Os oceanos constituem 71% da superfície da Terra e muitos microrganismos que provém deste ambiente podem secretar enzimas de alto interesse biotecnológico. As enzimas produzidas por microrganismos marinhos podem ser mais estáveis, devido à alta complexidade deste ambiente. Por este motivo, objetivou-se isolar e identificar os fungos presentes no sedimento marinho e verificar a atividade enzimática da amilase, celulase, lípase e protease dos mesmos em diferentes temperaturas (15°C, 20°C, 25°C e 30°C). As amostras de sedimento foram coletadas com auxilio de seringas estéreis à 10 metros de profundidade, e transportadas até o laboratório em caixas isotérmicas. Após o crescimento, fragmentos de micélio foram repicados no centro de placa de petri para isolamento das amostras. Diferentes substratos foram utilizados para avaliar a atividade da amilase, celulase, lipase e protease dos isolados. A capacidade de hidrolisar os substratos foi verificada em diferentes temperaturas (15°C, 20°C, 25°C e 30°C). Foram isoladas 22 cepas fúngicas, de 14 diferentes espécies, pertencentes a nove gêneros, dentre eles Helicosporium, Helicomyces, Paecilomyces, Phoma, Chaetomium, Geotrichum, duas espécies de Cladosporium sp., Aspergillus sp., Penicillium sp., Penicillium chrysogenum e Penicillium aurantiogriseum. O maior índice de positividade enzimática ocorreu na temperatura de 25°C, na qual 71% dos isolados hidrolisaram o substrato lipídico, seguido de protease com 50%, celulase 43% e amilase 36%. Todos os isolados produziram no mínimo um tipo de enzima, nas temperaturas testadas, ficando evidente a que o ambiente marinho é para descoberta de novas enzimas. / Oceans constitute 71% of the Earth's surface, and many microorganisms from this environment may secrete enzymes of high biotechnological interest. Enzymes produced by marine microorganisms might be more stable due to the great complexity of this environment. Therefore, we aimed at isolating and identifying the fungi present in marine sediment and at verifying the corresponding enzymatic activities of amylase, cellulase, lipase, and protease. Sediment samples were collected with sterile syringes at a depth of 10 meters and transported to the laboratory in cool boxes. After their growth, mycelial fragments were sub-cultured and inoculated in the center of Petri dishes for isolating the samples. Different substrates were used to evaluate the activity of amylase, cellulase, lipase and protease. The ability to hydrolyse the substrates was observed at different temperatures (15°C, 20°C, 25°C e 30°C).Twenty-two fungal strains were isolated from 14 different species belonging to nine genera, including Helicosporium, Helicomyces, Paecilomyces, Phoma, Chaetomium, Geotrichum, two different types of Cladosporium sp., Aspergillus sp., Penicillium sp., Penicillium chrysogenum, and Penicillium aurantiogriseum. The highest enzymatic production rate was found for lipase, produced by 71% of the isolated fungi, followed by protease (50%), cellulase (43%), and amylase (36%). All isolates produced at least one type of enzyme, evidencing that the sea is a environment for discovering new enzymes.

Os oceanos constituem 71% da superfície da Terra e muitos microrganismos que provém deste ambiente podem secretar enzimas de alto interesse biotecnológico. As enzimas produzidas por microrganismos marinhos podem ser mais estáveis, devido à alta complexidade deste ambiente. Por este motivo, objetivou-se isolar e identificar os fungos presentes no sedimento marinho e verificar a atividade enzimática da amilase, celulase, lípase e protease dos mesmos em diferentes temperaturas (15°C, 20°C, 25°C e 30°C). As amostras de sedimento foram coletadas com auxilio de seringas estéreis à 10 metros de profundidade, e transportadas até o laboratório em caixas isotérmicas. Após o crescimento, fragmentos de micélio foram repicados no centro de placa de petri para isolamento das amostras. Diferentes substratos foram utilizados para avaliar a atividade da amilase, celulase, lipase e protease dos isolados. A capacidade de hidrolisar os substratos foi verificada em diferentes temperaturas (15°C, 20°C, 25°C e 30°C). Foram isoladas 22 cepas fúngicas, de 14 diferentes espécies, pertencentes a nove gêneros, dentre eles Helicosporium, Helicomyces, Paecilomyces, Phoma, Chaetomium, Geotrichum, duas espécies de Cladosporium sp., Aspergillus sp., Penicillium sp., Penicillium chrysogenum e Penicillium aurantiogriseum. O maior índice de positividade enzimática ocorreu na temperatura de 25°C, na qual 71% dos isolados hidrolisaram o substrato lipídico, seguido de protease com 50%, celulase 43% e amilase 36%. Todos os isolados produziram no mínimo um tipo de enzima, nas temperaturas testadas, ficando evidente a que o ambiente marinho é para descoberta de novas enzimas. / Oceans constitute 71% of the Earth's surface, and many microorganisms from this environment may secrete enzymes of high biotechnological interest. Enzymes produced by marine microorganisms might be more stable due to the great complexity of this environment. Therefore, we aimed at isolating and identifying the fungi present in marine sediment and at verifying the corresponding enzymatic activities of amylase, cellulase, lipase, and protease. Sediment samples were collected with sterile syringes at a depth of 10 meters and transported to the laboratory in cool boxes. After their growth, mycelial fragments were sub-cultured and inoculated in the center of Petri dishes for isolating the samples. Different substrates were used to evaluate the activity of amylase, cellulase, lipase and protease. The ability to hydrolyse the substrates was observed at different temperatures (15°C, 20°C, 25°C e 30°C).Twenty-two fungal strains were isolated from 14 different species belonging to nine genera, including Helicosporium, Helicomyces, Paecilomyces, Phoma, Chaetomium, Geotrichum, two different types of Cladosporium sp., Aspergillus sp., Penicillium sp., Penicillium chrysogenum, and Penicillium aurantiogriseum. The highest enzymatic production rate was found for lipase, produced by 71% of the isolated fungi, followed by protease (50%), cellulase (43%), and amylase (36%). All isolates produced at least one type of enzyme, evidencing that the sea is a environment for discovering new enzymes.

Os oceanos constituem 71% da superfície da Terra e muitos microrganismos que provém deste ambiente podem secretar enzimas de alto interesse biotecnológico. As enzimas produzidas por microrganismos marinhos podem ser mais estáveis, devido à alta complexidade deste ambiente. Por este motivo, objetivou-se isolar e identificar os fungos presentes no sedimento marinho e verificar a atividade enzimática da amilase, celulase, lípase e protease dos mesmos em diferentes temperaturas (15°C, 20°C, 25°C e 30°C). As amostras de sedimento foram coletadas com auxilio de seringas estéreis à 10 metros de profundidade, e transportadas até o laboratório em caixas isotérmicas. Após o crescimento, fragmentos de micélio foram repicados no centro de placa de petri para isolamento das amostras. Diferentes substratos foram utilizados para avaliar a atividade da amilase, celulase, lipase e protease dos isolados. A capacidade de hidrolisar os substratos foi verificada em diferentes temperaturas (15°C, 20°C, 25°C e 30°C). Foram isoladas 22 cepas fúngicas, de 14 diferentes espécies, pertencentes a nove gêneros, dentre eles Helicosporium, Helicomyces, Paecilomyces, Phoma, Chaetomium, Geotrichum, duas espécies de Cladosporium sp., Aspergillus sp., Penicillium sp., Penicillium chrysogenum e Penicillium aurantiogriseum. O maior índice de positividade enzimática ocorreu na temperatura de 25°C, na qual 71% dos isolados hidrolisaram o substrato lipídico, seguido de protease com 50%, celulase 43% e amilase 36%. Todos os isolados produziram no mínimo um tipo de enzima, nas temperaturas testadas, ficando evidente a que o ambiente marinho é para descoberta de novas enzimas. / Oceans constitute 71% of the Earth's surface, and many microorganisms from this environment may secrete enzymes of high biotechnological interest. Enzymes produced by marine microorganisms might be more stable due to the great complexity of this environment. Therefore, we aimed at isolating and identifying the fungi present in marine sediment and at verifying the corresponding enzymatic activities of amylase, cellulase, lipase, and protease. Sediment samples were collected with sterile syringes at a depth of 10 meters and transported to the laboratory in cool boxes. After their growth, mycelial fragments were sub-cultured and inoculated in the center of Petri dishes for isolating the samples. Different substrates were used to evaluate the activity of amylase, cellulase, lipase and protease. The ability to hydrolyse the substrates was observed at different temperatures (15°C, 20°C, 25°C e 30°C).Twenty-two fungal strains were isolated from 14 different species belonging to nine genera, including Helicosporium, Helicomyces, Paecilomyces, Phoma, Chaetomium, Geotrichum, two different types of Cladosporium sp., Aspergillus sp., Penicillium sp., Penicillium chrysogenum, and Penicillium aurantiogriseum. The highest enzymatic production rate was found for lipase, produced by 71% of the isolated fungi, followed by protease (50%), cellulase (43%), and amylase (36%). All isolates produced at least one type of enzyme, evidencing that the sea is a environment for discovering new enzymes.

Información extraída del <a href="http://www.cindefi.org.ar/?page_id=61&language=es">sitio web del CINDEFI</a>

Orientador: Claudio Airoldi / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-07-21T02:13:26Z (GMT). No. of bitstreams: 1 Critter_SilvanaAuxiliadoraMissola_D.pdf: 3877415 bytes, checksum: 32c5ed6dba42714fbf28309305a85f13 (MD5) Previous issue date: 1996 / Doutorado

Os agentes infecciosos têm sido considerados iniciadores da desestabilização da placa de ateroma. Este mecanismo pode estar relacionado a uma intensificação do processo inflamatório através da interação dos receptores de membrana CD14 e TLR com os microorganismos. Para avaliar esta hipótese, estudou-se a participação da resposta inflamatória induzida por Chlamydophila pneumoniae (Cp) e Mycoplasma pneumoniae (Mp) em indivíduos com infarto agudo do miocárdio (IAM). Avaliou-se também, a possível associação entre polimorfismos dos genes CD14, TLR2, TLR4 e TNFA e a expressão dos genes IL6, TLR2, TLR4 e TNFA em leucócitos do sangue periférico, assim como a sua associação com o IAM. Para isso, foi realizado um estudo caso-controle constituído por pacientes com IAM e por indivíduos sem evidência de doença cardiovascular (grupo controle). As imunoglobulinas IgM e IgG séricas anti-Cp foram detectadas por imunofluorescência indireta. O DNA dos agentes infecciosos foi detectado no sangue periférico pela PCR em tempo real. A genotipagem dos polimorfismos TNFA -308G>A, IL6 -174G>C, CD14 -260C>T, TLR4 (Asp299Gli e Thr39911e) e TLR2 Arg753Gln e a quantificação relativa da expressão gênica nas células sanguíneas foram analisados pela PCR em tempo real. A porcentagem de positividade para DNA de Cp foi de 18,0% e 8,1% nos grupos IAM e controle (p=0,071), respectivamente, (p=0,071). Foram positivos para DNA de Mp, 5,0% e 11,2% dos indivíduos nos grupos IAM e controle, respectivamente (p=0,318). Sete indivíduos (7,1%) do grupo IAM tiveram títulos anti-Cp IgG positivos (1:512) e 3,9% dos indivíduos do grupo controle (p=0,718). A expressão do TLR4 foi significantemente menor no grupo IAM (0,00113±0,00102) comparado ao grupo controle (0,00144±0,000806; p=0,003). As frequências genotípicas e alelicas dos polimorfismos TNFA -308G>A, CD14 -260C>T, TLR4 (Asp299GIi e Thr39911e) e TLR2 Arg753Gln foram similares entre os grupos estudados (p>0,05) sugerindo que esses polimorfismos não estão associados com IAM nesta amostra populacional. No grupo IAM, houve associação entre o genótipo -260CT+TI CD14 com títulos IgG anti-Cp detectados na diluição 1:16 (p=0,042). Da mesma forma, o alelo A do polimorfismo -308G>A TNF-&#945; foi associado com títulos positivos de IgG anti-Cp na diluição 1:512 (p=0,0058). No grupo IAM, pacientes positivos para DNA de Cp tiveram maiores concentrações de fibrinogênio do que pacientes negativos para este agente infeccioso (541,8±161,5mg/dL e 450,5±196,8mg/dL, respectivamente; p=0.043). Os agentes infecciosos Chlamydophila pneumoniae e Mycoplasma pneumoniae não foram significantemente mais frequentes em indivíduos que tiveram infarto agudo do miocárdio em relação ao grupo controle, porém houve uma associação, no grupo IAM, entre positividade para DNA de C. pneumoniae e concentrações mais elevadas de fibrinogênio. / Atheroma plaque instability has been attributed to the presence of some infectious agents. This mechanism may be related with increased stimulus of inflammatory process through interactions of CD14 and TLR with infectious agents. In this present study, it was evaluated the association of the presence of Chlamydophila pneumoniae and Mycoplasma pneumonia with acute myocardial infarction (AMI). A case-control study was conducted with AMI patients and non-AMI individuais as controls. Immunoglobulin G (lgG) and IgM antibodies anti-Chlamydophila pneumoniae were detected by indirect immunifluorescent assay and the Cp DNA and Mp DNA were detected by real time PCR (RT-PCR) in peripheral blood cells. Using the same method, the individuals were genotyped and the gene expressions of TLR2, TLR4, IL-6 e TNF-&#945; were evaluated by RT-qPCR. In AMI patients, Cp DNA and Mp DNA were positive in 18,0% and 5,0% samples, respectively. In controls, 8,1% and 11,2% were positive for Cp DNA and Mp DNA, respectively. TLR4 expression was significantly decreased in AMI patients (0.00113±0.001 02) compared with controls (0.00144±0.000S06; p=0.003). The frequencies of -308G>A TNF-&#945;., -260C>T CD14, Asp299Gli TLR4, Thr39911e TLR4e Arg753Gln TLR2 SNPs in AMI group were similar to those found in controls. On the other hand, In AMI group, the -260CT+TT CD14 genotype was associated with anti-CP IgG antibody titer of 1/16. Likewise, the rare allele of -308G>A TNF-&#945; was associated with anti-CP IgG antibody titer of 1/16. Cp DNA positive patients had high concentration of fibrinogen when compared with negative patients. In conclusion, Cp DNA and Mp DNA positivity were not associated with AMI.

Escherichia coli produtoras de toxina de Shiga (STEC) são patógenos veiculados por alimentos capazes de causar desde diarréia branda até severa e sanguinolenta e evoluir para complicações graves como colite hemorrágica, síndrome hemolítica urêmica e púrpura trombótica trombocitopênica. Esses microrganismos têm sido associados a numerosos surtos e vários casos esporádicos de infecções em todo o mundo devido ao consumo de alimentos contaminados. O sorogrupo O157 desse grupo de microrganismos é considerado o principal devido ao seu envolvimento em surtos de doença por STEC, entretanto, muitos casos vêm ocorrendo em todo o mundo devido a cepas patogênicas de STEC não-O157, como O26, O103, O111 e O145. Os objetivos do presente trabalho foram avaliar a ocorrência de STEC em três pontos da linha de abate de bovinos destinados à exportação e em cortes refrigerados de aves e de bovinos comercializados na região da Grande São Paulo; pesquisar a presença dos fatores de virulência dos isolados através dos genes stx1, stx2, eaeA e ehxA; identificar isolados de E. coli O157:H7 pela pesquisa dos genes uidA, rfbO157 e flicH7; verificar a citotoxicidade em células Vero; pesquisar a atividade enterohemolítica dos isolados; avaliar o perfil de suscetibilidade a antimicrobianos; identificar os sorotipos e avaliar a diversidade genética dos isolados de STEC obtidos. Na linha de abate, 201 animais foram amostrados, obtendo-se 603 amostras que compreenderam 201 amostras provenientes do couro, 201 de carcaça e 201 de meia carcaça. No varejo, foram analisadas 100 amostras de cortes de carne bovina e 100 de cortes de frango. A metodologia utilizada para detecção de E. coli sorogrupo O157 foi a preconizada pela ISO 16654, enquanto para os sorogrupos O26, O103, O111 e O145 foi empregada a metodologia descrita pelo \"Surveillance Group for Diseases and Infections of Animals\" (NRM 006). Os isolados obtidos foram confirmados como STEC pela técnica de PCR. Dos 201 animais amostrados, dois (1,0%) foram positivos para STEC, obtendo-se sete isolados (três do animal número 399 e quatro do animal 401) do couro. Não houve o isolamento do microrganismo nas amostras de carcaça e meia carcaça. Os sete isolados apresentaram o perfil stx2, uidA, eaeA, ehxA, rfbO157 e fliCH7 podendo, assim, ser considerados E. coli enterohemorrágica (EHEC) pertencentes ao sorotipo O157:H7. Na avaliação da atividade enterohemolítica, nenhum dos isolados expressou essa proteína e, com relação ao teste de suscetibilidade antimicrobiana, três (42,8%) isolados apresentaram resistência ao ácido nalidíxico e um (14,3%) ao cloranfenicol. O PFGE revelou que as sete cepas de STEC isoladas apresentaram dois perfis genéticos distintos, com similaridade entre eles de 75,3%. STEC não foi detectada nas amostras de carne bovina e de aves comercializadas no varejo. Estes resultados sugerem que, apesar de presente no couro dos animais, o emprego de medidas sanitárias eficientes ao longo da cadeia de produção da carne bovina até sua comercialização na forma de corte, contribui para que o isolamento de STEC nas etapas posteriores seja raro. / Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that can cause since mild or severe and bloody diarrhea to serious complications, such as hemorrhagic colitis, hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. These microorganisms have been associated with numerous outbreaks and several sporadic cases worldwide due to consumption of contaminated food, especially meat. E. coli O157 is considered the main serogroup involved in disease outbreaks of STEC, however, many cases have been occurred worldwide due to non-O157, such as O26, O103, O111 and O145 strains. The aims of the present study were to determine the occurrence of STEC at three points of cattle slaughter for exporting and in refrigerated beef and poultry cuts commercialized in the Metropolitan area of Sao Paulo, Brazil; identify the genes that code for the virulence factors stx1, stx2, eaeA and ehxA; detect E. coli O157:H7 strains using uidA, rfbO157 and flicH7 genes; verify the citotoxicity in Vero cells and the enterohemolytic activity; evaluate the antimicrobial susceptibility profile; identify the STEC serotypes and evaluate the genetic diversity of STEC isolates. A total of 603 samples were collected from 201 animals at slaughter. The samples were taken from hide (201), carcass (201) and half-carcass (201) and were always collected from the breast region. At retail, 100 refrigerated beef cuts and 100 chicken cuts were analyzed. The detection of E. coli O157 samples were conducted according to the ISO methodology (16654) and for detection of O26, O103, O111 e O145 serogroups the Surveillance Group for Diseases and Infections of Animals methodology (NRM 006) was used. The isolates were confirmed as STEC by PCR technique. Among 201 animals sampled, two (1,0%) were positive for STEC, obtaining seven isolates from hide (three from animal number 399 and four from animal number 401). The microrganism was not detect in carcass and half carcass samples. The seven isolates carried stx2, uidA, eaeA, ehxA, rfbO157 and fliCH7 genes, so, they can be considered as enterohaemorrhagic E. coli (EHEC) O157:H7 serotype. None of the isolates produced the enterohemolytic activity and three (42,8%) isolates showed resistence to nalidixic acid and one (14,3%) to chloramphenicol. PFGE revealed that the seven STEC strains showed two distinct genetic profiles, with 75.3% of similarity between them. STEC was not detected from beef and poultry cuts commercialized at retail. These results suggest that, although present in animals hides, the STEC isolation at later stages of food chain was rare, probably due to effective sanitary measures to control contamination and transmission of this pathogen along the beef production chain until commercialization.

Resumo: A soja se destaca com um dos principais produtos do agronegócio brasileiro. No entanto, o ataque de insetos desfolhadores como Anticarsia gemmatalis comprometem a produtividade. O controle dessa praga é baseado na aplicação de inseticidas químicos e no uso de plantas Bt. Contudo, a seleção de populações resistentes é uma séria limitação para o manejo dessa praga. Em vista disso, torna-se necessário um sistema de manejo que também auxilie a retardar a evolução da resistência. Uma alternativa é o uso de agentes de controle microbiano, com o uso de bioinseticidas à base da bactéria Bacillus thuringiensis (Bt). Entretanto, a principal limitação do uso desses bioinseticidas é elevada sensibilidade a fatores climáticos, como radiação UV, temperatura e umidade. A maioria dos estudos disponíveis sobre persistência de Bt são antigos e foram realizados no hemisfério Norte em espécies ornamentais e florestais, onde as condições climáticas diferem do hemisfério Sul. Os bioinseticidas a base de Bt são utilizados em todo o mundo, visando o controle de pragas agrícolas. Dentre as suas principais vantagens em relação aos inseticidas convencionais se destacam a seletividade aos inimigos naturais e risco reduzido ao meio ambiente. Apesar de algumas limitações, esses bioinseticidas foram muito importantes para suprimir surtos recentes de lepidópteros praga no Brasil, onde os inseticidas convencionais falharam. Diante disso, faz-se necessário estudos de persistência em condições de campo. Os ens... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The soybean stands out as one of the main products of Brazilian agribusiness. However the attack of defoliating insects such as Anticarsia gemmatalis compromises productivity. Control of this pest is based on the application of chemical insecticides and the use of Bt plants. However, the selection of resistant populations is a serious limitation for the management of this pest. Given this, a management system that also helps to retard the evolution of resistance becomes necessary. An alternative is the use of microbial control agents with the use of Bacillus thuringiensis (Bt) -based Biopesticide. However, the main limitation of the use of these bioinsecticides is their high sensitivity to climatic factors such as UV radiation, temperature, and humidity. Most of the available studies on Bt persistence are old and have been carried out in the northern hemisphere on ornamental and forest species, where climatic conditions differ from the southern hemisphere. Bt B. thuringiensisbased Bioinsecticides are used worldwide to agricultural pest control. Among its main advantages over conventional insecticides are selectivity to natural enemies and reduced risk to the environment. Despite some limitations, these bioinsecticides have been very important in suppressing recent outbreaks of pest Lepidoptera in Brazil, where conventional insecticides have failed. Therefore, persistence studies under field conditions are necessary. Field Bt persistence tests were conducted on soybean crop in... (Complete abstract click electronic access below) / Mestre

El objetivo principal de esta Tesis Doctoral fue evaluar la inactivación bacteriana usando Ultra Alta Presión de Homogeneización (UHPH) a 300 + 30 MPa de las cepas Listeria innocua ATCC 33090, Escherichia coli ATCC 10536, Escherichia coli O157:H7 CCUG 44857, Staphylococcus aureus ATCC 13565 y Staphylococcus carnosus CECT 4491 inoculadas en leche entera, leche desnatada y zumo de naranja. También, intentamos estudiar el efecto de la temperatura de entrada de la leche y del zumo en la máquina de alta presión sobre la letalidad y producción de daños subletales en estos microorganismos, así como la habilidad de estos para sobrevivir, recuperarse y crecer en condiciones de refrigeración después del tratamiento de UHPH. Dado que no existían protocolos previos de limpieza y desinfección de la máquina de UHPH que garantizara la correcta desinfección entre un ensayo y otro, el experimento 1 tuvo como objetivo evaluar la eficacia bactericida como reducción decimal (DR) de una mezcla de ácido peracético y peróxido de hidrógeno (PAHP) contra cepas de Staphylococcus, Listeria y Escherichia coli en presencia de diferentes tipos de materia orgánica, tiempos de exposición y concentración de desinfectante. También se desarrollo de un procedimiento de limpieza y desinfección eficiente adaptado a la máquina de UHPH. Una vez ajustado y evaluado, el proceso de desinfección, el experimento 2 tuvo como finalidad evaluar la eficacia bactericida de los tratamientos de UHPH contra Listeria innocua ATCC 33090 inoculada en leche entera UHT y zumo de naranja UHT, así como estudiar el efecto de la temperatura de entrada en la máquina sobre la letalidad y la producción de daños subletales en este microorganismo. Así mismo se estudió su habilidad de sobrevivir, repararse y crecer almacenado en refrigeración después del tratamiento de UHPH. Los experimentos 3 y 4 tuvieron como objetivo evaluar la inactivación por UHPH de Escherichia coli ATCC 10536 y Escherichia coli O157:H7 CCUG 44857 inoculadas en leche entera UHT, leche desnatada UHT y zumo de naranja UHT, considerando el efecto de la temperatura de entrada de la leche y el zumo, en los valores de letalidad y la capacidad del tratamiento de producir daños subletales en estas cepas. También estudiamos la habilidad de estos microorganismos de sobrevivir, repararse y crecer durante su conservación a 4,0 ºC tras el tratamiento de UHPH. El experimento 5 tuvo como propósito evaluar la inactivación inducida por UHPH en Staphylococcus aureus ATCC 13565 y Staphylococcus carnosus CECT 4491 inoculados en leche entera UHT y zumo de naranja UHT, considerando el efecto de la temperatura de entrada de la muestra en los valores de letalidad, así como la producción de daños subletales en estas cepas, y su capacidad de sobrevivir, repararse y crecer almacenados a bajas temperaturas tras el tratamiento de UHPH. Con respecto a la evaluación del desinfectante, Staphylococcus spp. mostró mayor resistencia a bajas concentraciones de desinfectante que las cepas de Escherichia coli y Listeria spp. El huevo fue la materia orgánica con mayor capacidad interferente mientras que el zumo de naranja presentó la menor capacidad interferente. Sin embargo, el PAHP fue efectivo (reducciones > 5 log UFC/mL) a concentraciones superiores a 0,1 % y 10 minutos de exposición, en todos los casos. No fueron encontradas diferencias estadísticas entre cepas patógenas y no patógenas del mismo grupo. En cuanto a los tratamientos de inactivación por UHPH, en Listeria innocua ATCC 33090, tanto la temperatura de entrada como el tipo de matriz influenciaron significativamente (P &#8804; 0,05) su inactivación, que fue mayor a una temperatura de entrada de 20 ºC. Los tratamientos por UHPH no causaron daños subletales en Listeria innocua. Durante el almacenamiento a 4 ºC después de los tratamientos, los recuentos de Listeria innocua en leche aumentaron alrededor de 2 unidades logarítmicas entre los días 0 y 9, mientras que en zumo de naranja disminuyeron 2,5 unidades logarítmicas entre los días 0 y 18. El nivel de inactivación fue similar para ambas cepas de Escherichia coli en leche y no fueron detectados daños subletales después de los tratamientos tanto en leche como en zumo de naranja. La temperatura de entrada así como el tipo de matriz influenciaron (P &#8804; 0,05) el grado de inactivación en las cepas de Escherichia coli, alcanzando su máximo nivel a una temperatura de entrada de 20 ºC. La evolución de los recuentos viables durante el almacenamiento a 4,0 ºC de muestras presurizadas fue similar en leche entera y desnatada, aunque la cepa E. coli ATCC 10536 no mostró diferencias significativas entre los días 0 y 9 de almacenamiento, mientras que la cepa E. coli CCUG 44857 mostró una tendencia decreciente de aproximadamente 0,3 unidades logarítmicas. En zumo de naranja, las cepas de Escherichia coli mostraron una mayor tendencia decreciente y la cepa CCUG 44857 fue más resistente al bajo pH, manteniendo recuentos altos para el día 27. En cepas de Staphylococcus, la temperatura de entrada, la matriz y la cepa influenciaron significativamente (P &#8804; 0,05) el nivel de inactivación, alcanzando sus mayores niveles a 20 ºC. No se observaron daños subletales tras los tratamientos en ninguno de los casos. La evolución de los recuentos viables de ambas cepas mostró una fuerte tendencia decreciente después del día 3 de almacenamiento a 4,0 ºC en zumo de naranja, mientras que la cepa Staphylococcus carnosus CECT 4491 mostró una baja tendencia y una gran resistencia cuando fue inoculada en leche y presurizada a bajas temperaturas. El PAPH fue efectivo contra todas las cepas probadas a concentraciones por encima de 0,1 % y tiempos de exposición superiores a 10 minutos para todos los tipos de materias orgánicas estudiadas. Los tratamientos de UHPH de leche entera, desnatada y zumo de naranja a 300 + 30 MPa y una temperatura de entrada de 6 y 20 ºC fueron eficientes para reducir los recuentos viables de Listeria innocua ATCC 33090, Escherichia coli ATCC 10536, Escherichia coli O157:H7 CCUG 44857, Staphylococcus aureus ATCC 13565 y Staphylococcus carnosus CECT 4491. La letalidad incrementó con la temperatura de entrada, sugiriendo un claro efecto de la temperatura en los valores de letalidad. El tipo de matriz influenció significativamente tanto la eficacia de los tratamientos de UHPH como la capacidad de crecimiento durante el subsiguiente periodo de almacenamiento a bajas temperaturas. Los tratamientos de UHPH no causaron daños subletales aparentes en ninguno de los casos. La tecnología de las UHPH puede ofrecer una prominente alternativa para la pasteurización de la leche, zumos y otros alimentos líquidos combinado con la posibilidad de incrementar la temperatura de entrada de las muestras antes del proceso. / The main objective of this Doctoral Thesis was to evaluate the bacterial inactivation using Ultrahigh-pressure Homogenisation (UHPH) to 300 + 30 MPa of Listeria innocua ATCC 33090, Escherichia coli ATCC 10536, Escherichia coli O157:H7 CCUG 44857, Staphylococcus aureus ATCC 13565 and Staphylococcus carnosus CECT 4491 inoculated into whole milk, skim milk and into orange juice. Also, we intended to study the effect of inlet temperature of the milk and orange juice in the machine on the lethality and production of sublethal injuries in these microorganisms and its ability for survival, repair and growth in refrigerated storage after UHPH treatment. The objective of experiment 1 was to evaluate the bactericidal efficacy as decimal reduction (DR) of a mixture of peracetic acid and hydrogen peroxide (PAHP) against strains of Staphylococcus, Listeria and Escherichia coli in presence of different organic matter types, exposure times and disinfectant concentrations. Also, we develop an efficient procedure of cleaning and disinfection adapted to the UHPH machine. Experiment 2 had the purpose of evaluating the bactericidal efficacy of UHPH treatments against Listeria innocua ATCC 33090 inoculated into UHT milk and UHT orange juice. We also studied the effect of inlet temperature on the degree of inactivation of this microorganism and the capacity of this UHPH treatment to cause sublethal injuries when inoculated into whole milk and orange juice. We also determined its ability for survival, repair and growth under refrigeration after the UHPH treatment. The experiments 3 and 4 had as objective to evaluate the inactivation by UHPH of Escherichia coli ATCC 10536 and Escherichia coli O157:H7 CCUG 44857 inoculated into UHT whole milk, UHT skim milk and UHT orange juice considering the effect of inlet temperature on the lethality values and the production of sublethal injuries in these strains. We also studied the ability of the microorganisms to repair and growth during storage at 4.0 ºC after treatment. The experiment 5 had as purpose to evaluate the inactivation induced by UHPH of Staphylococcus aureus ATCC 13565 and Staphylococcus carnosus CECT 4491 inoculate into UHT whole milk and UHT orange juice considering the effect of inlet temperature of sample on the lethality values reached and the production of sublethal injuries in these strains, as well as their ability to survive, repair and growth when stored at low temperatures after UHPH treatments. In the evaluation of the disinfectant, Staphylococcus spp. showed more resistance at low concentrations of disinfectant than strains of E. coli and Listeria spp. Egg was the organic matter with the greatest interfering capacity and orange juice was the one with least interfering capacity. However PAHP was effective (reductions > 5 log CFU/mL) in all cases from 0.1 % PAHP and 10 min of exposure. No statistical differences were found between pathogenic and non pathogenic strains in the same group. Concerning the effect of UHPH treatment, in Listeria innocua ATCC 33090 both the inlet temperature and the food matrix influenced significantly (P < 0.05) the level of inactivation reached, which was higher in whole milk with inlet at 20 ºC. The UHPH treatment caused few or no sublethal injuries in L. innocua. During storage at 4.0 ºC after treatments, counts increased by approximately two logarithmic units from days 0 to 9 in whole milk, while in orange juice counts diminished by approximately 2.5 logarithmic units from days 0 to 18. The level of inactivation was similar for both strains of E. coli and no sublethal injuries were detected after treatments in milk as in juice of orange. Both the inlet temperature and the type of milk influenced significantly (P < 0.05) the degree of inactivation reached in both strains of E. coli, being higher at 20 ºC. The evolution of viable counts during storage at 4.0 ºC was similar in whole and skim milk but although ATCC 10536 strain counts did not show significant differences between the days 0 and 9 of storage, the strain CCUG 44857 showed a decreasing tendency of approximately 0.3 logarithmic units. In orange juice the strains of Escherichia coli showed a greater decreasing tendency and the strain CCUG 44857 was more resistant at low pH, maintaining high viable counts until 27th of storage. In Staphylococcus strains the inlet temperature, food matrix and the strain influenced significantly (P < 0.05) the lethality level, which was higher with 20 ºC of inlet temperature. We not observed sublethal injuries after treatments in any case. The evolution of viable counts showed for both strains a very strong decreasing tendency after 3 days of storage at 4 ºC for orange juice, although S. carnosus CECT 4491 showed a low decreasing tendency and great resistance when was inoculated in milk and pressurized at low temperatures. The PAHP was effective against all strains tested at concentrations above 0.1 % and exposure times over 10 minutes for all the organic matters studied. The UHPH treatments of whole milk, skim milk and orange juice at 300 + 30 MPa at inlet temperatures of 6 and 20 ºC were efficient to reduce the viable counts of Listeria innocua ATCC 33090, Escherichia coli ATCC 10536, Escherichia coli O157:H7 CCUG 44857, Staphylococcus aureus ATCC 13565 and Staphylococcus carnosus CECT 4491. The lethality increased with the inlet temperature, suggesting a clear effect of the temperature on the lethality values. The kind of matrix significantly influenced both the efficacy of the treatment and the growing capability during the subsequent storage period at low temperatures. Apparently UHPH treatment (300 + 30 MPa) did not cause significant sublethal injuries in any of the cases. UHPH technology may offer a promising alternative to the pasteurization of milk, juices and others liquid foods in combination with an increase of the inlet temperature of the sample before the process.

Esta investigación se enmarca en una de las principales líneas que en la actualidad movilizan las reflexiones en la didáctica de las ciencias: la influencia del uso de modelos en el desarrollo del conocimiento científico de los estudiantes. En particular, el proceso de construcción y revisión del modelo ser vivo por niños y niñas de segundo curso del primer ciclo en una escuela pública de Barcelona durante la realización de una secuencia didáctica sobre los microorganismos. El objetivo principal consistió en analizar cómo los distintos modos comunicativos y las actividades experimental, lectora y comparativa contribuyen a la construcción del modelo ser vivo por estos alumnos. La implementación en el aula de la unidad didáctica diseñada permitió la obtención de datos de la investigación. Los instrumentos de recogida de datos fueron: cuestionarios KPSI (inicial y final), producciones gráficas, escritas y orales. El análisis de los datos se hizo a partir del análisis del contenido de las producciones de los alumnos, lo que permitió identificar la contribución de cada modo comunicativo y de cada actividad a partir del desempeño alcanzado en la progresión de aprendizaje al construir el modelo ser vivo. Así mismo, desde el marco teórico de la multimodalidad fue posible reconocer la relación de cooperación o de especialización de los distintos modos comunicativos en este proceso. Los resultados analizados revelaron que cada modo comunicativo contribuyó de forma específica. La representación gráfica permitió al alumnado expresar el proceso de evolución gradual de las caries, los elementos que creían esenciales para este proceso, el tamaño y cantidad del bicho, su morfología (siendo la más frecuente la similar a un insecto o humanoide) y su ubicación. La representación textual y oral permitió al alumnado enunciar procesos de interacción entre el alimento, la caries y el ser vivo, así mismo a evidenciar la confusión entre los conceptos caries y bacteria. Analizando los tres modos comunicativos se observa que la relación más frecuente entre ellos es la especialización, en las representaciones individuales el dibujo alcanzó un mayor nivel de desempeño en la progresión de aprendizaje con relación a los demás modos comunicativos. Así mismo, la cooperación entre los modos comunicativos fue encontrada en otros casos, cuando las representaciones del alumnado se encontraban en el mismo nivel de desempeño. En las discusiones entre iguales, constatamos que a través del modo oral se alcanza un nivel mayor de desempeño, en tres de los cuatro grupos, caracterizando por tanto una relación de especialización con el dibujo y el texto y mostrando el potencial del discurso oral en la construcción de conocimiento. A través del análisis del contenido de los elementos argumentativos generados en la discusión entre iguales, se observó que el alumnado dio mucha importancia a aspectos formales del dibujo, siendo el color el más representativo. Los aspectos más fundamentales del modelo se produjeron una vez decididos los aspectos formales. La influencia de las tres actividades propuestas fue diversa. El planteamiento de la actividad experimental – elaboración del yogur – fue demasiado ambicioso y los alumnos no fueron capaces de reconocer la importancia del medio en la vida de las bacterias, aunque si permitió discutir el efecto de la temperatura. La actividad lectora permitió reconocer de manera explícita la relación entre consumo de dulces, reproducción y crecimiento de las bacterias y desarrollo de las caries. Así mismo, la actividad comparativa de la bacteria con otro ser vivo (conejo) permitió a los estudiantes generalizar el modelo ser vivo construido en el curso anterior, aplicándolo a un organismo microscópico. Esta actividad permitió al alumnado progresar en su nivel de desempeño al reconocer la presencia de células en ambos seres vivos. Analizar el proceso de construcción del modelo ser vivo a través de la identificación de niveles de desempeño en la progresión de aprendizaje facilita el reconocimiento de dificultades y puede orientar en el diseño de las secuencias de aprendizaje implementadas en el aula. / This research falls within one of the main lines that currently move the reflections on science education: the influence of the use of models in the development of scientific knowledge of the students. In particular, in the process of construction and revision of the living being model by children in the second year of primary education in a public school in Barcelona during the performance of a learning sequence on microorganisms. The main objective was to analyze to what extent the different communicative modes and experimental, reading and comparative activities contribute to the construction of the living being model of these students. The implementation of the designed learning sequence allowed obtaining research data. Data was collected using KPSI questionnaires (collected at the beginning and at the end of the sequence), drawings of the students as well as their written and oral productions. Data analysis consisted in content analysis of the worksheets of the students, which allowed to identify the contribution of each communicative mode as well as the contribution of each activity from the performance achieved in the learning progression in the construction of the living being model. Likewise, the theoretical framework of multimodality was used to analyze and recognize the relationship between the different communicative modes in this process, which could be cooperation or specialization. The results revealed that each communicative mode contributed in a specific way to the construction of the living being model. The graphic representation (drawings) allowed students to express the gradual process of evolution of dental caries process as well as the components they believed essential to this process: size and quantity of the bug, its morphology (most frequently being similar to an insect or humanoid) and its location. Oral and textual representations allowed students to spell out the processes of interaction between food, dental caries and a living being, it also allowed to highlight the conceptual confusion between dental caries and bacteria. By analyzing the three communicative modes it is observed that the most common relation between them is the specialization: in the individual representations the drawing reached a higher level of performance in learning progression than the other communicative modes. Furthermore, the relation of cooperation between communicative modes was found in other cases, where representations of students reached the same level of performance. In peer discussion activities, it can be noted that through the oral mode a higher level of performance is reached in three of the four groups, therefore characterizing a relationship of specialization between drawing and text, and showing the potential of oral discourse in the construction of knowledge. Through the content analysis of the argumentative elements generated during the peer discussion, it was noted that students gave great importance to formal aspects of the drawing, the color being the more representative. The most fundamental aspects of the model ocurred once the formal features were set. The influence to the construction of the living being model of the three proposed activities was different. The approach of the experimental activity - preparation of yogurt - was too ambitious and the students were not able to recognize the importance of the environment in the life of the bacteria, but it allowed to discuss the effect of temperature. The reading activity allowed to recognize explicitly the relationship between consumption of candies, reproduction and growth of bacteria and the development of dental caries. Furthermore, the comparative activity between bacteria and another living being (a rabbit) allowed students to generalize the living being model (which they had worked in the previous year), applying it to a microscopic organism. This activity allowed the students to progress in their level of performance to recognize the presence of cells in both living beings. Analyzing the process of construction of the living being model by the identification of levels of performance in a learning progression, facilitates the recognition of difficulties and can guide the design of learning sequences to be implemented in the classroom.