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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

ProduÃÃo de Proteases por Aspergillus em FermentaÃÃo Semi-sÃlida utilizando Torta de Canola / PRODUCTION PROTEASES FOR ASPERGILLUS BY SOLID-STATE FERMENTATION USING THE CANOLA OIL CAKE

Adriana Crispim de Freitas 19 February 2009 (has links)
As proteases fazem parte da classe de enzimas com a capacidade de hidrolisar ligaÃÃes peptÃdicas em proteÃnas e fragmentos de proteÃnas, modificando os substratos com grande seletividade e especificidade. A fermentaÃÃo semi-sÃlida à um processo fermentativo no qual o crescimento do microrganismo e a formaÃÃo dos produtos ocorrem na superfÃcie do substrato sÃlido prÃximo à ausÃncia de Ãgua livre, geralmente utilizando matÃria-prima natural como fonte de carbono e energia. Realizou-se neste estudo o cultivo semi-sÃlido de fungos filamentosos, visando à produÃÃo de proteases utilizando como substrato a torta de canola. Avaliando-se, em um primeiro estudo, o potencial de diferentes linhagens de Aspergillus para a produÃÃo de proteases. As linhagens estudadas foram Aspergillus niger (CNPAT 001, IOC 207, IOC 4222, IOC 4220 e IOC 3883) e Aspergillus oryzae IV. A melhor linhagem obtida para a produÃÃo de protease foi a linhagem de Aspergillus oryzae IV. Em seguida, estudou-se a influÃncia da adiÃÃo de diferentes volumes de Ãgua a torta, onde a melhor produÃÃo foi obtida nos meios com proporÃÃo 40 mL de Ãgua para 100 g de torta. Determinada a melhor umidade do meio, avaliou-se a temperatura de incubaÃÃo, os melhores resultados obtidos foram em meios incubados a 20ÂC. Na sequÃncia, foram feitos estudos para determinaÃÃo da melhor concentraÃÃo de inÃculo, as concentraÃÃes testas foram 1Ã104, 1Ã105, 1Ã106 e 1Ã107 esporos por grama de meio, sendo 1Ã107 a melhor concentraÃÃo analisada. Avaliou-se a influÃncia da suplementaÃÃo do meio fermentativo com fontes de fÃsforo, carbono e nitrogÃnio. A suplementaÃÃo da torta de canola, com 1% (m/m) de fosfato de sÃdio monobÃsico nÃo influenciou positivamente na produÃÃo de proteases. A suplementaÃÃo do meio com diferentes fontes de nitrogÃnio foram estudadas na proporÃÃo de 1,0 % (m/m) em relaÃÃo ao peso do substrato, sendo o extrato de levedura a melhor fonte avaliada, apresentando maior produÃÃo de proteases. Em seguida, testou-se o perÃodo de incubaÃÃo do meio sem suplementaÃÃo de nutrientes, entre 0 e 240 horas de fermentaÃÃo com amostras sendo retiradas a cada 24 h de processo fermentativo. A maior produÃÃo ocorreu em 96 h com produÃÃo de 336 U.g-1 de proteases. ApÃs esta etapa, avaliou-se a suplementaÃÃo do meio com diferentes fontes de carbono em diferentes concentraÃÃes, 1,0; 2,0; 3,0; 4,0; 5,0; 7,5; 10,0; 12,5 15,0 % (m/m) em relaÃÃo ao peso do substrato, a maior produÃÃo de proteases ocorreu no meio com adiÃÃo de glicose na concentraÃÃo de 7,5 % obtendo 452 U.g-1. Na Ãltima etapa, fez-se a caracterizaÃÃo do meio ao longo do processo fermentativo, atravÃs de determinaÃÃes analÃticas de ordem quÃmicas e fÃsico-quÃmicas. A maior produÃÃo de proteases obtida foi de 452 U.g-1 em 96 horas de processo fermentativo, nos meios suplementados com 7,5 % de glicose, nas seguintes condiÃÃes fermentativas: temperatura de incubaÃÃo do meio a 20ÂC, torta umedecida com 40 mL de Ãgua por 100 g de substrato e concentraÃÃo de inÃculo de 1Ã107 esporos.g-1. / The proteases belong to the class of enzymes with the capacity to hydrolyze peptidic connections into protein and protein fragments, modifying the substrate by great selectivity and specification. The semi-solid fermentation is a fermentation process in which the growth of microorganisms and the formation of the products occur on the surface of the solid substrate next to the absence of free water, usually using natural raw materials as a source of carbon and energy. On this study, it was done the semi solid cultivation of filament fungi, in order to produce proteases using as a substrate a cake of canola. It was evaluated, in a first study, the potential of different strains of Aspergillus for the production of proteases. The strains studied were Aspergillus niger (CNPAT 001, IOC 207, IOC 4222, IOC 4220 and IOC 3883) and Aspergillus oryzae IV. The best strain obtained for the production of protease was a strain of Aspergillus oryzae IV. Later on, it was studied the influence of the different quantities of water and coke canola added; the best production was obtained in proportion with 40 mL of water to 100 g of cake. After determination to medium of moisture, was evaluated the temperature of incubation, the best results were obtained in media incubated at 20 C. Further, studies were done to determine the best concentration of inoculum, the concentrations tested were 1 à 104, 1 à 105, 1 à 106 and 1 à 107 spores per gram of medium, being 1 à 107 to better concentration analyzed. The influence of supplementation of fermentation medium with sources of phosphorus, carbon and nitrogen was evalueted. Supplementation of the cake of canola, with 1% (w/w) monobasic sodium phosphate did not influence positively the production of proteases. Supplementation of medium with different sources of nitrogen were studied in the proportion of 1.0% (w/w) in the weight of the substrate, being the yeast extract of the best source evaluated, showing increased production of proteases. Then, it was tested the incubation period of the medium without supplementation of nutrients, between 0 and 240 hours of fermentation with samples being removed every 24 h of fermentation process. The highest production occurred at 96 h with production of 336 U.g-1 protease. After this stage, was evaluated the supplementation of medium with different carbon sources at different concentrations, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5, 10.0, 12, 5 15.0 % (w/w) in the weight of the substrate; the increased production of proteases occurred in the medium with the addition of glucose at a concentration of 7.5 % obtaining 452 Ug-1. In the last stage, was performed the characterization of the medium during the fermentation process by of a chemical and physicochemical analytical determinations. The increased production of proteases obtained was 452 U.g-1 in 96 hours of fermentation process, in media supplemented with 7.5 % glucose, at the fermentation conditions: temperature of incubation of the medium at 20 C, damp cake with 40 mL water per 100 g of substrate and concentration of inoculum of 1 à 107 spores.g-1.

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