Lisozimas são enzimas que fazem parte do mecanismo de defesa contra bactérias, no entanto lisozimas com função digestiva também são encontradas no trato digestivo de vertebrados e no intestino médio de insetos. As lisozimas digestivas de insetos são do tipo c e assim compartilham semelhanças estruturais e mecanísticas com a lisozima da clara de ovo de galinha (HEWL). Entretanto, para desempenhar sua função digestiva, as lisozimas de insetos apresentam algumas propriedades particulares entre as quais se destaca um pH ótimo mais ácido em relação às lisozimas não-digestivas. Para elucidar as bases moleculares dessa diferença no pH ótimo, duas lisozimas digestivas (lisozima 1 AAQ20048 e lisozima 2 AAQ20047) da larva de Musca domestica (mosca Diptera Cyclorrhapha), clonadas em Pichia pastoris e purificadas, foram caracterizadas estruturalmente e cineticamente com o substrato sintético (MUQ3) e natural (cápsulas de Micrococcus lysodeikticus). Foi observado que o efeito do pH na atividade das lisozimas 1 e 2 sobre o MUQ3 é uma curva com formato de sino e pH ótimo mais ácido que o da HEWL. Essas curvas foram reflexos da diminuição simultânea dos valores de pKas do nucleófilo e do doador de prótons. Estruturas cristalográficas das lisozimas digestivas de Musca domestica foram obtidas a 1,9 Å e análise comparativa com a estrutura terciária da HEWL revelou resíduos de aminoácidos no ambiente do nucleófilo (N46) e do doador de prótons (S106 e T107) que podem estar envolvidos na modulação das constantes de ionização dos resíduos essenciais à catálise. Esses resíduos foram substituídos via mutagênese sítio-dirigida por D, V e A respectivamente e três mutantes simples (N46D, S106V e T107A) e um triplo (N46DS106V- T107A) foram produzidos e purificados. Caracterização revelou que as contribuições individuais da N46, S106 e T107 foram pequenas e próximas do limite de detecção da técnica utilizada. Por outro lado, o conjunto dos 3 aminoácidos foi responsável pelo pH ótimo ácido frente ao substrato sintético, elevando os valores de pKas do nucleófilo e doador de prótons para valores muito semelhantes ao da HEWL. Diferentemente, essa tripla mutação não foi suficiente para elevar o pH ótimo da lisozima 2 sobre cápsulas de Micrococcus lysodeikticus para valores próximos àqueles de HEWL, sugerindo que as bases moleculares do pH ótimo frente ao substrato natural e sintético são diferentes. Uma comparação estrutural entre lisozima 1 e HEWL sugere que os resíduos de aminoácidos carregados na superfície dessas lisozimas sejam importantes para determinação do pH ótimo. A investigação dessa hipótese foi feita substituindo 5 aminoácidos neutros e 1 ácido, via mutagênese sítio-dirigida, por resíduos básicos. A caracterização do mutante sêxtuplo revelou um aumento significativo nos valores de pH ótimo da lisozima 1, indicando que a redução da basicidade da superfície das lisozimas digestivas é determinante para seus pHs ótimos ácidos. / Lysozymes are enzymes that are part of the defence mechanism against bacteria, however lysozymes with digestive function are also found in the digestive tract of vertebrates and in the insect midgut. The digestive lysozymes from insects are c type, so they share similar structural and mechanistic characteristics with hen egg-white lysozyme (HEWL). However, to perform their digestive function, insect lysozymes present some particular properties among them a more acidic pH optimum than that of non-digestive lysozymes. To elucidate the molecular basis of this pH optimum difference, two digestive lysozymes (lysozyme 1 AAQ20048 and lysozyme 2 AAQ20047) from Musca domestica larvae (housefly Diptera Cyclorrhapha), cloned in Pichia pastoris and purified, were structurally and kinecticly characterized with synthetic (MUQ3) and natural (lyophilized cells of Micrococcus lysodeikticus) substrates. It was observed that the pH effect on the activity of lysozymes 1 and 2 upon MUQ3 is a bell shaped curve exhibiting a more acidic pH optimum than that of HEWL. These curves result from simultaneous decrease of pKas values of the nucleophile and proton donor. Crystallographic structures of these digestive lysozymes from Musca domestica were obtained at 1.9 Å and comparative analysis with the terciary structure of HEWL revealed amino acid residues in the catalytic nucleophile (N46) and proton donor environment (S106 and T107) that may be involved in the modulation of ionization constants of those catalytic residues. N46, S106, and T107 were replaced via site-directed mutagenesis by D, V and A respectively and three simple (N46D, S106V and T107A) and one triple (N46D-S106V-T107A) mutants were produced and purified. Their characterization revealed that the individual contributions of N46, S106 and T107 were small and close to the detection borderline of the technique utilized. On the other hand, a set of these 3 amino acids was responsible by acidic pH optimum upon synthetic substrate, increasing the pKas values of nucleophile and proton donor to similar values to that of the HEWL. Differently, this triple mutation was not enough to increase the pH optimum of lysozyme 2 upon lyophilized cells of Micrococcus lysodeikticus to values close to those of HEWL, suggesting that the molecular bases of pH optimum upon natural and synthetic substrates are different. A structural comparison between lysozyme 1 and HEWL suggests that the charged amino acid residues on the surface of these lysozymes are important for pH optimum determination. The investigation of this hypothesis was done replacing 5 neutral and 1 acidic amino acids, via site-directed mutagenesis, by basic residues. The characterization of this mutant revealed a significant increase in the pH optimum values of lysozyme 1, suggesting that the reduction of basicity on the surface of the digestive lysozymes is a important factor in the determination of their acidic pH optimum.

Lisozimas são enzimas que fazem parte do mecanismo de defesa contra bactérias, no entanto lisozimas com função digestiva também são encontradas no trato digestivo de vertebrados e no intestino médio de insetos. As lisozimas digestivas de insetos são do tipo c e assim compartilham semelhanças estruturais e mecanísticas com a lisozima da clara de ovo de galinha (HEWL). Entretanto, para desempenhar sua função digestiva, as lisozimas de insetos apresentam algumas propriedades particulares entre as quais se destaca um pH ótimo mais ácido em relação às lisozimas não-digestivas. Para elucidar as bases moleculares dessa diferença no pH ótimo, duas lisozimas digestivas (lisozima 1 AAQ20048 e lisozima 2 AAQ20047) da larva de Musca domestica (mosca Diptera Cyclorrhapha), clonadas em Pichia pastoris e purificadas, foram caracterizadas estruturalmente e cineticamente com o substrato sintético (MUQ3) e natural (cápsulas de Micrococcus lysodeikticus). Foi observado que o efeito do pH na atividade das lisozimas 1 e 2 sobre o MUQ3 é uma curva com formato de sino e pH ótimo mais ácido que o da HEWL. Essas curvas foram reflexos da diminuição simultânea dos valores de pKas do nucleófilo e do doador de prótons. Estruturas cristalográficas das lisozimas digestivas de Musca domestica foram obtidas a 1,9 Å e análise comparativa com a estrutura terciária da HEWL revelou resíduos de aminoácidos no ambiente do nucleófilo (N46) e do doador de prótons (S106 e T107) que podem estar envolvidos na modulação das constantes de ionização dos resíduos essenciais à catálise. Esses resíduos foram substituídos via mutagênese sítio-dirigida por D, V e A respectivamente e três mutantes simples (N46D, S106V e T107A) e um triplo (N46DS106V- T107A) foram produzidos e purificados. Caracterização revelou que as contribuições individuais da N46, S106 e T107 foram pequenas e próximas do limite de detecção da técnica utilizada. Por outro lado, o conjunto dos 3 aminoácidos foi responsável pelo pH ótimo ácido frente ao substrato sintético, elevando os valores de pKas do nucleófilo e doador de prótons para valores muito semelhantes ao da HEWL. Diferentemente, essa tripla mutação não foi suficiente para elevar o pH ótimo da lisozima 2 sobre cápsulas de Micrococcus lysodeikticus para valores próximos àqueles de HEWL, sugerindo que as bases moleculares do pH ótimo frente ao substrato natural e sintético são diferentes. Uma comparação estrutural entre lisozima 1 e HEWL sugere que os resíduos de aminoácidos carregados na superfície dessas lisozimas sejam importantes para determinação do pH ótimo. A investigação dessa hipótese foi feita substituindo 5 aminoácidos neutros e 1 ácido, via mutagênese sítio-dirigida, por resíduos básicos. A caracterização do mutante sêxtuplo revelou um aumento significativo nos valores de pH ótimo da lisozima 1, indicando que a redução da basicidade da superfície das lisozimas digestivas é determinante para seus pHs ótimos ácidos. / Lysozymes are enzymes that are part of the defence mechanism against bacteria, however lysozymes with digestive function are also found in the digestive tract of vertebrates and in the insect midgut. The digestive lysozymes from insects are c type, so they share similar structural and mechanistic characteristics with hen egg-white lysozyme (HEWL). However, to perform their digestive function, insect lysozymes present some particular properties among them a more acidic pH optimum than that of non-digestive lysozymes. To elucidate the molecular basis of this pH optimum difference, two digestive lysozymes (lysozyme 1 AAQ20048 and lysozyme 2 AAQ20047) from Musca domestica larvae (housefly Diptera Cyclorrhapha), cloned in Pichia pastoris and purified, were structurally and kinecticly characterized with synthetic (MUQ3) and natural (lyophilized cells of Micrococcus lysodeikticus) substrates. It was observed that the pH effect on the activity of lysozymes 1 and 2 upon MUQ3 is a bell shaped curve exhibiting a more acidic pH optimum than that of HEWL. These curves result from simultaneous decrease of pKas values of the nucleophile and proton donor. Crystallographic structures of these digestive lysozymes from Musca domestica were obtained at 1.9 Å and comparative analysis with the terciary structure of HEWL revealed amino acid residues in the catalytic nucleophile (N46) and proton donor environment (S106 and T107) that may be involved in the modulation of ionization constants of those catalytic residues. N46, S106, and T107 were replaced via site-directed mutagenesis by D, V and A respectively and three simple (N46D, S106V and T107A) and one triple (N46D-S106V-T107A) mutants were produced and purified. Their characterization revealed that the individual contributions of N46, S106 and T107 were small and close to the detection borderline of the technique utilized. On the other hand, a set of these 3 amino acids was responsible by acidic pH optimum upon synthetic substrate, increasing the pKas values of nucleophile and proton donor to similar values to that of the HEWL. Differently, this triple mutation was not enough to increase the pH optimum of lysozyme 2 upon lyophilized cells of Micrococcus lysodeikticus to values close to those of HEWL, suggesting that the molecular bases of pH optimum upon natural and synthetic substrates are different. A structural comparison between lysozyme 1 and HEWL suggests that the charged amino acid residues on the surface of these lysozymes are important for pH optimum determination. The investigation of this hypothesis was done replacing 5 neutral and 1 acidic amino acids, via site-directed mutagenesis, by basic residues. The characterization of this mutant revealed a significant increase in the pH optimum values of lysozyme 1, suggesting that the reduction of basicity on the surface of the digestive lysozymes is a important factor in the determination of their acidic pH optimum.

Thesis (MScAgric)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The objective of this study was to evaluate the use of Musca domestica (common house fly) larvae meal, as protein source, for broiler production. This was done by investigating the nutritive value of house fly larvae meal together with its total tract digestibility, potential toxicity and carcass characteristics of the broilers supplemented with house fly larvae meal. The proximate analysis of house fly larvae meal show that it contained, on a dry matter basis, a gross energy value of 20.10 MJ/kg, 60.38% crude protein, 14.08% crude fat and 10.68% ash and that the house fly pupae contained a gross energy of 20.42 MJ/kg, 76.23% crude protein, 14.39% crude fat and 7.73% ash. House fly pupae meal had the closest match of amino acid profile when compared with the ideal amino acid profile required by broilers and it has arginine relative to the lysine content closer to the ideal amino acid profile than the house fly larvae meal. The essential fatty acid, linoleic acid, was found at levels of 26.25 and 36.27% of the total fats for the house fly larvae and pupae meal respectively. House fly larvae meal supplementation did not induce gizzard erosion or showed toxicity (regarding the gastro intestinal tract, immune system and organ stress) in broilers. Results revealed that house fly pupae meal had higher total tract digestibilities for most nutrients than of the house fly larvae meal. House fly larvae meal had a crude protein total tract digestibility of 69% and that of pupae meal was 79%. Both larvae and pupae meal had high amino acid total tract digestibilities of all the amino acids analysed. The house fly larvae and pupae meal had an apparent metabolizable energy (AME) value of 14.23MJ/kg and 15.15MJ/kg respectively. The larvae meal total tract crude fat and crude fibre digestibilities were 94% and 62% respectively. The pupae meal total tract crude fat and crude fibre digestibilities were 98% and 58% respectively. House fly larvae meal supplementation in a three phase feeding system significantly increased average broiler live weights at slaughter, total feed intake, cumulative feed intake as well as average daily gain (ADG) when compared to commercial maize: soya oil cake meal diet. In direct comparison of larvae inclusion levels with fishmeal in isonitrogenous and isoenergetic diet, no significant differences were observed between a 10% house fly larvae and a 10% fish meal diets regarding performance characteristic. The 25% house fly larvae meal diet yielded significantly better average broiler live weights at slaughter, total feed intake, cumulative feed intake (from the second week until slaughter) as well as average daily gain when compared to the 25% fish meal diet in the growth phases. Carcass characteristics of the 10% larvae, 10% fishmeal and commercial diets were compared. Chicks that received either the 10% house fly larvae meal or 10% fish meal supplementation produced significantly heavier carcasses and breast muscle portions than the chicks that received the commercial maize: soya oil cake meal. No treatment differences were found regarding breast and thigh muscle colour or pH. This study showed that house fly larvae meal can be regarded as a safe protein source that can be used to replace other protein sources and that has the ability to promote broiler performance without having any detrimental effects on carcass characteristics. / AFRIKAANSE OPSOMMING: Die evaluasie van Musca domestica (gewone huisvlieg) larwe meel as ‘n proteien bron vir braaikuiken produksie Die doel van die studie was om die effek van Musca domestica (gewone huisvlieg) larwe meel, as ‘n protein bron, in braaikuikens te evalueer. Dit was gedoen deur die nutrient waarde van huisvlieg larwe meel saam met die totale spysvertering verteerbaarheid, moontlike toksiesiteit en karkas-eienskappe van braai kuikens te evalueer. Laboratoruim analiese toon dat huisvlieg larwe meel 20.10 MJ/kg bruto energie, 60.38% ru- protein, 14.08% ru- vet en 10.68% as bevat en huisvlieg papie meel 20.42 MJ/kg bruto energie, 76.23% ru- protein, 14.39% ru- vet en 7.73% as bevat. Huisvlieg papie meel stem die meeste ooreen met die idiale amino suur profiel soos wat benodig word deur braaikuikens en dit het ‘n arginien tot lisien verhouding wat die meeste ooreenstem met die idiale amino suur profiel in vergelyking met huis vlieg larwe meel. Die essensiele vet suur, linolien suur, was geanaliseer teen vlakke van 26.25- en 36.27% van die totale vette onderskeidelik vir huisvlieg larwe- en papie meel. Huisvlieg larwe meel vervanging het nie spiermaag erosie of enige ander toksiese effekte te veroorsaak nie. Resultate het getoon dat huisvlieg papie meel, in vergelyking met larwe meel, het ‘n hoër totale spysvertering verteerbaarheid vir meeste van die nutrient. Die huisvlieg larwe meel het ‘n totale ru- protein spysvertering verteerbaarheid van 69% en die van papie meel van 79%. Beide larwe en papie meel het hoë amino suur spysvertering verteerbaarheid. Larwe meel en papie meel het skynbare metaboliseerbare energie waardes van 14.23MJ/kg en 15.15%MJ/kg onderskeidelik. Die larwe meel het ‘n ru-vet en ru- vesel spysvertering verteerbaarheid van 94% en 62% onderskeidelik, waar die papies ‘n ru-vet en ru- vesel spysvertering verteerbaarheid van onderskeidelik 98% en 58% het. Huisvlieg larwe meel vervanging in ‘n drie fase voer stelsel het getoon om die gemiddelde braaikuiken lewende gewigte by slag, totale voer iname, sowel as die gemiddelde daaglikse toename te verhoog waneer dit vergelyk word met ‘n kommersiele mielie- soya olie koek dieet. Geen mekwaardige verskille was waargeneem toe die 10% larwe meel dieet direk met die 10% vismeel diet vergelyk was rakende enige produksie einskappe gemeet nie. Die 25% larwe meel dieet het merkwaardig beter gemiddelde braaikuiken lewende gewigte by slag, totale voer iname, sowel as die gemiddelde daaglikse toename getoon wanneer vergelyk word met die 25% vismeel dieet gedurende die verskeie groei fases. Karkas eienskappe van die 10% larwe meel, 10% vismeel en die kommersiele diete was gevergelyk. Kuikens wat 10% larwe meel en 10% vismeel in die diete ontvang het, het swaarder karkasse gelewer met swaarder borsie massas wanneer vergelyk word met die kommersiele mielie- soya olie koek dieet. Geen behandelings verskille was gevind rakende die borsie- en dy spier kleure of pH nie. Die studie toon dat huisvlieg larwe meel as ‘n veillige protein bron kan beskou word, wat gebruik kan word om ander protein bronne te vervang. Huisvlieg larwe meel het ook die vermoë om braaikuiken produksie te verhoog sonder om enige negitiewe effekte rakende die karkas eienskappe te toon nie.

Thesis (MScAgric (Animal Sciences))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: The objective of this study was to evaluate the use of Musca domestica (common house fly) larvae meal, as protein source, for broiler production. This was done by investigating the nutritive value of house fly larvae meal together with its total tract digestibility, potential toxicity and carcass characteristics of the broilers supplemented with house fly larvae meal. The proximate analysis of house fly larvae meal show that it contained, on a dry matter basis, a gross energy value of 20.10 MJ/kg, 60.38% crude protein, 14.08% crude fat and 10.68% ash and that the house fly pupae contained a gross energy of 20.42 MJ/kg, 76.23% crude protein, 14.39% crude fat and 7.73% ash. House fly pupae meal had the closest match of amino acid profile when compared with the ideal amino acid profile required by broilers and it has arginine relative to the lysine content closer to the ideal amino acid profile than the house fly larvae meal. The essential fatty acid, linoleic acid, was found at levels of 26.25 and 36.27% of the total fats for the house fly larvae and pupae meal respectively. House fly larvae meal supplementation did not induce gizzard erosion or showed toxicity (regarding the gastro intestinal tract, immune system and organ stress) in broilers. Results revealed that house fly pupae meal had higher total tract digestibilities for most nutrients than of the house fly larvae meal. House fly larvae meal had a crude protein total tract digestibility of 69% and that of pupae meal was 79%. Both larvae and pupae meal had high amino acid total tract digestibilities of all the amino acids analysed. The house fly larvae and pupae meal had an apparent metabolizable energy (AME) value of 14.23MJ/kg and 15.15MJ/kg respectively. The larvae meal total tract crude fat and crude fibre digestibilities were 94% and 62% respectively. The pupae meal total tract crude fat and crude fibre digestibilities were 98% and 58% respectively. House fly larvae meal supplementation in a three phase feeding system significantly increased average broiler live weights at slaughter, total feed intake, cumulative feed intake as well as average daily gain (ADG) when compared to commercial maize: soya oil cake meal diet. In direct comparison of larvae inclusion levels with fishmeal in isonitrogenous and isoenergetic diet, no significant differences were observed between a 10% house fly larvae and a 10% fish meal diets regarding performance characteristic. The 25% house fly larvae meal diet yielded significantly better average broiler live weights at slaughter, total feed intake, cumulative feed intake (from the second week until slaughter) as well as average daily gain when compared to the 25% fish meal diet in the growth phases. Carcass characteristics of the 10% larvae, 10% fishmeal and commercial diets were compared. Chicks that received either the 10% house fly larvae meal or 10% fish meal supplementation produced significantly heavier carcasses and breast muscle portions than the chicks that received the commercial maize: soya oil cake meal. No treatment differences were found regarding breast and thigh muscle colour or pH. This study showed that house fly larvae meal can be regarded as a safe protein source that can be used to replace other protein sources and that has the ability to promote broiler performance without having any detrimental effects on carcass characteristics. / AFRIKAANSE OPSOMMING: Die doel van die studie was om die effek van Musca domestica (gewone huisvlieg) larwe meel, as ‘n protein bron, in braaikuikens te evalueer. Dit was gedoen deur die nutrient waarde van huisvlieg larwe meel saam met die totale spysvertering verteerbaarheid, moontlike toksiesiteit en karkas-eienskappe van braai kuikens te evalueer. Laboratoruim analiese toon dat huisvlieg larwe meel 20.10 MJ/kg bruto energie, 60.38% ru- protein, 14.08% ru- vet en 10.68% as bevat en huisvlieg papie meel 20.42 MJ/kg bruto energie, 76.23% ru- protein, 14.39% ru- vet en 7.73% as bevat. Huisvlieg papie meel stem die meeste ooreen met die idiale amino suur profiel soos wat benodig word deur braaikuikens en dit het ‘n arginien tot lisien verhouding wat die meeste ooreenstem met die idiale amino suur profiel in vergelyking met huis vlieg larwe meel. Die essensiele vet suur, linolien suur, was geanaliseer teen vlakke van 26.25- en 36.27% van die totale vette onderskeidelik vir huisvlieg larwe- en papie meel. Huisvlieg larwe meel vervanging het nie spiermaag erosie of enige ander toksiese effekte te veroorsaak nie. Resultate het getoon dat huisvlieg papie meel, in vergelyking met larwe meel, het ‘n hoër totale spysvertering verteerbaarheid vir meeste van die nutrient. Die huisvlieg larwe meel het ‘n totale ru- protein spysvertering verteerbaarheid van 69% en die van papie meel van 79%. Beide larwe en papie meel het hoë amino suur spysvertering verteerbaarheid. Larwe meel en papie meel het skynbare metaboliseerbare energie waardes van 14.23MJ/kg en 15.15%MJ/kg onderskeidelik. Die larwe meel het ‘n ru-vet en ru- vesel spysvertering verteerbaarheid van 94% en 62% onderskeidelik, waar die papies ‘n ru-vet en ru- vesel spysvertering verteerbaarheid van onderskeidelik 98% en 58% het. Huisvlieg larwe meel vervanging in ‘n drie fase voer stelsel het getoon om die gemiddelde braaikuiken lewende gewigte by slag, totale voer iname, sowel as die gemiddelde daaglikse toename te verhoog waneer dit vergelyk word met ‘n kommersiele mielie- soya olie koek dieet. Geen mekwaardige verskille was waargeneem toe die 10% larwe meel dieet direk met die 10% vismeel diet vergelyk was rakende enige produksie einskappe gemeet nie. Die 25% larwe meel dieet het merkwaardig beter gemiddelde braaikuiken lewende gewigte by slag, totale voer iname, sowel as die gemiddelde daaglikse toename getoon wanneer vergelyk word met die 25% vismeel dieet gedurende die verskeie groei fases. Karkas eienskappe van die 10% larwe meel, 10% vismeel en die kommersiele diete was gevergelyk. Kuikens wat 10% larwe meel en 10% vismeel in die diete ontvang het, het swaarder karkasse gelewer met swaarder borsie massas wanneer vergelyk word met die kommersiele mielie- soya olie koek dieet. Geen behandelings verskille was gevind rakende die borsie- en dy spier kleure of pH nie. Die studie toon dat huisvlieg larwe meel as ‘n veillige protein bron kan beskou word, wat gebruik kan word om ander protein bronne te vervang. Huisvlieg larwe meel het ook die vermoë om braaikuiken produksie te verhoog sonder om enige negitiewe effekte rakende die karkas eienskappe te toon nie.

Orientador: Arnaldo Yoshiteru Kuaye / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-08T04:17:50Z (GMT). No. of bitstreams: 1 Cardozo_GinaMariaBuenoQuirino_M.pdf: 1157718 bytes, checksum: 2612bc7c01ab1e98484d19addbdcb0b7 (MD5) Previous issue date: 2007 / Resumo: As moscas são consideradas responsáveis pela transmissão de doenças de origem alimentar ao atuarem como vetores mecânicos de microrganismos patogênicos, como Shigella sp, Salmonella Enteriditis, Escherichia coli O157:H7, Campylobacter jejuni desde o reservatório até os alimentos. Os produtos lácteos, principalmente os produzidos artesanalmente quase sempre em condições inadequadas de higiene, são os mais susceptíveis a estes tipos de vetores, representando sérios riscos à saúde do consumidor. A contagem total de bactérias aeróbias mesofílicas e de coliformes totais e fecais, a presença de microrganismos patogênicos em população de moscas domésticas e o potencial de transmissão dos mesmos para queijos Minas frescal foram estudados neste trabalho. Os resultados confirmaram a presença de Musca domestica em todos os ambientes de fabricação de queijo Minas frescal aqui estudados. As análises microbiológicas das amostras de moscas domésticas, matéria-prima (leite) e produto final (queijo Minas frescal), coletadas em local de fabricação de queijo Minas frescal artesanal, demonstraram uma correlação entre os microrganismos patogênicos presentes no ¿pool¿ de moscas, no leite e no queijo. As análises microscópicas das amostras de leite e queijo artesanal apresentaram matérias prejudiciais à saúde humana como mosca inteira, fragmentos de mosca e pêlos de roedor, indicando condições higiênico-sanitárias precárias nas etapas de obtenção do leite e processamento dos queijos. Foi verificado também o potencial de transmissão dos microrganismos patogênicos (Salmonella spp., Staphylococcus aureus, Listeria monocytogenes e Escherichia coli) das moscas em amostras comerciais de queijo Minas frescal ultrafiltrado. Através do processamento do queijo Minas frescal em planta piloto, pode-se verificar tanto a efetiva eliminação das matérias estranhas pela utilização da etapa de filtração do leite antes da fase de pasteurização, quanto o possível carreamento de microrganismos indesejáveis através do contato com homogeneizado de moscas. A capacidade de transmissão de microrganismos patogênicos de habitat contaminado artificialmente, via Musca domestica, para queijo Minas frescal pode ser verificado, comprovando assim o potencial de risco da presença de moscas domésticas em ambientes de fabricação de produtos lácteos/queijo / Abstract: The flies are considered responsible for the transmission of foodborne diseases origin when acting as mechanical vectors of pathogenic microrganisms, such as Shigella sp., Salmonella Enteriditis, Escherichia coli O157: H7, Campylobacter jejuni from the reservoir to food. The artisan dairy products are produced almost always in a non totally adequate conditions of hygiene. Therefore, the handmade dairy products are the most suitable to these types of vectors, representing serious risks to the consumer¿s health. The total counting of mesophilic microrganisms and total and fecal coliforms, the presence of pathogenic microrganisms in relation to the population of houseflies and the potential of transmission of the same for ¿Minas frescal¿ cheese were studied in this work. The results confirmed the presence of Musca domestica in all environments of manufacturing of ¿Minas frescal¿ cheese studied here. The microbiological analyses of the samples of houseflies, raw material (milk) and last item (¿Minas frescal¿ cheese), collected in places of manufacturing of handmade ¿Minas frescal¿ cheese, demonstrated the direct correlation between pathogenic microrganisms present in ¿pool¿ of flies, in milk and cheese. The microscopical analyses of the milk and handmade cheese, that characterized themselves for presenting harmful material to human health such as the whole fly, fly fragments and rodent hairs, indicating precarious hygienical-sanitary conditions in the stages of the attainment of milk and the processing of cheese. It was verified that there is a real potential of transmission of the pathogenic microrganisms (Salmonella spp., Staphylococcus aureus, Listeria monocytogenes and Escherichia coli) from the flies to a commercial sample of ¿Minas frescal¿ cheese ultrafiltered. Through the processing of the ¿Minas frescal¿ cheese in pilot laboratory, it could be verified the effective elimination of extraneous materials by the use of the stage of filtration of milk before the pasteurization phase, although there is a possible undesirable carrying of microrganisms through the homogeneized contact with flies. The capacity of transmission of pathogenic microrganisms of habitat contaminated artificially, through Musca domestica, to ¿Minas frescal¿ cheese can be verified, thus proving the risk potential of the presence of houseflies in environments of manufacturing of dairy products/cheese / Mestrado / Mestre em Tecnologia de Alimentos

Submitted by (edna.saturno@ufrpe.br) on 2016-10-19T17:11:18Z No. of bitstreams: 1 Marcia Paula Oliveira Farias.pdf: 458390 bytes, checksum: 177ae1686037c9ba17a91bd3068a2d56 (MD5) / Made available in DSpace on 2016-10-19T17:11:18Z (GMT). No. of bitstreams: 1 Marcia Paula Oliveira Farias.pdf: 458390 bytes, checksum: 177ae1686037c9ba17a91bd3068a2d56 (MD5) Previous issue date: 2007-02-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This study was performed with the aim of to evaluate the “in vitro” acaricidal activity of the oil of the seed of andiroba against Boophilus microplus, Rhipicephalus sanguineus and Anocentor nitens; the biological activity of the third stage larvae (L3) of Musca domestica and the activity of the oil of the seed of andiroba on the larval culture of gastrointestinal nematodes of goat and sheep. The acaricidal activity in the related species of tick was carried by means of the engorged females immersion test, using groups of 10 engorged females with three repetitions for treatment; for the test with the third stage larvae (L3) of Musca domestica it had been formed four repetitions with 10 larvae for treatment. Strips of filter paper measuring 8.5 x 1.5cm (L x W) were soaked in 0.3ml of the solutions and controls, and next, they were introduced in test tubes, adding 1.30g of vermiculite, being the tubes closed with absorbent cotton. Daily observations were made for detection of mortality of larvae, formation of pupae and adults emergence. The activity of the oil of andiroba on larval culture of gastrointestinalnematodes was determined by the calculation of the percentages of reduction of infective larvae for gram of faeces, using three repetitions. For each experiment it had been used dilutions of 100%, 50%, 30%, 25% and 10% (T1, T2, T3, T4 and T5 respectively) of the oil of the seed of andiroba, using tween 80 as detergent. Two groups had been formed as controls, one with deionized water and another one with tween 80 + deionized water. For the test of reduction of gastrointestinal infective larvae of nematodes, another group was added as positive control (doramectina). The results showed effectiveness of 100% for all the dilutions tested in the three species of ticks. In the test with L3 of de M. domestica, eight days after the accomplishment of the test, it was observed in the 100% concentration 20.0% of larval mortality and 62.5% of inhibition of emergency of adults, values significantly higher than those got in the other treated groups.In the test of reduction of gastrointestinal infective larvae of nematodes, effective reduction (> 90%) was obtained for 100%, 50% and 30% treatments in goat, and for all the treatments for sheep. / Avaliou-se “in vitro” a atividade acaricida do óleo da semente da andiroba contra Boophilus microplus, Rhipicephalus sanguineus e Anocentor nitens; a atividade biológica do óleo da semente de andiroba em larvas de terceiro estágio (L3) de Musca domestica e a atividade do óleo da semente da andiroba sobre o cultivo de larvas de nematóides gastrintestinais caprinos e ovinos. A atividade acaricida nas referidas espécies de carrapatos foi realizada por meio do teste de imersão de fêmeas ingurgitadas, utilizando-se grupos de 10 fêmeas ingurgitadas com três repetições por tratamento; para o teste com L3 de Musca domestica quatro repetições compostas por 10 larvas de terceiro estágio foram formadas. Tiras de papel filtro medindo 8,5 x 1,5cm (C x L) foram embebidas em 0,3ml das soluções do óleo de andiroba e controles, em seguida, colocadas dentro de tubos de ensaio, acrescentando-se 1,30g de vermiculita, introduzindo-se, então, as 10 larvas e vedando-se com algodão hidrófilo. Fez-se observação diária para detecção de mortalidade de larvas, pupação e emergência de adultos. A atividadedo óleo de andiroba sobre cultivos de L3 de nematóides gastrintestinais foi determinada pelo cálculo dos percentuais de redução de larvas infectantes por gramas de fezes (LPG), utilizando-se três repetições. Para cada experimento empregaram-se diluições de 100%, 50%, 30%, 25% e 10% (T1, T2, T3, T4 e T5 respectivamente) do óleo da semente de andiroba, utilizando-se tween 80 como dispersante. Formaram-se dois grupos controle, um com água destilada e outro com tween 80 + água destilada. Para o teste de redução de larvas infectantes de nematóides gastrintestinais, acrescentou-se um grupo controle positivo (doramectina). Os resultados revelaram eficácia de 100% para todas as diluições testadas nas três espécies de carrapatos. No teste com L3 de Musca domestica, após oito dias da realização do teste, na concentração de 100% obteve-se 20,0% de mortalidade larval e 62,5% de inibição de emergência de adultos, valoressignificativamente superiores aos obtidos nos demais tratamentos. No teste de redução do LPG de nematóides gastrintestinais, os resultados revelaram redução altamente efetiva (> 90%) no número de larvas totais para os tratamentos 100%, 50% e 30% em caprinos e em todos os tratamentos para a espécie ovina.

<div>Forensic entomology is a subdiscipline of entomology that involves the use of insect behavior and developmental data to aid in criminal investigations. Genetic data has become increasingly important to the field as there has been a push for DNA-based species identification methods of forensically relevant insects. Genetic data can also elucidate population structure and relatedness of these insects, and such knowledge can contribute to the development of more specific datasets for insects in different regions. The first study presented here investigated the phylogenetics of sister species <i>Lucilia cuprina</i> and <i>Lucilia sericata</i> to identify possible subspecies divisions and issues with DNA-based identifications in the United States. The initial aim of this study was to identify genetic differences between specimens of <i>L. cuprina</i> that preferred live versus carrion flesh. Flies collected from Indiana, USA and South Africa were sequenced and analyzed. Upon sequencing of the genes <i>COI, Period,</i> and <i>28s,</i> our results indicated that <i>L. cuprina</i> from Indiana possess a unique combination of nuclear and mitochondrial haplotypes that suggest a unique lineage, possibly indicating modern hybridization with <i>L. sericata. </i>The inability of both nuclear and mitochondrial genes to distinguish between <i>L. cuprina</i> and <i>L. sericata</i> raises questions about the capabilities of DNA-based species identifications within this genus. Additionally, the inability of these genes to distinguish between specimens that preferred live versus carrion flesh highlights a need for continued research of these behavioral differences. The second study presented here investigated the population structure and relatedness of house flies in the American southwest in relation to a civil lawsuit where neighbors of a poultry farm alleged that flies were emanating from the farm to their homes. <i>Musca domestica</i> (house fly) specimens were collected from the chicken farm and from locations in varying directions and distances from the farm. Amplified fragment length polymorphism (AFLP) analysis was performed and the data were used in a number of analyses. Population reallocation simulations generally indicated that samples from different locations were not genetically different enough from other locations to allocate to their true origin population over others. Kinship analysis showed differences in samples collected in a later season that indicate a genetic bottleneck over time. Population structure analysis indicated the presence of two intermixing genetic populations in the dataset. AMOVA revealed that the majority of genetic variation laid within, rather than among, populations. A Mantel test revealed no significant correlation between genetic and geographic distances. These results indicate that the <i>M. domestica</i> population in this region of southwestern America is large and intermixing, with no clear genetic distinctions between specimens collected at the poultry farm versus the surrounding locations. In regard to the civil lawsuit, it was not possible to conclude that the flies did not emanate from the poultry farm. In a broader perspective, these data can be utilized to develop pest management strategies in this region. Overall, the data from both studies presented here will be useful to forensic investigations, development of more specific and detailed data and identification techniques, and pest control measures.</div>