganismes, excepte per a les espores de G. stearothermophilus. Respecte als tractaments de PEF i HPU, no es va aconseguir la inactivació completa de les emulsions amb els tractaments individuals. No obstant això, quan el PEF (152,3-176,3 kJ / kg) va ser seguit de HPU (3 min), es van obtindre nivells d'inactivació de 8,2, 6,6 i 1,0 cicles- log per a E. coli, A. niger i B. pumilus. A més, la inactivació reeixida pel tractament amb PEF- HPU va ser major que la de la suma dels tractaments individuals per a tots els microorganismes. Per contra, la inactivació reeixida pel tractament HPU- PEF va ser menor que la de la suma dels tractaments individuals. Per tant, la seqüència més eficaç va ser aquella en la qual el PEF va ser seguit dels HPU. Es pot concloure que, la combinació de HPU amb SC-CO¿ o PEF generalment va millorar la inactivació microbiana. En conseqüència, es podrien utilitzar temps de / [EN] Thermal treatments are generally used for the sterilization of emulsions. However, heating has demonstrated its ability to induce the hydrolysis of lipids and lecithin. In this sense, non-thermal technologies are emerging in the industry with the aim of achieving microbial stability while avoiding the loss of quality related to heat. Supercritical carbon dioxide (SC-CO2) and pulsed electric fields (PEF) are non-thermal technologies for microbial inactivation. However, these techniques have demonstrated to require high treatment intensities or long treatment times to guarantee the product's safety. Therefore, there is still room for the improvement in the use of these technologies. Literature has illustrated the capacity of high power ultrasound (HPU) for the intensification of mass and/or heat transfer phenomena. Therefore, its application to non-thermal technologies could be an interesting approach to enhance the microbial inactivation effectiveness. In this context, the objective was to evaluate the effect of SC-CO2, PEF and HPU treatments, applied in individual and combined form, on the inactivation of different microorganisms in emulsions. In order to achieve this goal, on the one hand, the influence of the implementation of HPU to the SC-CO2 treatments was studied on different types of microorganisms and on media with different oil content. On the other hand, the effect of the individual and combined PEF and HPU treatments was assessed on different microorganisms. Results showed that, generally, the application of HPU intensified the inactivation capacity of SC-CO2. HPU probably enhanced the solubilization of CO2 into the medium and provoked damages in the cells. In this regard, the microscopy analysis of the inactivated cells revealed important morphological changes, including damaged cell walls and an important loss of the cytoplasmic content. Nevertheless, HPU did not improved the SC-CO2 inactivation of A. niger spores in emulsion. The increase of the pressure led to a higher inactivation, except for E. coli in water, where no effect of pressure was found. However, pressures above 350 bar did not seem to exert any additional inactivation. The increase of the temperature had a significant effect for all treatments and microorganisms. Regarding the effect of the medium, the presence of oil is known to protect microorganisms, as was observed in the SC-CO2 inactivation of bacteria in water and in emulsions with different oil content. However, the application of HPU masked the protective effect exerted by the oil in the emulsions. On the contrary, for A. niger spores no effect of the media was found on the effectiveness of the treatments In relation to the effect of the SC-CO2 + HPU treatments on the quality of the treated emulsions, only a mild effect of the process conditions was found and by the selection of suitable SC-CO2 + HPU conditions, minimal changes on the quality of the emulsions and a satisfactory inactivation for all the microorganisms, except for G. stearothermophilus spores, can be obtained. Regarding PEF and HPU treatments, the complete inactivation in the emulsions was not achieved with the individual treatments. However, when PEF (152.3-176.3 kJ/kg) was followed by HPU (3 min), inactivation levels of 8.2, 6.6 and 1.0 log-cycles were obtained for E. coli, A. niger and B. pumilus, respectively. Moreover, the inactivation achieved by the PEF-HPU treatment was higher than the sum of the individual treatments for all microorganisms. On the contrary, the inactivation achieved by HPU-PEF treatment was lower than that of the sum of the individual treatments. Thus, the most effective sequence for the combined treatment was the one in which PEF was followed by HPU. It can be concluded that, the combination of HPU with SC-CO2 or PEF generally improved the microbial inactivation. Consequently, reasonable processing times and mild process conditions could be used. / Gómez Gómez, Á. (2021). Pasteurization of Lipid Emulsions with Supercritical CO2 and High Power Ultrasound [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/175486 / TESIS / Compendio

No description available.

<p dir="ltr">Mass spectrometry has been at the forefront of complex mixture analysis and, as a result, has greatly advanced the understanding of biological systems with its application in the biological sciences. One area in which mass spectrometry has succeeded is the area of microbiology and the identification of pathogens and has gained much attention from the biothreat detection community. Although this technology has matured in the past decade, very few systems have been developed for point-of-need analysis in cases such as the detection of biothreats. Current MS systems for the analysis of microbes utilizing MALDI-TOF-MS require large instruments to accommodate a drift tube long enough for high resolution mass analysis and high vacuum which is not amenable to the miniaturization requirements of point-of-need analysis. The previously mentioned methods also require extensive manipulation of the sample which takes time and can pose a risk to instrument operators in the biothreat detection space. Additionally, most mass spectrochemical instruments provide only one-dimension of data which can limits classification accuracy when using classification algorithms to provide an identity on a microbiological sample which could consist of any of the numerous common bacterial pathogens or biothreats.</p><p dir="ltr">A possible solution to this problem is the implementation of two-dimensional tandem mass spectrometry (2D MS/MS) which allows the analysis of the product ions of all precursor ions representing the result in the 2D MS/MS data domain. This methodology is possible with a linear quadrupolar ion trap mass analyzer and can be applied to miniature ion trap technology for portability. In this dissertation, a progression of mass spectrochemical analysis of biological systems from conventional methods to the implementation of 2D MS/MS is demonstrated: by (i) the development of a rapid biomolecule extraction method to analyze bacterial spores, using a (ii) modified linear quadrupolar ion trap mass spectrometer, (iii) then a miniature ion trap mass spectrometer, and (iv) finally adding numerical methods to discriminate between biological systems using data acquired on each 2D MS/MS instrument. This work is then taken a step further by developing a high throughput experimentation method in which DESI is coupled to 2D MS/MS to analyze a moderate number of samples rapidly, automatically, and with high reproducibility.</p>

No description available.

No description available.

<p dir="ltr">Liquid hydrogen peroxide (LHP) and vapor hydrogen peroxide (VHP) efficacy as a sterilant for <i>Bacillus atrophaeus</i> and <i>Geobacillus stearothermophilus</i> spores in aseptic packaging systems under commercial sterilization conditions were evaluated in this research. The work centers on quantifying and modeling the kinetic parameters that impact peroxide sterilization efficacy, including the D and z values, that relate to the change in concentration required for a 1-log reduction in spore population and a novel Z_conc parameter This comprehensive study is divided into four key investigations, each contributing critical insights to the overall understanding of peroxide sterilization processes.</p><p dir="ltr">The first study examined the inactivation kinetics of <i>B. atrophaeus</i> spores in liquid hydrogen peroxide. By evaluating different concentrations (20%, 28%, and 33%) and temperatures (up to 82.2°C), the study revealed that higher concentrations and elevated temperatures significantly enhanced spore inactivation. The Weibull model provided a more accurate fit for the data, indicating a non-linear relationship between spore reduction and exposure time.</p><p dir="ltr">The second part of the research explored the use of VHP for sterilizing <i>B. atrophaeus</i> spores. With VHP concentrations of 2500 ppm and 4450 ppm at various temperatures, the study demonstrated that higher concentrations and temperatures are highly effective for spore inactivation. Both log-linear and Weibull models accurately described the inactivation kinetics, with the Weibull model showing a slightly better fit, emphasizing the potential of VHP in achieving commercial sterility.</p><p dir="ltr">The third investigation focused on developing predictive models for VHP concentration and its efficacy in spore inactivation. The study evaluated VHP concentrations ranging from 2.32 mg/L to 7.35 mg/L and their impact on spore inactivation rates. The Weibull model proved particularly effective in predicting the inactivation of <i>G. stearothermophilus</i> and <i>B. atrophaeus</i> spores, offering a robust tool for optimizing sterilization protocols in aseptic packaging.</p><p dir="ltr">The fourth and final study of the research investigated the influence of surface roughness on spore survival during VHP sterilization cycles on plastic packaging materials. Artificial roughness on high-density polyethylene (HDPE) coupons was created using sandpaper with different grits. <i>B. atrophaeus</i> spores were applied to both roughened and smooth HDPE surfaces. The study finds that rougher surfaces provide more shelter for spores, reducing sterilization efficacy. For example, surfaces roughened with P-36 grit showed a 2.75 log reduction in spore count, whereas smoother surfaces with P-220 grit achieved a 4.42 log reduction. Contact angle measurements indicated that increased roughness led to more hydrophilic surfaces, with water contact angles decreasing from 149.7° for the pristine sample to 105.4° for the P-36 sample. Scanning electron microscopy (SEM) images confirmed that spores were more likely to reside in the valleys of rough surfaces, highlighting the importance of surface characteristics in optimizing VHP sterilization protocols.</p><p dir="ltr">The findings of this dissertation underscore the significant impact of hydrogen peroxide concentration, application conditions, and packaging material surface properties on the efficacy of spore inactivation during sterilization. By providing a comprehensive understanding of these factors, the research contributes to the development of optimized aseptic sterilization protocols, enhancing the reliability and safety of aseptically packaged food and pharmaceutical products. This work will ensure compliance with regulatory standards and improve food safety in commercial manufacturing, laying a solid foundation for future research and practical applications in VHP sterilization technology.</p>

Le recours au plasma pour stériliser des dispositifs médicaux (DM) est un domaine de recherche ne datant véritablement que de la fin des années 1990. Les plasmas permettent, dans les conditions adéquates, de réaliser la stérilisation à basse température (≤ 65°C), tel qu’exigé par la présence de polymères dans les DM et ce contrairement aux procédés par chaleur, et aussi de façon non toxique, contrairement aux procédés chimiques comme, par exemple, l’oxyde d’éthylène (OEt). Les laboratoires du Groupe de physique des plasmas à l’Université de Montréal travaillent à l’élaboration d’un stérilisateur consistant plus particulièrement à employer les effluents d’une décharge N2-%O2 basse pression (2-8 Torrs) en flux, formant ce que l’on appelle une post-décharge en flux. Ce sont les atomes N et O de cette décharge qui viendront, dans les conditions appropriées, entrer en collisions dans la chambre de stérilisation pour y créer des molécules excitées NO*, engendrant ainsi l’émission d’une quantité appréciable de photons UV. Ceux-ci constituent, dans le cas présent, l’agent biocide qui va s’attaquer directement au bagage génétique du micro-organisme (bactéries, virus) que l’on souhaite inactiver. L’utilisation d’une lointaine post-décharge évite du même coup la présence des agents érosifs de la décharge, comme les ions et les métastables. L’un des problèmes de cette méthode de stérilisation est la réduction du nombre de molécules NO* créées par suite de la perte des atomes N et O, qui sont des radicaux connus pour interagir avec les surfaces, sur les parois des matériaux des DM que l’on souhaite stériliser. L’objectif principal de notre travail est de déterminer l’influence d’une telle perte en surface, dite aussi réassociation en surface, par l’introduction de matériaux comme le Téflon, l’acier inoxydable, l’aluminium et le cuivre sur le taux d’inactivation des spores bactériennes. Nous nous attendons à ce que la réassociation en surface de ces atomes occasionne ainsi une diminution de l’intensité UV et subséquemment, une réduction du taux d’inactivation. Par spectroscopie optique d’émission (SOE), nous avons déterminé les concentrations perdues de N et de O par la présence des matériaux dans le stérilisateur, ainsi que la diminution de l’émission UV en découlant. Nous avons observé que cette diminution des concentrations atomiques est d’autant plus importante que les surfaces sont catalytiques. Au cours de l’étude du phénomène de pertes sur les parois pour un mélange N2-%O2 nous avons constaté l’existence d’une compétition en surface entre les atomes N et O, dans laquelle les atomes d’oxygènes semblent dominer largement. Cela implique qu’au-delà d’un certain %O2 ajouté à la décharge N2, seuls les atomes O se réassocient en surface. Par ailleurs, l’analyse des courbes de survie bi-phasiques des micro-organismes a permis d’établir une étroite corrélation, par lien de cause à effet, entre la consommation des atomes N et O en surface et la diminution du taux d’inactivation des spores dans la première phase. En revanche, nous avons constaté que notre principal agent biocide (le rayonnement ultraviolet) est moins efficace dans la deuxième phase et, par conséquent, il n’a pas été possible d’établir un lien entre la diminution des concentrations et le taux d’inactivation de cette phase-là. / The use of plasmas to sterilize medical devices (MDs) is a research field, which really started only at the end of the 90’s. Plasmas under adequate conditions allow achieving low-temperature (≤ 65°C) sterilization, as required by MDs made from polymers, in contrast to heat-driven sterilization methods, and provide a non-toxic method, in contrast to chemical processes such as performed, for example, with ethylene oxide (EtO). The Groupe de physique des plasmas laboratories at Université de Montréal is working on the design and testing of a sterilizer, which has the peculiarity of utilizing the species outflowing from a N2-%O2 discharge at reduced pressure (2-8 Torrs), which is called a plasma flowing-afterglow. It is the N and O atoms of this discharge mixture that, under appropriate conditions, interact in the sterilization chamber to form NO* excited molecules, generating a significant level of UV photons. These are, in the present case, the actual biocidal agent which will create lethal lesions on the genetic material of the microorganisms (bacteria, viruses) that should be inactivated. Using a flowing late afterglow instead of the discharge itself enables us to avoid the presence of the erosive agents of the discharge (ions, metastable-state particles). A major problem of this sterilization method is the reduction in the concentration of NO* molecules resulting from the losses of the N and O atoms on the surfaces of the MD materials that we want to sterilize. These radicals are, in fact, well-known to interact with surfaces and recombine on them. The main aim of our work is to determine the loss level of such atoms following their surface recombination on materials such as Teflon, stainless steel, aluminum and copper and the corresponding influence of such losses on the inactivation rate of bacterial spores. We can expect that surface recombination of these atoms leads to a reduction in the UV emission intensity and, as a result, in a reduction in the inactivation rate. Using optical emission spectroscopy (OES), we have determined the loss of N and O concentrations resulting from the presence of various materials in the sterilizer chamber as well as the corresponding decrease in UV emission intensity. We have observed that this reduction in atomic concentrations increases with the catalytic properties (recombination coefficient) of these materials. While examining the surface recombination phenomenon on these various materials, we have noticed a competition between the surface recombination of N and O atoms where the latter appear to play the main role. This implies that above a certain percentage of O2 added to N2, only the O atoms do recombine on these surfaces. On the other hand, the analysis of the bi-phasic survivor curves has enable us to show a strong correlation between the consumption of N and O atoms on surfaces and the reduction in the inactivation rate coefficient in the first phase of the survivor curve. We have also observed that our main biocidal agent is less efficient in the second phase of the survivor curve and, as a result, it was not possible to make a connection between the reduction in N and O atom concentration and the inactivation rate of the second phase.

Le recours au plasma pour stériliser des dispositifs médicaux (DM) est un domaine de recherche ne datant véritablement que de la fin des années 1990. Les plasmas permettent, dans les conditions adéquates, de réaliser la stérilisation à basse température (≤ 65°C), tel qu’exigé par la présence de polymères dans les DM et ce contrairement aux procédés par chaleur, et aussi de façon non toxique, contrairement aux procédés chimiques comme, par exemple, l’oxyde d’éthylène (OEt). Les laboratoires du Groupe de physique des plasmas à l’Université de Montréal travaillent à l’élaboration d’un stérilisateur consistant plus particulièrement à employer les effluents d’une décharge N2-%O2 basse pression (2-8 Torrs) en flux, formant ce que l’on appelle une post-décharge en flux. Ce sont les atomes N et O de cette décharge qui viendront, dans les conditions appropriées, entrer en collisions dans la chambre de stérilisation pour y créer des molécules excitées NO*, engendrant ainsi l’émission d’une quantité appréciable de photons UV. Ceux-ci constituent, dans le cas présent, l’agent biocide qui va s’attaquer directement au bagage génétique du micro-organisme (bactéries, virus) que l’on souhaite inactiver. L’utilisation d’une lointaine post-décharge évite du même coup la présence des agents érosifs de la décharge, comme les ions et les métastables. L’un des problèmes de cette méthode de stérilisation est la réduction du nombre de molécules NO* créées par suite de la perte des atomes N et O, qui sont des radicaux connus pour interagir avec les surfaces, sur les parois des matériaux des DM que l’on souhaite stériliser. L’objectif principal de notre travail est de déterminer l’influence d’une telle perte en surface, dite aussi réassociation en surface, par l’introduction de matériaux comme le Téflon, l’acier inoxydable, l’aluminium et le cuivre sur le taux d’inactivation des spores bactériennes. Nous nous attendons à ce que la réassociation en surface de ces atomes occasionne ainsi une diminution de l’intensité UV et subséquemment, une réduction du taux d’inactivation. Par spectroscopie optique d’émission (SOE), nous avons déterminé les concentrations perdues de N et de O par la présence des matériaux dans le stérilisateur, ainsi que la diminution de l’émission UV en découlant. Nous avons observé que cette diminution des concentrations atomiques est d’autant plus importante que les surfaces sont catalytiques. Au cours de l’étude du phénomène de pertes sur les parois pour un mélange N2-%O2 nous avons constaté l’existence d’une compétition en surface entre les atomes N et O, dans laquelle les atomes d’oxygènes semblent dominer largement. Cela implique qu’au-delà d’un certain %O2 ajouté à la décharge N2, seuls les atomes O se réassocient en surface. Par ailleurs, l’analyse des courbes de survie bi-phasiques des micro-organismes a permis d’établir une étroite corrélation, par lien de cause à effet, entre la consommation des atomes N et O en surface et la diminution du taux d’inactivation des spores dans la première phase. En revanche, nous avons constaté que notre principal agent biocide (le rayonnement ultraviolet) est moins efficace dans la deuxième phase et, par conséquent, il n’a pas été possible d’établir un lien entre la diminution des concentrations et le taux d’inactivation de cette phase-là. / The use of plasmas to sterilize medical devices (MDs) is a research field, which really started only at the end of the 90’s. Plasmas under adequate conditions allow achieving low-temperature (≤ 65°C) sterilization, as required by MDs made from polymers, in contrast to heat-driven sterilization methods, and provide a non-toxic method, in contrast to chemical processes such as performed, for example, with ethylene oxide (EtO). The Groupe de physique des plasmas laboratories at Université de Montréal is working on the design and testing of a sterilizer, which has the peculiarity of utilizing the species outflowing from a N2-%O2 discharge at reduced pressure (2-8 Torrs), which is called a plasma flowing-afterglow. It is the N and O atoms of this discharge mixture that, under appropriate conditions, interact in the sterilization chamber to form NO* excited molecules, generating a significant level of UV photons. These are, in the present case, the actual biocidal agent which will create lethal lesions on the genetic material of the microorganisms (bacteria, viruses) that should be inactivated. Using a flowing late afterglow instead of the discharge itself enables us to avoid the presence of the erosive agents of the discharge (ions, metastable-state particles). A major problem of this sterilization method is the reduction in the concentration of NO* molecules resulting from the losses of the N and O atoms on the surfaces of the MD materials that we want to sterilize. These radicals are, in fact, well-known to interact with surfaces and recombine on them. The main aim of our work is to determine the loss level of such atoms following their surface recombination on materials such as Teflon, stainless steel, aluminum and copper and the corresponding influence of such losses on the inactivation rate of bacterial spores. We can expect that surface recombination of these atoms leads to a reduction in the UV emission intensity and, as a result, in a reduction in the inactivation rate. Using optical emission spectroscopy (OES), we have determined the loss of N and O concentrations resulting from the presence of various materials in the sterilizer chamber as well as the corresponding decrease in UV emission intensity. We have observed that this reduction in atomic concentrations increases with the catalytic properties (recombination coefficient) of these materials. While examining the surface recombination phenomenon on these various materials, we have noticed a competition between the surface recombination of N and O atoms where the latter appear to play the main role. This implies that above a certain percentage of O2 added to N2, only the O atoms do recombine on these surfaces. On the other hand, the analysis of the bi-phasic survivor curves has enable us to show a strong correlation between the consumption of N and O atoms on surfaces and the reduction in the inactivation rate coefficient in the first phase of the survivor curve. We have also observed that our main biocidal agent is less efficient in the second phase of the survivor curve and, as a result, it was not possible to make a connection between the reduction in N and O atom concentration and the inactivation rate of the second phase.

O Brasil é um dos maiores produtores mundiais de suco de laranja, da mesma forma que a produção é elevada, a geração de resíduos também é significativa. Sabe-se que estes resíduos, os quais incluem sementes, cascas e restos de polpa são ricos em nutrientes que poderiam ser utilizados como substrato por micro-organismos, seja para o crescimento ou para a obtenção de subprodutos. Esporos de Bacillus atrophaeus ATCC 9372 são utilizados como indicadores biológicos, IBs, em processos térmicos por formarem esporos termorresistentes. O objetivo deste trabalho foi avaliar o uso de resíduos do processamento de suco de laranja como um meio de cultura alternativo para obtenção de esporos de B. atrophaeus, para serem aplicados em processos industriais. Ao bagaço de laranja (de 1,0 g a 20,0 g), obtido por processamento em centrífuga de frutas, foram adicionados 100 mL de água, e incubados a 150 rpm / 37 ºC por até 6 dias. Evidenciada a viabilidade de crescimento celular (&#181;máx = 0,0238 h-1 e Px = 0,0787 g/L.h, para 5,0 g de bagaço) procedeu-se ao estudo de planejamento experimental fatorial 22 em formato estrela com 6 pontos centrais, considerando a concentração de bagaço e o volume de meio. Foram determinados os valores de pH, de biomassa, de esporos viáveis e a resistência térmica dos mesmos a 102 ºC. Observou-se que houve aumento nos valores de pH após o cultivo e que as maiores concentrações de esporos foram de 1,73 x 109 esporos /mL e 5,75 x 109 esporos /mL após 3 e 6 dias de cultivo e os tempos de redução decimal determinados variaram de D102C = 0,92 min a D102C = 2,71 min e de D102C = 1,34 min a D102C = 3,98 min após 3 e 6 dias de cultivo, respectivamente. Com base no planejamento proposto e a análise de regressão, o desenvolvimento de esporos em bagaço segue a relação: Esporos = {-1,15 + 0,0303* [bagaço (g)] - 0,00611* [volume (mL)] + 0,611* [tempo (dias)]}, p=0,000, R2 =0,452, sendo o tempo (p=0,000) o fator de maior influência na formação de esporos. Os meios preparados com bagaço de laranja apresentaram-se viáveis para a produção de esporos de B. atrophaeus termorresistentes, produto de interesse farmacêutico e industrial, agregando valor ao resíduo que seria descartado. / Brazil is one of the world´s largest producers of oranges juice, in the same way that the production is high the amount of generated waste is also significant. It is well known that these residues, which include seeds, peel and pulp, are rich in nutrients that could be used as substrate by microorganisms whether for growth or for obtaining by-products. Bacillus atrophaeus ATCC 9372 spores are used as biological indicators, BIs, in thermal processes due to their ability to form heat-resistant spores. This study aimed to evaluate the use of orange juice processing waste as an alternative culture media to obtain B. atrophaeus spores, to be applied in industrial processes. To orange\'s bagasse (from 1.0 g to 20.0 g), obtained by processing in a fruit\'s centrifuge, 100 mL of water was added, and sterilized at 121 ºC. An aliquot of 0.1g/L of Bacillus atrophaeus spores was inoculated to bagasses\'s media and incubated at 150 rpm / 37 ºC up to 6 days. As cells (&#181;máx = 0.0238 h-1 and Px = 0.0787 g/L.h, for 5.0 g of bagasse) were obtained, a factorial experimental design 22, with star-shaped model and 6 central points, was performed considering the bagasse concentration and the media volume used. Values of pH, biomass, viable spores and their thermal resistance at 102 ºC were determined. It was observed that pH increased after cultivation and major values of spore concentration achieved were 1.73 x 109 spores /mL and 5.75 x 109 spores /mL after 3 and 6 days, respectively. Decimal reduction times determined ranged from D102C = 0.92 min to D102C = 2.71 min and from D102C = 1.34 min to D102C = 3.98 min after 3 and 6 days of incubation, correspondingly. The regression analysis showed that the development of spores in bagasse can be defined by the equation: Spores = , p=0.000, R2 =0.452 and time has a positive influence in the spore formation. Results demonstrated media prepared with oranges\' bagasse were capable to grow and to develop B. atrophaeus heat-resistant spores, being an alternative to add value to a waste that would be discarded, generating a product of great importance in the pharmaceutical field.

Leaky buildings are those that show elevated moisture contents of the framing timber, which can subsequently lead to the establishment of fungal and bacterial decay. Prior to this study, the causative agents of the decay in these leaky buildings were unknown, though it was suspected to be one or more species of decay fungi. Therefore, the overall goal of this multi-disciplinary PhD thesis research was to determine the causative agents of decay in leaky buildings of New Zealand in an effort to develop solutions for both their remediation and future prevention. Use of molecular biology methodology and classical mycological techniques based on morphology enabled identification of decay fungi from framing timber and air samples of leaky New Zealand buildings and provided insight into relative importance based on isolation frequency. In most cases, fungi colonising Pinus radiata D. Don were isolated to produce pure cultures. Mycelia from these cultures on agar media were collected to extract DNA. To identify the fungi to the species level, polymerase chain reaction (PCR) with fungal specific DNA primer pairs were performed followed by DNA sequencing of the internal transcribed spacer (ITS) region. Identification was by BLAST (Basic Local Alignment Search Tool) search on sequences in known GenBanks. In total, 421 samples from leaky buildings were processed, predominately untreated P. radiata decayed framing timber and also fibre cement boards and building paper. From these, sixty-eight fungal identifications were made. The only taxa that were isolated with significant frequency were identified as 4 basidiomycete species, as follows, along with the number of times they were isolated from the 421 samples: • Gloeophyllum sepiarium (Wulf.: Fr.) Karst. 13x • Oligoporus placenta (Fries 1865) Gilb. In Ryv.1985 11x • Antrodia sinuosa (Fr.) Karst. 8x • Gloeophyllum trabeum (Fr.) Murr. 4x Although these species were identified repeatedly, in total they represent less than 10% of the total samples and, therefore, it is concluded that the leaky building decay samples represent high fungal biodiversity. An aerial spore study of internal air, wall cavity air and exterior air of leaky buildings was carried out using a Merck MAS-100 instrument which collects spores directly onto selective media plates. Viable fungal aerial spores were detected at every sampling location tested at the leaky buildings, by the criteria of culturing, with a highest mean of 3714 colony-forming units (CFU) per cubic metre found in the cavities of water-damaged walls. This aerial spore study in conjunction with isolation from decayed wood samples from the same leaky buildings enabled identification of G. sepiarium and A. sinuosa at the same test site. The use of carboxymethylcellulose medium further demonstrated the presence of potential cellulose-degrading fungi within and around the location. Overall, the combination of direct sampling of timber and air sampling proved useful for detection of fungal species variability at a multi-unit building. Four decay fungi isolated from New Zealand leaky buildings and two standard control decay fungi (Coniophora puteana and Serpula lacrymans) were submitted to laboratory wood block testing to determine the effectiveness of currently used wood framing preservatives under laboratory conditions before and after a standard leaching regime. P. radiata blocks were treated with water based boron copper azole and solvent based IPBC propiconazole plus tebuconazole (1:1) preservatives and exposed to the basidiomycetes for 12 weeks. Mass loss for the fungal decay-infected samples was recorded of up to 55% for preservative-treated samples, up to 62% mass loss for leached samples and up to 58% mass loss for un-preservative treated samples. Additionally, well defined dosage responses and approximate toxic thresholds were obtained for all preservatives tested. Results suggested that the minimum IPBC retention specified by Hazard Class 1.2 of NZS3640:2003 (0.025% m/m) is on the low side, and demonstrated after the 2 week leaching regime complete loss of efficacy of boron at 0.4% m/m boric acid equivalent (BAE). This PhD research gave a first overview of fungi occurring in New Zealand leaky buildings, and it demonstrated the following key aspects of wood preservation: 1. The isolated test fungus Antrodia sinuosa was more difficult to control with propiconazole plus tebuconazole at retention 0.007% m/m than the known tolerant fungus Oligoporus placenta; 2. Boron at Hazard Class 1.2 retention of 0.4% m/m BAE was not toxic to Oligoporus placenta; 3. Serpula lacrymans exhibited tolerance to the highest retention of 0.06 %m/m tebuconazole plus propiconazole; and 4. Gloeophyllum species appeared susceptible to all wood preservatives. In order to correlate fungal colonisation and wood decay, colonised wood blocks were studied using light microscopy (LM) and field- emission scanning electron microscopy (FE-SEM). Microscopic observations of P. radiata wood blocks following a standard wood decay test of twelve weeks of fungal colonisation by Serpula lacrymans, Antrodia sinuosa, Oligoporus placenta and Gloeophyllum sepiarium revealed that the two microscopic techniques employed were complementary by allowing features such as pit membranes, chlamydospores or S3/S2 compound middle lamella interface to be photographed in greater detail, allowing for more precise analyses and interpretation of key findings, as follows: 1. Brown rot fungi directly target their apical growth towards degraded pit apetures; 2. Reliance on light microscopy and observed birefringence as a tool to record changes in cell wall crystallinity associated with brown rot decay alone could be misleading; 3. Presence of fine (≤ 1 m) to wide (≥ 3.5 m) bore-hole and hyphal size ranges, and nearly unchanged cell wall thickness of all wood/test fungal combinations, confirmed active decay at moderate to late stages; 4. Some ray parenchyma cells for Antrodia sinuosa, Oligoporus placenta and Gloeophyllum sepiarium colonised blocks were intact throughout late stages of decay, outlining that they were not preferentially degraded early in the brown rot decay process, and 5. Presence of bore-holes, clamp and medallion clamp formation and resting spores (chlamydospores and arthrospores) are fungal specific, can aid in their differentiation and identification, and should be recorded during wood decay studies, as especially resting spores are an important factor when planning remediation strategies. In summary, this PhD thesis research provided the first comprehensive investigation into the biodiversity of fungi from leaky New Zealand buildings, identified the dominant species and presented details about their micromorphology and their decay patterns. It also demonstrated substantial differences in efficacy of preservative formulations currently (December 2008) approved for framing treatments in New Zealand and possible deficiencies where framing may be subjected to severe leaching. This study also provided the first comparative analyses of viable fungal aerial spores between leaky wall cavities and the surrounding air environment. Subsequently, this research added to the knowledge of the decay fungal species diversity in and around New Zealand leaky buildings, outlined their capabilities to degrade treated and un-treated P. radiata framing timber and illustrated the efficacy of New Zealand approved wood preservatives for their potential as remedial treatment and future prevention.