<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>

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No description available.

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 / Compendio

<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>

Ziel der vorliegenden Arbeit war zum einen die Ermittlung des im Grünfutter notwen-digen Mindest-Nitratgehaltes zur Erzielung buttersäurefreier Silagen, wobei TS-Gehalt und Z/PK-Quotient des Ausgangsmaterials sowie dessen Belastungsgrad mit Clostridiensporen berücksichtigt wurden. Dazu wurden zwei mehrfaktorielle Laborsi-lierversuche durchgeführt, bei denen eine Variationsreihe des Nitratgehaltes (0,01...0,3 % NO3-N in TS) mit Variationsreihen des TS-Gehaltes (ca. 14...40 %) und des Z/PK-Quotienten (1,5...3,1) systematisch kombiniert wurden. Jede Wertekombi-nation wurde sowohl mit sauber geerntetem Grünfutter als auch mit Clostridienspo-ren kontaminiertem Grünfutter geprüft. Die Silagen wurden nach 180 Tagen Lage-rungsdauer untersucht. Zum anderen wurde die Dynamik der Clostridienentwicklung im Gärungsverlauf in Abhängigkeit von TS-Gehalt, Säuerungsintensität und Nitratge-halt geprüft. Jede Stufe des TS-Gehaltes (20, 30, 40 und 50 %) wurde mit Zusätzen von Nitrat, Milchsäurebakterien (MSB) bzw. MSB + Glucose und MSB + Nitrat ange-setzt. Das Ausgangsmaterial (Dac. glomerata, nitratfrei) war durchgehend mit Clostridiensporen kontaminiert. Die Untersuchung der Silagen erfolgte 3, 7, 14, 28, 56 und 180 Tage nach dem Ansatz. Der Konservierungserfolg bei der Silierung hängt nicht nur vom TS-Gehalt und Z/PK-Quotienten sondern auch vom Nitratgehalt und Clostridiensporenbesatz des Aus-gangsmaterials ab. Bei sehr niedrigem Nitratgehalt des Grünfutters liegt ein erhöhtes Risiko für die Entstehung von Buttersäure in der Silage vor, auch bei dem als leicht vergärbar geltendem Grünfutter (VK > 45) und auch dann, wenn es im sauber geern-tetem Zustand einsiliert worden ist. Bei weiter erhöhten VK-Werten, > 45 (durch Er-höhung des TS-Gehaltes und/oder Z/PK-Quotienten), wird die Höhe der Buttersäure-gehalte zwar eingeschränkt. Zur sicheren Ausschaltung von Buttersäuregärung ist jedoch auch hier ein gewisser Nitratgehalt notwendig. Bei der Silierung nitratfrei-en/nitratarmen Grünfutters nimmt das Fehlgärungsrisiko mit dem Belastungsgrad an Clostridiensporen zu. Der notwendige Mindest-Nitratgehalt (MNG) hängt sowohl vom VK-Wert als auch vom Kontaminationsgrad des Grünfutters mit Clostridiensporen ab. Er ist um so niedriger, je höher der VK-Wert und geringer der Kontaminationsgrad ist, und umgekehrt. (MNG (% NO3-N in TS) für sporenarmes Grünfutter = 0,24 - 0,0035 . VK MNG (% NO3-N in TS) für sporenreiches Grünfutter = 0,20 - 0,0021 . VK) Hohe Clostridiensporengehalte lagen vor allem in buttersäurehaltigen Silagen vor und insbesondere dann, wenn das Grünfutter sehr geringe Nitratgehalte aufwies. Zwischen der Höhe der Buttersäuregehalte und dem Clostridiensporengehalt besteht jedoch kein direkter Zusammenhang. Erhöhung des TS-Gehaltes bewirkt eine Ein-schränkung der Clostridienentwicklung. Ein Rückgang des Sporengehaltes im Ver-gleich zum Ausgangsmaterial (nitratfrei) lag aber erst bei einem TS-Gehalt von etwa 50 % vor. Durch Zusätze von MSB sowie MSB + Glucose konnte die Milchsäuregä-rung deutlich intensiviert werden. Ein sehr geringer pH-Wert war schon am 3. oder 7. Lagerungstag erreicht. Buttersäuregärung war aber erst bei TS-Gehalten > 40 % ausgeschaltet. Eine Einschränkung der Sporenbildung lag ebenfalls erst bei TS-Gehalten über 40 % vor. Bei Nitratzusatz blieben die Silagen aller TS-Stufen bis zur Auslagerung buttersäurefrei. Die Sporengehalte gingen in allen TS-Stufen während des Gärungsverlaufes kontinuierlich zurück. Bei steigenden TS-Gehalten war der Rückgang der Sporengehalte verlangsamt. Durch die Kombination von MSB und Nit-rat konnte nicht nur ein sicherer Erfolg bei der Unterbindung von Buttersäurebildung und Laktatabbau sondern auch eine starke Verminderung der Sporengehalte erreicht werden. / The first aim of this work was to determine the minimum content of nitrate (MCN) which is required to get silage free of butyric acid. For it, two multi-factorial experi-ments with orchardgrass were carried out under laboratory condition. In this experi-ments, nitrate content (0.01 ... 0.3 % N in DM) was systematically combined with staggered levels of the dry matter (DM) content (14 ... 40 %) and of the ratio of water-soluble carbohydrate to buffering capacity (WSC/BC: 1.5 ... 3.1). All variants were tested with forage without or with addition of clostridial spores. The silages were ana-lysed after 180 days of incubation. The second aim of this study was to explain the dynamic of clostridial development during ensilage, depending on DM content, intensity of lactic acid formation, and ni-trate content. orchardgrass (free of nitrate) with 4 levels of DM (20, 30, 40, and 50 %) was firstly contaminated with clostridial spores about 104 / g FM. Then it was ensi-laged with following treatments: without additives, with inoculation of lactic acid bac-teria (LAB) alone or in combination with 2 % Glucose in FM (LAB+G), with nitrate addition (0.1 and 0.15 % N in DM), and with LAB plus nitrate 0.1 % N in DM. The si-lages were analysed 3, 7, 14, 28, 56, and 180 days after ensiling. Results showed that silage quality not only depends on DM content and ratio of WSC to BC, but also depends on nitrate content and extent of clostridial spores in forage. With an extremely low content of nitrate a high risk of butyric acid formation is given in silages, even if the ensiling material had a high ensilability (FC ³ 45) and a very low content of clostridial spores. The butyric acid concentration decreased with in-creasing DM content from 14 to 40 % or with increasing WSC/BC-ratio. But to get the silages free of butyric acid, a certain amount of nitrate was required. By adding clos-tridial spores in fodder the risk of butyric acid formation was increased, especially in case of material lacking in nitrate. The value of MCN depends on ensilability of the forage, as measured by DM-content and WSC/BC-ratio or by fermentability coefficient (FC) = DM+8WCS/BC, as well as depends on content of clostridial spores in the material used. The higher the FC-value and the lower the spores content is, the less nitrate is required to get silage free of butyric acid. MNC (% N in DM) for very low contaminated forage = 0.24 - 0.0035.FC MNC (% N in DM) for high contaminated forage = 0.20 - 0.021.FC High content of clostridial spores was especially found in silages containing butyric acid, which were made from forage with very low nitrate content (£ 0.02 % N in DM). But a strong relationship was not found between butyric acid and spores content. By increasing DM content the development of clostridia during ensiling was limited. A continuous decrease of spores content, in comparison with the forage before ensiled, was observed at first by increasing DM content to 50 %. By inoculation with LAB or LAB+G the lactic acid formation was strongly stimulated. A very low pH was reached 3 or 7 days after ensiling. But the butyric acid formation could be firstly prevented by increasing DM content to over 40 %. For all levels of DM, by nitrate addition the silages remained no butyric acid during the whole period of incubation. The concentration of clostridial spores decreased continuously during ensilage. This decrease was slower with increasing DM content . By combination of LAB with nitrate a reliable prevention of butyric acid formation and a fast decrease in spores concentration were reached.

Ziel der Arbeit war es, die Besonderheiten des Gärungsverlaufes bei der Silierung von nitratarmem Grünfutter aufzuklären. Dazu wurden sechs Silierversuche zur Untersuchung des Gärungsverlaufes mit unterschiedlichem Clostridiensporenbesatz durchgeführt. Dabei wurde auch die Wirkung eines Zusatzes von 0,05 bzw. 0,1 % N / TS als Nitrat und Nitrit sowie Zusätze von Inoculantien und Ameisensäure geprüft. Weiterhin wurde untersucht, ob die unter Laborbedingungen gefundenen Auswirkungen des Fehlens von Nitrat ebenso bei Grünfutter, das unter praxisnahen Bedingungen geerntet wurde, auftreten. Zur Fragestellung, welche Siliermittel bei nitratarmem Grünfutter eingesetzt werden können, wurde auch hier der Zusatz von zwei MSB-Präparaten und Ameisensäure, sowie eines nitrithaltigen Siliermittels bei geringem und erhöhtem Clostridiensporengehalt des Siliergutes geprüft. Als Ergebnis dieser mehrjährigen, umfangreichen Untersuchungen mit verschiedenen Futterpflanzen und unterschiedlicher Clostridiensporenbelastung hatte sich gezeigt, daß der Verlauf der Stoffumsetzungen und das Gärproduktmuster am Ende der Gärung in Abhängigkeit vom Nitratgehalt wesentlich unterschiedlich ist. In Silagen aus nitratarmem Grünfutter trat Buttersäure bereits von Gärbeginn und parallel zur Milchsäuregärung auf. Die Essigsäuregehalte waren stets sehr niedrig. Anaerob stabile und instabile Silagen aus nitratarmem Grünfutter weisen in allen Stadien des Gärungsprozesses ein völlig anderes Verhältnis zwischen Buttersäure und den übrigen Merkmalen des unerwünschten Stoffabbaus - Essigsäure, Ammoniak, pH - auf als Silagen aus nitrathaltigem Grünfutter. Für die Einschätzung der Vergärbarkeit sind außer TS und Z/PK auch der Nitratgehalt sowie epiphytischer Keimbesatz und Clostridiensporengehalt des AM zu berücksichtigen. Bei Fehlen von Nitrat besteht, unabhängig von der nach TS und Z/PK vorhergesagten Vergärbarkeit, ein besonderes Risiko für das Auftreten von Buttersäure. In Abhängigkeit vom Aufwuchs war die Einschätzung der Vergärbarkeit des Grünfutters verschieden und es trat eine unterschiedliche Gärqualität der Silagen auf. Bei nitratarmem Grünfutter ist der strategische Einsatz von MSB-Präparaten zu empfehlen. Das nitrithaltige Siliermittel hatte sich insbesondere bei erhöhter Clostridiensporenbelastung und/oder niedrigem TS-Gehalt des Grünfutters als sehr wirksam erwiesen. Bei Anwendung des derzeit gültigen, für Silagen aus nitratreichem Grünfutter entwickelten DLG-Beurteilungsschlüssels auf Silagen aus nitratarmem Ausgangsmaterial ist mit einer Fehlbewertung zu rechnen. / The object of this work is to explain the distinctive feature of ensilage of green forage low in nitrate. The fermentation process of ensiling green forage low in nitrate was proved in 6 experiments with different content of spores of clostridia. In all experiments the green forage was ensiled with following treatments: without additives (control), with 0,05 and 0,1% N / DM as nitrate or nitrite, with lactic acid bacteria and formic acid. Furthermore the effects of absence of nitrate, proved under laboratory conditions, has been investigated in experiments with green forage produced under practical conditions. The treatments were the same as mentional above. As a result of this several years and extensive investigations with different green fodder and different content of spores of clostridia it was shown that metabolism during fermentation process and pattern of fermentation products in the end of fermentation are significant different depending on content of nitrate in green forage. In ensiling material low in nitrate butyric acid was formed already at the beginning of the fermentation process, parallel to the lactic acid fermentation. The content of acetic acid was always extremely low. In comparison with silages from green forage high in nitrate anaerobe stable or unstable silages show a different ratio between butyric acid and other characteristics of undesirable decomposition during fermentation - acetic acid, ammonia, pH - in equal fermentation stages. In summary, one can say that epiphytic lactic acid bacteria, content of clostridia spores and nitrate of herbage take into account to judge the fermentability more than previous, together with DM and WSC/BC. Furthermore the judgement of herbage fermentability and the fermentation quality of silages were different depending on number of growth. In silages low in nitrate is an especially risk for occurrence of butyric acid, independing on judgement of fermentability on the basis of DM and ratio of watersoluble carbohydrates to buffering capacity. It`s advisable to use lactic acid bacteria additives (inoculants) always for ensiling green forage low in nitrate. The additive with nitrite has proved as most effectively especially for green forage high in clostridia spores and/ or low content of dry matter of green forage. It is very probably, that the use of current DLG- evaluation system to estimate the fermentation quality, developed for silages from green forage high in nitrate, is not correct and leads to error of judgement of silages from green forage low in nitrate.

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.