Efficient disposal methods are needed to manage manure produced by industrial animal production. Saprophagous fly larvae could potentially convert manure into fertilizer and produce protein; however, the process is not well studied. Musca domestica, Hydrotaea aenescens, and Coproica hirtula were investigated to determine the most suitable species and conditions that facilitate efficient poultry manure conversion. The objectives were to (1) develop laboratory protocols and timelines for fly production; (2) identify environmental conditions that affect conversion; and (3) determine the ideal manure moisture content, depth, and fly egg-to-manure ratio for manure conversion and protein production. Mass-production was possible for every species and timelines were established for all species except C. hirtula. The most promising species for use in a conversion system was M. domestica and the presence of C. hirtula facilitated complete conversion. When using these species simultaneously the ideal initial conditions were: 77.5% moisture, 2.9cm deep and 0.82g eggs/kg manure. / OMAFRA/ University of Guelph partnership

Orientador: João Alexandre Ribeiro Gonçalves Barbosa / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T20:09:24Z (GMT). No. of bitstreams: 1 Valerio_AmandaAbdalla_D.pdf: 3553304 bytes, checksum: 97ba97062f1c23d4b29447c0b9a7316d (MD5) Previous issue date: 2007 / Resumo: Glicosídeo hidrolases (EC 3.2.1.-) são enzimas que hidrolisam ligações glicosídicas. Neste trabalho realizamos estudos funcionais e estruturais de duas glicosídeo hidrolases: a celulase XF0810 de Xylella fastidiosa e a lisozima digestiva 1 de Musca domestica (MdL1). A celulase XF0810 está anotada no genoma da X. fastidiosa como membro da família 5 das glicosídeo hidrolases (EC 3.2.1.4). Após a amplificação, clonagem, expressão e purificação da mesma; prosseguimos com os experimentos de filtração em gel analítica, dicroísmo circular, DLS, ensaio enzimático e cristalização desta proteína. Foi feito um estudo de modelagem onde ficou evidenciado que a XF0810 não pertenceria à família 5 pois quatro dos sete resíduos conservados que caracterizam esta família foram substituídos, incluindo os dois resíduos catalíticos de glutamato essenciais para o mecanismo de clivagem de retenção. Isto foi comprovado através da ausência de atividade nos ensaios feitos com a proteína purificada. A MdL1 pertence à família 22 das glicosídeo hidrolases (EC 3.2.1.17) e foi cristalizada com o ligante Nacetilquitotetraosídeo para a difração de raios X. A resolução da estrutura (2H5Z no PDB) foi realizada por meio do método de substituição molecular tendo como modelo a estrutura nativa. A análise comparativa da MdL1 com outras lisozimas de quatro classes diferentes de animais mostrou grande semelhança e pequenas diferenças apenas na região das voltas. Estas diferenças foram utilizadas para explicar as características especiais de uma lisozima com função digestiva. A volta na região definida pelos resíduos 98-100 apresenta uma deleção na MdL1, tornado-a menos exposta ao solvente, podendo justificar a resistência à proteólise. O resíduo Gln100 participa de uma interação com o ligante. Os resíduos Thr107 e Asn46 são apontados como responsáveis pelo decréscimo dos pKa dos grupos carboxilas dos resíduos catalíticos Glu32 e Asp50, respectivamente. A diminuição dos pKa explica o pH ótimo mais ácido característico de lisozimas digestivas / Abstract: Glycoside hydrolases (EC 3.2.1.-) are enzymes that hydrolyze glycoside bonds. In this work we studied functional and structural features of two glycoside hydrolases: the cellulase XF0810 of Xylella fastidiosa and the digestive lysozyme 1 of Musca domestica (MdL1). The cellulase XF0810 is annotated in the genome of X. fastidiosa as member of family 5 of glycoside hydrolases (EC 3.2.1.4). After the amplification, cloning, expression and purification of XF0810; we continued with the experiments of analytical gel filtration, circular dichroism, DLS, enzymatic assay and crystallization of this protein. A homology model was built which showed that XF0810 did not belong to family 5 because four of seven conserved residues that characterize the family were substituted, including the two catalytic residues of glutamate that are essential for the retention hydrolysis mechanism. This was further confirmed by the absence of activity in the assays (exocellulase with PNPc) performed with the purified protein. The MdL1 belongs to the family 22 of glycoside hydrolases (EC 3.2.1.17) and was crystallized with the ligand N-acetilchitotetraose for X-ray diffraction. The resolution of the structure (2H5Z in PDB) was accomplished by molecular replacement with the native structure as the searching model. The comparative analysis of MdL1 with other lysozymes of four different classes of animals showed a high similarity and few differences appeared only in the loops¿ regions. These differences were used to explain the special characteristics of the lysozyme with a digestive function. The loop in the region defined by residues 98-100 presents one deletion in the MdL1, becoming less exposed to the solvent, this might justify the proteolysis resistance. The residue Gln100 participates directly in an interaction with the ligand. The residues Thr107 and Asn46 are pointed out as responsible for a reduction in the pKa of the carboxyl groups of catalytic residues Glu32 e Asp50, respectively. The reduction in pKa explains the more acidic pH optimum that characterizes the digestive lysozymes / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2017-08-24T18:58:04Z No. of bitstreams: 2 Dissertação - Marcos Daniel Filgueiras Gomes - 2017.pdf: 1627510 bytes, checksum: 4f9bbc1cf7e9457ee3eb0b398bfffc42 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Rejected by Erika Demachki (erikademachki@gmail.com), reason: subtituir PDF on 2017-09-01T17:18:15Z (GMT) / Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2017-09-05T14:58:59Z No. of bitstreams: 2 Dissertação - Marcos Daniel Filgueiras Gomes - 2017.pdf: 1627510 bytes, checksum: 4f9bbc1cf7e9457ee3eb0b398bfffc42 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Rejected by Erika Demachki (erikademachki@gmail.com), reason: on 2017-09-05T17:01:40Z (GMT) / Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2017-09-05T17:03:46Z No. of bitstreams: 2 Dissertação - Marcos Daniel Filgueiras Gomes - 2017.pdf: 1628086 bytes, checksum: 7ab87fcdc18cfc95d88131b8b9272ecc (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-09-15T15:11:58Z (GMT) No. of bitstreams: 2 Dissertação - Marcos Daniel Filgueiras Gomes - 2017.pdf: 1628086 bytes, checksum: 7ab87fcdc18cfc95d88131b8b9272ecc (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-09-15T15:11:59Z (GMT). No. of bitstreams: 2 Dissertação - Marcos Daniel Filgueiras Gomes - 2017.pdf: 1628086 bytes, checksum: 7ab87fcdc18cfc95d88131b8b9272ecc (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-03-09 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The current study investigated the virulence of Metarhizium spp., Beauveria bassiana and Isaria javanica against Musca domestica larva; it also compared the virulence of different propagules, and evaluated the efficacy of matrine, a plant alkaloid, and the association of matrine and entomopathogenic fungi against larvae of Musca domestica. In addition, the study reports the conidial viability and radial growth of different fungal isolates in culture medium supplemented or not with matrine. Conidia produced on BDAL medium were suspended in Tween 80 0.01% and adjusted to 1 × 108 conidia / mL, and spread on filter paper for indirect treatment of third-instar larvae of M. domestica. The efficacy of treatment ranged from 77,44% (IP 119) to 87,35% (IP 146) among Metarhizium spp. isolates. The efficacy of treatment ranged from 39,37% (ARSEF 9588) to 70,60% (IP 361) among the isolates of B.bassiana; variations between 26,75% (CG 1316) and 53,28% (CG 1283) was reported in groups treated with isolates of I. javanica. Conidia and blastospores of fungi were produced on Adamek medium, and suspensions were prepared and adjusted to 1 × 108 propagules/mL. Aliquots were used in indirect treatments by spreading the suspension to sterile filter paper or to comminuted ration to evaluate virulence in third or second instar larvae, respectively. Only the treatment with IP 146 blastospores in ration was effective against M. domestica larva. Larvae were immersed on matrine solutions (direct treatment) or exposed to sterile-filter paper treated with matrine (indirect treatment) in different concentrations (1, 5, 10 and 20 mg/mL). Ten larvae were assayed in each treatment and control group. Matrine concentrations higher than 1 mg/mL were effective against M. domestica larva in both treatments. Germination of conidia of Metarhizium spp., B. bassiana and I. javanica isolates was assessed at 24 and 48 honto BDAL medium plus chloramphenicol and benomyl, supplemented or not (control) with matrine. All isolates had germination around 100%, in both incubation times evaluated. In addition, the radial growth of isolates was evaluated on BDAL medium supplemented or not (control) with matrine. The isolates of Metarhizium spp. and B. bassiana, with the exception of CG 138, had a reduced radial growth on medium containing matrine. Isolates of I. javanica did not reduce their growth when exposed to matrine. The association of entomopathogenic fungi [IP 119 (Metarhizium anisopliae s.s.), CG 307 (Beauveria bassiana) or CG 1285 (Isaria javanica)] with matrine (1; 0,5; 0,1; 0,05; 0,01 mg/mL), was investigated against larva of M. domestica in indirect tests on sterile filter paper. The association of fungal conidia and matrine was more efficient against M. domestica larva than conidia, and the association was more efficient than matrine alone. All experiments were incubated at 27 ± 1 oC and RH ≥ 80%. In conclusion, Metarhizium sp. is indicated as a promising microbial agent to control larva of M. domestica; also, IP 146 blastospores applied to ration were effective in the control of second instar larvae. In addition, the alkaloid matrine was effective to control M. domestica larva; it did not affect the viability of the conidia of the fungi investigated in this study, but it slowed down the growth of most of the Metarhizium and Beauveria isolates tested. Radial growth of isolates of Isaria sp. was not affected by supplementation of medium with matrine. No increased efficiency against M. domestica larva was detected when fungi was associated with matrine. / O presente estudo investigou a virulência de Metarhizium spp., Beauveria bassiana e Isaria javanica contra larvas de Musca domestica; comparou também a virulência de diferentes propágulos fúngicos, e avaliou a eficácia de matrine, um alcaloide vegetal, e sua associação com fungos entomopatogênicos contra larvas de M. domestica. Além disso, o estudo relata a viabilidade conidial e o crescimento radial de diferentes isolados fúngicos em meio de cultura suplementado ou não com matrine. Nesse sentido, conídios produzidos em meio BDAL foram suspensos em Tween 80 a 0,01% e ajustados a 1 × 108 conídios/mL e espalhados em papel filtro para o tratamento indireto de larvas de terceiro ínstar de M. domestica. A eficácia do tratamento dos isolados de Metarhizium spp. variou entre 77,44% (IP 119) e 87,35% (IP 146). A eficácia do tratamento variou de 39,37%(ARSEF 9588) e 70,60% (IP 361) entre os isolados de B.bassiana, e variações entre 26,75% (CG 1316) e 53,28% (CG 1283) foram observadas em grupos tratados com isolados de I. javanica. Conídios e blastosporos de IP 146 (Metarhizium robertsii) e IP 361(Beauveria bassiana) foram produzidos em meio Ádamek, em seguida foram preparadas suspensões e estas ajustadas para 1 × 108 propágulos/mL. Tratamentos indiretos foram conduzidos espalhando-se a suspensão em papel filtro estéril ou em rações para avaliar a virulência em larvas de terceiro ou segundo ínstar de M. domestica, respectivamente. Apenas o tratamento com blastosporos IP 146 em ração foi eficaz contra larva de M. domestica. Larvas de M. domestica também foram imersas em soluções contendo matrine (tratamento direto) ou expostas a papel filtro estéril tratado com matrine (tratamento indireto) em diferentes concentrações (1, 5, 10 e 20 mg / mL); dez larvas foram utilizadas em cada tratamento e grupo de controle. Para ambos tratamentos concentrações de matrine superiores a 1 mg/mL foram eficazes contra larvas de M. domestica. A germinação de conídios dos isolados de Metarhizium spp., B. bassiana e I. javanica foi avaliada em 24 e 48 horas sobre meio BDAL acrescido de cloranfenicol e benomil, e suplementado ou não (controle) com matrine (2%). Todos os isolados tiveram germinação em torno de 100%, em ambos os tempos de incubação avaliados. O crescimento radial de isolados também foi avaliado em meio BDAL suplementado ou não (controle) com matrine. Nesse estudo, os isolados de Metarhizium spp. e B. bassiana, com exceção de CG 138, tiveram crescimento radial reduzido em meio contendo matrine. Os isolados de I. javanica, no entanto, não reduziram seu crescimento quando expostos à matrine em meio de cultura. Foi investigada a associação de fungos entomopatogênicos [IP 119 (Metarhizium anisopliae s.s.), CG 307 (Beauveria bassiana) ou CG 1285 (Isaria javanica)] com matrine (1; 0,5; 0,1; 0,05; 0,01 mg/mL) contra larvas de M. domestica. Em testes indiretos em papel filtro estéril, a associação de conídios fúngicos e matrine foi mais eficaz contra larva de M. domestica do que conídios apenas, e a associação foi mais eficiente do que matrine sozinho. Todos os experimentos foram incubados a 27 ± 1 oC e UR ≥ 80%. Concluindo, Metarhizium sp. é indicado como um agente microbiano promissor para controlar larvas de M. domestica; também, blastosporos do isolado IP 146 aplicados à ração foram eficazes no controle de larvas de segundo instar. Além disso, o alcaloide matrine foi eficaz para controlar larvas de M. domestica; não afetou a viabilidade dos conídios dos fungos investigados neste estudo, mas retardou o crescimento da maioria dos isolados de Metarhizium e Beauveria testados. O crescimento radial de isolados de Isaria sp. não foi afetado pela suplementação de meio com matrine. Não houve aumento da eficácia contra larva de M. domestica quando fungos foram associados ao matrine.

El previsible aumento de la población mundial, estimado para el año 2050 en 9.500-10.000 millones de personas, supondrá uno de los principales retos a los que se enfrente el siglo XXI. Además de los recursos e insumos necesarios (espacio, agua, residuos etc.), la seguridad alimentaria del planeta conllevará una elevada demanda de proteínas tanto para la alimentación animal como para la nutrición humana. Nuestras principales fuentes de proteína provienen de la agricultura, la ganadería (incluyendo la acuicultura) y la pesca. Es por ello por lo que la producción y rendimiento de las plantas y animales domesticados, ha aumentado exponencialmente a lo largo de las últimas décadas, estando en la actualidad muy cerca de sus límites biológicos. Sin embargo, esta elevada eficiencia esta inversamente relacionada con la sostenibilidad ambiental de estos sistemas de producción. Entre las consecuencias, la producción de piensos compuestos altamente nutritivos también ha aumentado. Para su elaboración son necesarias grandes cantidades de harina y aceite de pescado, así como de soja, entre otros componentes. El cultivo intensivo de soja conlleva graves problemas medioambientales. Por otro lado, el medio marino está cada vez más deteriorado como consecuencia de la contaminación derivada de la actividad humana y la sobreexplotación pesquera. Todo ello ha provocado una inestabilidad económica a largo plazo y un aumento continuado de los precios de estas materias primas. Pero ¿es técnicamente posible maximizar la sostenibilidad de estos sistemas de producción, optimizando simultáneamente la imprescindible eficiencia económica? Con este objetivo, se hace necesaria la búsqueda de nuevas fuentes de proteínas que puedan utilizarse como ingredientes en la elaboración de piensos compuestos destinados a la alimentación animal e indirectamente a la nutrición humana. Una de las más prometedoras, es la obtención de proteína animal a partir de la biomasa de ciertas especies de insectos domesticados. En particular, la utilización de proteínas y grasas de insectos a escala industrial, mejorará significativamente la sostenibilidad y eficiencia de la ganadería terrestre y la acuicultura. Indirectamente, permitirá mantener los niveles de pesca a unos niveles racionales y los subproductos derivados tienen un gran valor agronómico como fertilizantes de origen natural. Entre los candidatos más plausibles, los dípteros (Insecta: Diptera) presentan características biológicas y nutricionales que los hacen buenos candidatos para su producción a escala industrial. Una de las especies más prometedoras es la mosca soldado negra Hermetia illucens (Linnaeus, 1758) que junto con el coleóptero Tenebrio molitor Linnaeus, 1758 constituyen actualmente los principales integrantes de este nuevo sector de producción. La Unión Europea, también ha aprobado el uso de otras cinco especies de insectos como ingredientes de la alimentación animal, destacando entre estas la mosca común Musca domestica Linnaeus, 1758. Otras especies de dípteros no están todavía aprobadas con este fin, pero también resultan muy interesantes por su gran potencial para la cría artificial, como las especies de Calliphoridae Lucilia sericata (Meigen, 1826) o Chrysomya megacephala (Fabricius, 1794). Entre los componentes que forman parte de los piensos compuestos en la alimentación animal, los minerales suponen un importante grupo limitante desde un punto de vista nutritivo. Mediante un aporte óptimo de minerales, se puede aumentar considerablemente el rendimiento de la producción ganadera a un bajo coste. No obstante, los niveles de estos compuestos deben mantenerse dentro de unos niveles determinados por la legislación para evitar problemas nutricionales o provocar efectos nocivos en la salud animal y humana. En este sentido, existen muy pocos estudios sobre los perfiles minerales de la biomasa de insecto en general y en particular de la mosca soldado negro y otras especies de dípteros. Son especialmente escasos los datos sobre la influencia que pueda ejercer la composición del sustrato de desarrollo de las larvas, así como su importancia en las diferentes etapas de desarrollo del ciclo de vida. Por otro lado, existe la posibilidad de que se produzca bioacumulación de metales pesados en la biomasa del insecto a través de la ingesta. Así, debido a su alta toxicidad, existen diversos trabajos sobre las concentraciones de Cd y Pb en larvas maduras de mosca soldado negra, pero hay muy poca información sobre bioacumulación en el resto de etapas y estadios de desarrollo, así como de la posible influencia del medio de cría larvario. En este proyecto de tesis se analiza la posible influencia del sustrato de cría en las concentraciones de distintos minerales y metales pesados a lo largo de las etapas del ciclo de vida de Hermetia illucens y otras especies de dípteros. Para ello se usaron tres sustratos larvarios con diferentes orígenes: a) pienso comercial de gallina ponedora, b) bagazo derivado de la producción de cerveza y c) una mezcla de carne de origen porcino. En el caso de H. illucens se tomaron muestras de larvas prepupales, pupas, puparios, exuvias prepupales y adultos desarrollados en los tres medios de desarrollo. En todos los casos, tras su secado y digestión en horno microondas, se analizaron mediante espectrometría de emisión óptica de plasma acoplado por inducción (ICP-OES) para obtener las concentraciones de Ca, Cr, Cu, Fe, K, Mg, Mn, Na, Ni y Zn, y en espectrometría de masas con fuente de ionización de plasma (ICP-MS) para obtener las concentraciones de Ti, Co, Sr, Mo, Cd, Sn y Pb. Pocos insectos tienen la capacidad para desarrollarse en medios de tan diferente composición orgánica, por ello a modo comparativo se utilizó M. domestica con pienso comercial de gallina y el bagazo de cerveza, mientras que Lucilia sericata y Chrysomya megacephala se desarrollaron exclusivamente en el medio de origen cárnico. De esta manera no solamente se pudieron comparar diferentes tipos de medio procesados por diferentes tipos de insectos sino que también pudieron compararse los resultados obtenidos por diferentes especies de mosca con el mismo tipo de medio. En cuanto a relaciones entre patrones de acumulación (Factor de Bioacumulación) y concentraciones de los minerales, con la especie de díptero y el sustrato utilizado, en larvas prepupales y adultos no se observa ninguna relación entre resultados y la especie o el sustrato, en cambio, los resultados en pupas y puparios sí están influenciadas por la especie de estudio y el sustrato de alimentación usado. Por otro lado, Ca y Na se comportan de manera distinta en las especies estudiadas en esta investigación. La mosca soldado negra presenta un buen perfil mineral para su uso en alimentación animal, comparable con los de harina de pescado y soja. Los resultados con mosca doméstica son también comparables a las harinas de pescado y soja, en los dos medios estudiados. Los dos califóridos presentan un perfil mineral de menor calidad, pero comparable a la harina de soja y también aptos para su uso en la elaboración de piensos.

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.