The aim of this study was to determine the influence of harvesting time and water stress on the chemical composition of the essential oil and to test the activities against ticks and fungus of the essential oil of L. gracilis. For the analysis of harvest season the plant material was collected from seven genotypes of L. gracilis at the Research Farm "Campus Rural da UFS", in the rainy and dry seasons. The experiment testing water stress was conducted in the dry season. The extraction of essential oils was performed in the Laboratory of Phytotechnology of the UFS through hydrodistillation. Chemical analysis of the essential oil performed using GC-MS in the Laboratory of Chromatography of the UFS. For the activity tests against ticks package larvae and immersion of engorged tick Rhipicephalus microplus in different concentrations of essential oil, thymol or carvacrol. To test of fungicidal activity, the essential oil at different concentrations was added to PDA medium. Each plate was inoculated with mycelia culture of Thielaviopis paradoxa. The essential oil of L. gracilis presented two distinct chemotypes, one genotype LGRA-106 presenting as major compound thymol and the other genotypes presenting carvacrol as major compound. The leaves provided essential oil with an average grade of 1.55% in the rainy season and 2.09% in the dry season. In the rainy season there was no significant difference in both the yield and the content. The chemical composition of essential oils L. gracilis showed high levels of terpenes, 92% in the rainy season and 96% in the dry season. In the experiment with irrigation the values of content and yield of all genotypes were smaller when compared without irrigation. In general, the species L. gracilis, for the presence of water in the soil, provides stability in the chemical composition of the essential oil regardless of season, since plants subjected to irrigation, even in the dry season, the essential oil produced in quantity and quality similar to the rainy season. The essential oil of L. gracilis exhibits high activity against ticks, proven by lethal concentrations of genotypes LGRA-201 (1.31 mg.mL-1) and LGRA-106 (4.66 mg.mL-1), demonstrating efficiency in the control of this parasite. The tests showed that the concentrations 0.45; 0.91 and 2.75 mg.mL-1 of all genotypes of L. gracilis completely inhibited the development of the pathogen T. paradoxa, corresponding to a percentage of mycelium growth inhibition of 100%. The concentration of 0.18 mg.mL-1 of essential oil was sufficient to significantly reduce the number of spores of T. paradoxa. The minimal fungicidal concentration T. paradoxa was found between concentrations from 0.80 to 0.98 mg.mL-1 for the essential oils and 0.26 mg.mL-1 for carvacrol and 0.35 mg.mL-1 to thymol. / O presente trabalho teve como objetivo determinar a influencia da época de colheita e do estresse hídrico na composição química do óleo essencial e testar a atividade carrapaticida e fungicida do óleo essencial de L. gracilis. Para as análises de época de colheita o material vegetal foi colhido de sete genótipos de L. gracilis no Campus Rural da UFS , nas épocas chuvosa e seca. Já para o ensaio de estresse hídrico o experimento foi conduzido na época seca. A extração de óleos essenciais foi realizada no Laboratório de Fitotecnia da UFS, por meio de hidrodestilação. A análise química do óleo essencial foi realizada utilizando CG-EM, no Laboratório de Cromatografia da UFS. Para a atividade carrapaticida foram realizadas os testes de pacote de larvas e de imersão de fêmeas ingurgitadas do carrapato Rhipicephalus microplus em diversas concentrações de óleo essencial, timol ou carvacrol. Para o teste de atividade fungicida, o óleo essencial, em diferentes concentrações foi adicionado ao meio BDA. Cada placa foi inoculada com micélios da cultura monospórica de Thielaviopis paradoxa. O óleo essencial de L. gracilis apresentou dois quimiotipos distintos um com o genótipo LGRA-106 apresentando o composto timol como majoritário e os demais o carvacrol. As folhas forneceram óleos essenciais amarelados com teor médio de 1,55% na época chuvosa e 2,09% na seca. Na época chuvosa não houve diferença significativa tanto no rendimento como no teor. A composição química dos óleos essenciais de L. gracilis apresentou altos níveis de terpenos, 92% na época chuvosa e 96% na época seca. No ensaio com irrigação os valores de teor e rendimento de todos os genótipos são menores quando comparado com o ensaio sem irrigação. De maneira geral a espécie L. gracilis, quanto à presença de água no solo, apresenta estabilidade na composição química do óleo essencial independente da época do ano, uma vez que as plantas submetidas à irrigação, mesmo no verão, produziram óleo essencial em quantidade e qualidade semelhantes à época do inverno. O óleo essencial de L. gracilis apresenta alta atividade carrapaticida, comprovados pelas concentrações letais dos genótipos LGRA-201 (1,31 mg.mL-1) e LGRA-106 (4,66 mg.mL-1), demonstrando eficiência no controle desse parasita. Os ensaios demonstraram que as concentrações 0,45; 0,91 e 2,75mg.mL-1 de todos os genótipos de L. gracilis inibiram completamente o desenvolvimento do patógeno T. paradoxa, correspondendo a uma porcentagem de inibição do crescimento micelial de 100%. A concentração de 0,18mg.mL-1 de óleo essencial foi suficiente para reduzir significativamente o número de esporos de T. paradoxa. A concentração fungicida mínima de T. paradoxa foi encontrada entre as concentrações de 0,80 a 0,98mg.mL-1 para os óleos essenciais e 0,26mg.mL-1 para o carvacrol, e 0,35mg.mL-1 para o timol.

The aim of this study was to determine the influence of harvesting time and water stress on the chemical composition of the essential oil and to test the activities against ticks and fungus of the essential oil of L. gracilis. For the analysis of harvest season the plant material was collected from seven genotypes of L. gracilis at the Research Farm "Campus Rural da UFS", in the rainy and dry seasons. The experiment testing water stress was conducted in the dry season. The extraction of essential oils was performed in the Laboratory of Phytotechnology of the UFS through hydrodistillation. Chemical analysis of the essential oil performed using GC-MS in the Laboratory of Chromatography of the UFS. For the activity tests against ticks package larvae and immersion of engorged tick Rhipicephalus microplus in different concentrations of essential oil, thymol or carvacrol. To test of fungicidal activity, the essential oil at different concentrations was added to PDA medium. Each plate was inoculated with mycelia culture of Thielaviopis paradoxa. The essential oil of L. gracilis presented two distinct chemotypes, one genotype LGRA-106 presenting as major compound thymol and the other genotypes presenting carvacrol as major compound. The leaves provided essential oil with an average grade of 1.55% in the rainy season and 2.09% in the dry season. In the rainy season there was no significant difference in both the yield and the content. The chemical composition of essential oils L. gracilis showed high levels of terpenes, 92% in the rainy season and 96% in the dry season. In the experiment with irrigation the values of content and yield of all genotypes were smaller when compared without irrigation. In general, the species L. gracilis, for the presence of water in the soil, provides stability in the chemical composition of the essential oil regardless of season, since plants subjected to irrigation, even in the dry season, the essential oil produced in quantity and quality similar to the rainy season. The essential oil of L. gracilis exhibits high activity against ticks, proven by lethal concentrations of genotypes LGRA-201 (1.31 mg.mL-1) and LGRA-106 (4.66 mg.mL-1), demonstrating efficiency in the control of this parasite. The tests showed that the concentrations 0.45; 0.91 and 2.75 mg.mL-1 of all genotypes of L. gracilis completely inhibited the development of the pathogen T. paradoxa, corresponding to a percentage of mycelium growth inhibition of 100%. The concentration of 0.18 mg.mL-1 of essential oil was sufficient to significantly reduce the number of spores of T. paradoxa. The minimal fungicidal concentration T. paradoxa was found between concentrations from 0.80 to 0.98 mg.mL-1 for the essential oils and 0.26 mg.mL-1 for carvacrol and 0.35 mg.mL-1 to thymol. / O presente trabalho teve como objetivo determinar a influencia da época de colheita e do estresse hídrico na composição química do óleo essencial e testar a atividade carrapaticida e fungicida do óleo essencial de L. gracilis. Para as análises de época de colheita o material vegetal foi colhido de sete genótipos de L. gracilis no Campus Rural da UFS , nas épocas chuvosa e seca. Já para o ensaio de estresse hídrico o experimento foi conduzido na época seca. A extração de óleos essenciais foi realizada no Laboratório de Fitotecnia da UFS, por meio de hidrodestilação. A análise química do óleo essencial foi realizada utilizando CG-EM, no Laboratório de Cromatografia da UFS. Para a atividade carrapaticida foram realizadas os testes de pacote de larvas e de imersão de fêmeas ingurgitadas do carrapato Rhipicephalus microplus em diversas concentrações de óleo essencial, timol ou carvacrol. Para o teste de atividade fungicida, o óleo essencial, em diferentes concentrações foi adicionado ao meio BDA. Cada placa foi inoculada com micélios da cultura monospórica de Thielaviopis paradoxa. O óleo essencial de L. gracilis apresentou dois quimiotipos distintos um com o genótipo LGRA-106 apresentando o composto timol como majoritário e os demais o carvacrol. As folhas forneceram óleos essenciais amarelados com teor médio de 1,55% na época chuvosa e 2,09% na seca. Na época chuvosa não houve diferença significativa tanto no rendimento como no teor. A composição química dos óleos essenciais de L. gracilis apresentou altos níveis de terpenos, 92% na época chuvosa e 96% na época seca. No ensaio com irrigação os valores de teor e rendimento de todos os genótipos são menores quando comparado com o ensaio sem irrigação. De maneira geral a espécie L. gracilis, quanto à presença de água no solo, apresenta estabilidade na composição química do óleo essencial independente da época do ano, uma vez que as plantas submetidas à irrigação, mesmo no verão, produziram óleo essencial em quantidade e qualidade semelhantes à época do inverno. O óleo essencial de L. gracilis apresenta alta atividade carrapaticida, comprovados pelas concentrações letais dos genótipos LGRA-201 (1,31 mg.mL-1) e LGRA-106 (4,66 mg.mL-1), demonstrando eficiência no controle desse parasita. Os ensaios demonstraram que as concentrações 0,45; 0,91 e 2,75mg.mL-1 de todos os genótipos de L. gracilis inibiram completamente o desenvolvimento do patógeno T. paradoxa, correspondendo a uma porcentagem de inibição do crescimento micelial de 100%. A concentração de 0,18mg.mL-1 de óleo essencial foi suficiente para reduzir significativamente o número de esporos de T. paradoxa. A concentração fungicida mínima de T. paradoxa foi encontrada entre as concentrações de 0,80 a 0,98mg.mL-1 para os óleos essenciais e 0,26mg.mL-1 para o carvacrol, e 0,35mg.mL-1 para o timol.

Die Entwicklung einer mukosal verabreichbaren, effektiven DNA-Vakzine gegen Infektionskrankheiten oder Tumorerkrankungen auf der Basis invasiver attenuierter Bakterien ist eine vielversprechende Alternative zu bisherigen parenteralen Strategien der genetischen Vakzinierung. Innerhalb dieser Arbeit wurden Salmonellen-Impfstämme für die orale Übertragung eines eukaryontischen Expressionsplasmids mit dem kleinen Oberflächenantigen des Hepatitis-B-Virus (HBsAg) als Modellantigen optimiert. Die kontinuierliche Sezernierung von Plasmiden als filamentöse Phagenpartikel wurde als ein erster Ansatz getestet, um mit lebenden Bakterien eine DNA-Vakzine innerhalb infizierter Zellen freizusetzen. Die Salmonellen-vermittelte Phagensekretion in der Wirtszelle ist jedoch nicht effizient genug, die Expression des Transgens zu vermitteln. Alternativ wurde ein Ansatz gewählt, durch eine spontan induzierte Lyse der Impfbakterien, Plasmid-DNA in die Wirtszelle zu übertragen. Dazu wurde ein neuartiges bakterielles Autolysesystem etabliert, basierend auf einem Zwei-Phasen-Expressionssystem und von Bakteriophagen abgeleiteten Lysedeterminanten. Dieses System ermöglicht erstmals die kontinuierliche Freisetzung von Plasmid-DNA und Proteinen aus einzelnen, lysierenden Salmonellen innerhalb einer sonst gesunden bakteriellen Gesamtpopulation. Innerhalb infizierter COS7-Zellen führt die Freisetzung des porenformierenden Proteins Listeriolysin O durch autolytische Salmonellen zur Zerstörung der Vakuole, in der die Impfbakterien replizieren, und erleichtert somit den Transfer der Plasmid-DNA aus den Bakterien in das Zytoplasma der Wirtszelle. Die Lysedeterminante und die eukaryontische Expressionskassette für HBsAg wurden auf einem Plasmid kombiniert, sowie eine Kassette zur konstitutiven Expression des Histon-ähnlichen Proteins aus Thermotoga maritima (TmHU) in ein solches Konstrukt integriert. TmHU stabilisiert die Plasmiderhaltung unter nicht selektiven Bedingungen und besitzt das Potential, die Effizienz der DNA-Translokation innerhalb der Wirtszelle zu erhöhen. Durch die orale Gabe optimierter autolytischer Impfbakterien konnte eine potente HBsAg-spezifische Antikörperantwort sowie eine zytotoxische zelluläre Antwort induziert werden. Bereits die einmalige Gabe der autolytischen Bakterien induzierte eine höhere antigenspezifische Antikörperantwort, als die herkömmliche intramuskuläre DNA-Vakzine. Das im Rahmen dieser Arbeit entwickelte Konzept autolytischer Salmonellen stellt also eine neuartige, effiziente Strategie für den mukosalen DNA-Transfer dar. Die Übertragung des Konzeptes der Autolyse auf andere bakterielle Trägersysteme ist möglich und kann zur Erweiterung des Anwendungspektrums bakterieller Vektoren beitragen. / The development of an effective mucosal DNA vaccine against infectious diseases or tumors based on invasive attenuated bacteria is a very promising alternative to common parenteral routes of genetic vaccination. This work aimed at the optimization of Salmonella vaccine strains for the oral delivery of an eukaryotic expression plasmid encoding the small Hepatitis B Virus surface antigen (HBsAg), here used as model antigen. The continuous secretion of plasmids as filamentous phage particles was first tested as a mean for the delivery of the DNA vaccine by living bacteria inside infected host cells. However, Salmonella-mediated phage secretion inside cells did not suffice for the induction of transgene expression. As alternative approach, inducible spontanous lysis of bacteria was used to mediate the release of plasmid DNA into host cells. For this purpose a novel bacterial autolytic system was established on the basis of a two-phase expression system and lysis determinants derived from bacteriophages. This system allows for the first time the continuous release of plasmid DNA and proteins from only few lysing Salmonella within an otherwise healthy bacterial population. Inside COS7 cells the release of the pore-forming protein listeriolysin O by autolytic Salmonella mediates the destruction of the Salmonella-harbouring vacuole, thereby facilitating the transfer of plasmid DNA from bacteria into the host cell cytoplasm. The lysis determinant was combined with the eukaryotic expression cassette for HBsAg on one plasmid. In addition, a cassette for the constitutive expression of TmHU, a histon-like protein derived from Thermotoga maritima, was integrated in such vector. TmHU stabilizes the plasmid propagation in the absence of selective pressure and has the potential to increase the efficiency of plasmid translocation inside the host cell. The oral administration of the optimized autolytic bacteria stimulated a potent HBsAg-specific antibody response as well as a cytotoxic cellular response. Already a single inoculation of the oral vaccine induced a higher specific antibody response than the conventional intramuscular DNA vaccine. Therefore the concept of autolytic Salmonella carrier strains developed in this work constitutes a novel efficient strategy for mucosal DNA delivery. The transfer of this concept to other bacterial carriers is possible and may widen the application field for bacterial vectors.

Penicillium nordicum è un importante contaminante di salumi, rappresentanando il 10 % e il 26 % della popolazione di Penicillium spp . isolati , rispettivamente dall'aria e dai prodotti carnei stagionati in un'indagine gestita in Italia ( Battilani et al. , 2007). Diverse colonie di P. nordicum isolate dai salumi hanno dimostrato di essere importanti produttori di ocratossina A , OTA ( Sansom e Frisvad , 2004 . Pietri et al, 2006 ; . Battilani et al , 2010). Attualmente, l'impostazione appropriata delle condizioni ambientali (temperatura, umidità relativa e circolazione dell'aria ), è l'unico strumento accettato per impedire la crescita incontrollata di P. nordicum all'interno degli impianti di stagionatura attraverso una accurata analisi dei punti critici di controllo e l’ideazione di un relativo piano HACCP (Hazard Analysis and Critical Control Points) ben struttutato ( Asefa et al , 2011; Virgili et al , 2012). Anche se il sistema HACCP è stato applicato con successo nel settore alimentare ci sono rischi per la sicurezza alimentare non attentamente considerati. Questo è particolarmente vero per quanto riguarda i rischi micotossigeni associati ai prodotti alimentari di origine animale. Il termine "rischi micotossigeni" è utilizzato da Asefa et al. ( 2011) per descrivere lieviti patogeni e metaboliti secondari tossici prodotti da specie fungine tossigene che contaminano i prodotti alimentari e incidono sulla sicurezza alimentare. La maggior parte dei piani HACCP nelle attività di trasformazione alimentare, come ad esempio la produzione di formaggi e di prodotti carnei stagionati, tiene in considerazione principalmente il rischio derivante da agenti batterici (Arvanitoyannis e Mavropoulos, 2000; Barbuti e Parolari, 2002) anche se tali prodotti alimentari vengono spesso contaminati da funghi micotossigeni e dai loro metaboliti (Spotti et al 1989; Spotti et al , 2001a; Battilani et al 2007). Pertanto, dovrebbe essere cruciale definire un piano HACCP specificamente incentrato sui rischi micotossigeni. L'identificazione, il controllo e la standardizzazione della micoflora superficie dei salumi è fondamentale per preservare la sicurezza delle produzioni e la salute dei consumatori . Questo è il contesto in cui deve essere valutata l’efficacia e l’affidabilità per l’identificazione delle popolazioni di Penicillium spp di interessante per la produzione alimentare. In questo contesto , il progetto di ricerca di questa tesi di dottorato ha cercato di approfondire le conoscenze su tali tematiche con l'intento di limitare il rischio micotossigeno nella catena di produzione dei prodotti carnei stagionati. Sono stati affrontati i seguenti argomenti: 1 . studio della composizione e dinamica della microflora fungina presente sulla superficie dei salumi (prodotto testato, salame) e l'aria di ambienti di stagionatura tenendo conto dell'influenza di alcuni parametri di processo (inoculo starter, temperatura, fase produttiva). 2 . sviluppo di un metodo MALDI TOF MS per l'identificazione di Penicilium a livello di specie per le prospettive future di screening diretti della microflora presente sui salumi. 3 . confronto e integrazione di diverse tecniche, come l'analisi morfologica, l’analisi molecolare e l’analisi tramite spettrometria di massa, per l'identificazione delle specie di Penicillium presenti nei salumi. 4 . valutazione dei lieviti selezionati, isolati dalla superficie di prosciutto crudo, per competere con P. nordicum ed inibire l'accumulo di OTA nella prospettiva del loro uso come starter superficiali con funzione di agenti di biocontrollo. / Penicillium nordicum is an important contaminant of cured meat products, representing 10% and 26% of the Penicillium spp. isolated, respectively, from the air or the products in a survey managed in Italy (Battilani et al., 2007). Several P. nordicum cured meat isolates proved to be important producers of ochratoxin A, OTA (Sansom and Frisvad, 2004; Pietri et al., 2006; Battilani et al., 2010). Currently, the appropriate setting of environmental conditions (temperature, relative humidity and air circulation), is the only accepted tool to prevent the uncontrolled growth of P. nordicum inside dry-curing plants through a carefully structured Hazard Analysis Critical Control Point (HACCP) plan (Asefa et al., 2011; Virgili et al., 2012). Even if the HACCP system has been successfully applied in the food industry, there are food safety hazards not carefully considered. This is especially true with regard to mycotoxigenic hazards associated with animal food products. The term “mycotoxigenic hazards” is used by Asefa et al. (2011) to describe pathogenic yeasts and toxic secondary metabolites of toxigenic moulds that contaminate food products and affect food safety. Most HACCP plans in food processing activities, such as the production of cheese and dry-cured meat products, considered mainly bacterial agents (Arvanitoyannis and Mavropoulos, 2000; Barbuti and Parolari, 2002), even if such food products get often contaminated with mycotoxigenic fungi and their metabolites (Spotti et al 1989; Spotti et al., 2001a; Battilani et al 2007). Therefore, it should be crucial to define a HACCP plan specifically focused on the mycotoxigenic hazards. The identification, control and standardization of the surface mycoflora of cured meat products is mandatory to preserve the productions safety and the consumers health. This is the context of the effectiveness and reliability evaluation for the Penicillium spp. identification methods of interesting species for food production. In this context, the research project of this PHD thesis tried to fill some gaps of knowledge with the attempt to limit the mycotoxigenic risk in the cured meat products chain. The following topics were faced: 1. study of the composition and dynamic of fungal microflora present on the surface of cured meat products (salami) and the air of seasoning environments taking into account the influence of some process parameters (starter inoculum, curing temperature, stage of seasoning). 2. development of a MALDI TOF MS method for the identification of Penicilium at species level for future direct screening perspectives of the microflora present on cured meat products. 3. comparison and integration of different techniques, as morphological, molecular and mass spectral analysis, for the identification of Penicillium species in cured meat products. 4. evaluation of selected yeasts, isolated from dry-cured ham surface, to compete with P. nordicum and to inhibit OTA accumulation in the perspective of their use as surface starter biocontrol agents.

In my thesis, I focused on monitoring of microorganisms in the sample of red grape juice and on the interactions between yeasts, bacteria and filamentous fungi. Three different media were applied for the cultivation of microorganisms; firstly for monitoring of total volume of microorganisms, secondly for yeasts and third time for lactic acid bacteria. The indirect method was used for the determination of the amount of viable cells. This method consists in enumerating of visible macroscopic colonies grown up on agar plates. When the cells grew up, the forms of colonies were analyzed visually and the morphology of microorganisms was detected microscopically. The operating time of enzymes in grape juice in the production of red wine was monitored after application of commercial enzymatic preparation. The enzym action in grape juice was observed on the basis of the process of degradation of high – molecular substrate by enzymes through the use of Ubbelohd´s viscometer. The research findings provided a lot of knowledge about the occurance of microflora in the process of production of red wine. The commercial preparations added to grape juice played a significant role.