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Estudo da imobilização das proteases aspárticas da flor de Cynara cardunculus L. Reologia da coalhada
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Abstract(s)
O cardo, Cynara cardunculus L. cresce abundantemente em terrenos baldios e áreas não
cultivadas, em muitas regiões de Portugal. Estas flores apresentam propriedades
coagulantes, sendo por isso utilizado em fabricos tradicionais de vários tipos de queijo.
O extracto aquoso das flores de Cynara cardunculus L. possui proteases aspárticas,
denominadas por cardosinas. Estas proteases podem ser imobilizadas, com alguma
perda da sua actividade catalítica. Estas proteases apresentam elevado valor económico,
sendo a imobilização uma forma de aumentar a sua aplicabilidade à escala industrial,
visto permitir a sua reutilização. Os extractos enzimáticos de cardo, na forma livre e
imobilizada, foram caracterizados em termos de pH e de temperatura de reacção, assim
como em termos de parâmetros cinéticos para diferentes substractos (caseína bovina
comercial, K-caseína bovina comercial, caseína de cabra e K-caseína de cabra). Os
extractos aquosos de cardo foram imobilizados em celite por adsorção. Verificou-se um
pH óptimo de 5,4 e uma temperatura de reacção de 60 0C, para ambas as formas (livre e
imobilizada). Os queijos fabricados a parir deste coagulante vegetal, apresentam
distintas características bioquímicas, organolepticas e reológicas, tornando-os assim
muito apreciados. Neste trabalho estudaram-se as características reológicas da coalhada
a diferentes temperaturas de coagulação, adição de NaCl e de fermentos lácteos. Foi
igualmente estudado o comportamento reológico da coalhada obtida a partir de extracto
aquoso de cardo, imobilizado em celite. Em todos ensaios os desenvolvidos a coalhada
apresentou sempre propriedades de fluido dilatante.
Foram germinadas em condições de assepsia sementes de cardo, de modo a monitorizar
o seu crescimento, determinando-se alguns parâmetros fisiológicos. Avaliou-se a forma
como o teor de proteína total está associado ao crescimento, e a presença de prolina em
situações de stresse durante o crescimento e a evolução da actividade proteolítica.
Verificou-se que a actividade proteolítica se comporta de uma forma crescente ao longo
do período de crescimento, em todas as secções da plântula.
As flores de cardo secas foram ainda caracterizadas microbiologicamente, pela
quantificação de microrganismos heterotroficos aeróbicos, coliformes totais, coliformes
fecais, clostrídios sulfito-redutores, lipolíticos, bolores e leveduras. Neste estudo
utilizaram-se duas amostras de origens diferentes, que foram sujeitas a diferentes
tratamentos, para a redução da carga microbiana. Avaliou-se o efeito de exposição à
radiação ultravioleta durante 12 horas, a microfiltração (0,22p.m) e a congelação
(-20oC±2) sob as flores de cardo. Todos estes tratamentos provocaram uma diminuição
do número de microrganismos contabilizados. Contudo o tratamento por microfiltração
revelou-se como sendo o que melhor resultado apresentou, provocando a redução total
da carga microbiana. É sugerido a sua utilização de outros métodos ao nível industrial,
pois também provoca diminuição na contaminação e são de mais lácil aplicação. O
extracto aquoso de cardo imobilizado em celite, pode ser utilizado à escala industrial,
sem alteração das propriedades reológicas e organolepticas da coalhada.
The cardoon, Cynara cardunculus L. is a variety of thistle, which grows wild and abundantly in uncultivated areas of many regions ofPortugal. In these regions, the dried flowers are used as a milk coagulant for the manufacture oftraditional cheese varieties. Aqueous extracts of dried flowers of Cynara cardunculus L. possess aspartic proteases, currently termed cardosins. Proteases can he immobilized with some loss of catalytic activity. Since proteases are often expensive, especially at high degrees of purity, immobilization may increase their industrial applicability. Free and immobilized crude extracts of cardoon were characterized in terms of determination of optimum temperature an pH values, determination of kinetic parameters towards at differents substracts (bovine casein, bovine K-casein, goat casein and goat K-casein). The aqueous extracts of cardoon were immobilized on celite by adsorption. Optimum pH was 5 .4 and optimum temperature was 60oC, both for free and immobilized extracts. Cheeses elaborated with vegetal rennet have distinct biochemical, sensory and rheological characteristics, wich make them very popular. In this work, the rheological characteristics of the curd were studied at different coagulation temperatures, addition NaCl and cultures of startes. The coagulation and rheologycal ofthe curd obtained with Cynara cardunculus L. extract immobilized on celite were also studied characteristcs. During milk clotting the fluid shows dilatant properties in ali cases studied. Seeds of cardon were germinated in sterile conditions, to characterize the growth, thought the monitorization some physiological parmeters. It was evaluated how concentration ofprotein is anciated to the growth and the development ofthe plant, and the presence of proline in stress conditions during growth. The proteolytic activity was studied, as well registed that increase with growth was observed in ali sections of the plant. Cardoon flowers were microbiologic caracterized, by quantification of the number of aerobic heterotrofic microrganisms, total coliforms, fecal coliforms, sulphite-redutors clostrídes, lipolitic microrganisms, mould and yeast. Two samples of cardoon with different source and conservation were subjected to different treatments, in order to reduce the microbiologic value. The effect of exposition to ultraviolet radiation during 12 hours, membrane microfiltration (0,22|,im) and the freezing (-20oC±2) of the dried cardoon flowers were evalueted. Those treatments induced a reduction ofthe number of tested microrganisms contents. However, the treatment using microfiltration showed a full eficiency. The use of the other ones is recommended for industrial a applications, because they also provide a decrease on contamination and are easier to apply. An efficient immobilized infusion of the cardoon, with controled contamination can be used in industry, without alteration of the rheological properties and flavour in cheese.
The cardoon, Cynara cardunculus L. is a variety of thistle, which grows wild and abundantly in uncultivated areas of many regions ofPortugal. In these regions, the dried flowers are used as a milk coagulant for the manufacture oftraditional cheese varieties. Aqueous extracts of dried flowers of Cynara cardunculus L. possess aspartic proteases, currently termed cardosins. Proteases can he immobilized with some loss of catalytic activity. Since proteases are often expensive, especially at high degrees of purity, immobilization may increase their industrial applicability. Free and immobilized crude extracts of cardoon were characterized in terms of determination of optimum temperature an pH values, determination of kinetic parameters towards at differents substracts (bovine casein, bovine K-casein, goat casein and goat K-casein). The aqueous extracts of cardoon were immobilized on celite by adsorption. Optimum pH was 5 .4 and optimum temperature was 60oC, both for free and immobilized extracts. Cheeses elaborated with vegetal rennet have distinct biochemical, sensory and rheological characteristics, wich make them very popular. In this work, the rheological characteristics of the curd were studied at different coagulation temperatures, addition NaCl and cultures of startes. The coagulation and rheologycal ofthe curd obtained with Cynara cardunculus L. extract immobilized on celite were also studied characteristcs. During milk clotting the fluid shows dilatant properties in ali cases studied. Seeds of cardon were germinated in sterile conditions, to characterize the growth, thought the monitorization some physiological parmeters. It was evaluated how concentration ofprotein is anciated to the growth and the development ofthe plant, and the presence of proline in stress conditions during growth. The proteolytic activity was studied, as well registed that increase with growth was observed in ali sections of the plant. Cardoon flowers were microbiologic caracterized, by quantification of the number of aerobic heterotrofic microrganisms, total coliforms, fecal coliforms, sulphite-redutors clostrídes, lipolitic microrganisms, mould and yeast. Two samples of cardoon with different source and conservation were subjected to different treatments, in order to reduce the microbiologic value. The effect of exposition to ultraviolet radiation during 12 hours, membrane microfiltration (0,22|,im) and the freezing (-20oC±2) of the dried cardoon flowers were evalueted. Those treatments induced a reduction ofthe number of tested microrganisms contents. However, the treatment using microfiltration showed a full eficiency. The use of the other ones is recommended for industrial a applications, because they also provide a decrease on contamination and are easier to apply. An efficient immobilized infusion of the cardoon, with controled contamination can be used in industry, without alteration of the rheological properties and flavour in cheese.
Description
Mestrado em Biotecnologia, Faculdade de Engenharia de Recursos Naturais, Univ. do Algarve, 2005
Keywords
Cynara cardunculus L Proteases aspárticas Imobilização Caracterização cinética Reologia da coalhada Controlo microbiológico