Publication: Agregación de proteínas de músculo de pescado congelado. Acción del formaldehido
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2002
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Universidad Complutense de Madrid, Servicio de Publicaciones
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Abstract
El presente trabajo tuvo como objetivo fundamental el estudio de la desnaturalización y agregación proteica durante la conservación en congelación, prestando especial interés al estudio de los tipos de enlaces y proteínas implicadas en esta agregación. Se utilizo como muestra músculo picado de bacalao, merluza y bacaladilla y filetes de bacalao, para observar el efecto del grado de integridad del músculo en esta especie. Se midieron los cambios en funcionalidad del músculo (solubilidad proteica, viscosidad aparente, textura) y en formación de dimetilamina (dma) y formaldehido (fa) libre de forma periódica a lo largo de la conservación. Paralelamente, en cada uno de los controles se aisló la actomiosina natural (amn) del músculo en solución salina, determinándose algunos parámetros funcionales y bioquímicos de la misma (actividad atpasica, hidrofobicidad superficial, viscosidad aparente etc.). La amn extraída se analizó además por electroforesis (sds-page) y cromatografía de filtración en gel, para estudiar los posibles cambios en composición, presencia de agregados solubles y enlaces que los forman. Estos extractos se estudiaron por microscopía electrónica de transmisión en músculo picado de bacalao. Por otro lado, el agregado no extraído en solución salina se trato con distintos agentes para estudiar los tipos de enlaces implicados y sus cambios a lo largo de la conservación. Las fracciones extraídas por este tratamiento se analizaron por electroforesis (sds-page) para conocer la participación de las distintas proteínas miofibrilares en la formación del agregado no extraído en solución salina. Para estudiar el efecto del fa, tanto solo como combinado con el de la congelación y conservación en congelación, se prepararon dos sistemas modelo con amn de músculo de bacalao, merluza y bacaladilla. En el primero de ellos se estudio el efecto de distintas concentraciones de fa y en el segundo la extractibilidad en distintos agentes de los agregados de amn insolubles formados durante la congelación y conservación en congelación en presencia o no de fa. En las fracciones extraídas se detectaron agregados solubles, formados por enlaces secundarios y/o covalentes, cuyo tamaño aumento a lo largo de la conservación. En la formación del agregado no extraído en nac1 0,6 m participaron enlaces secundarios, disulfuro y covalentes. La proporción de los mismos cambio a lo largo del tiempo de conservación, dependiendo de la especie y del grado de integridad. Se observó una relación entre estos cambios y la cantidad de fa formado. También se observo mayor participación de la miosina en la formación de agregados no extraídos en nac1 0,6 m y mayor implicación de la misma en enlaces covalentes. El fa modifico el mecanismo de agregación de la amn conservada en congelación y causó cambios en la proporción de los distintos enlaces implicados en la formación de agregados insolubles dependiendo de la funcionalidad de la amn estudiada
The main objective of the present work was to study the denaturation and protein aggregation during freezing preservation, giving special interest to the study of the types of bonds and proteins involved in this aggregation. Chopped cod, hake and blue whiting muscle and cod fillets were used as a sample to observe the effect of the degree of muscle integrity in this species. Changes in muscle functionality (protein solubility, apparent viscosity, texture) and in formation of free dimethylamine (dma) and free formaldehyde (fa) were measured periodically throughout preservation. In parallel, in each of the controls, the natural actomyosin (amn) was isolated from the muscle in saline solution, determining some functional and biochemical parameters of the same (atpasic activity, surface hydrophobicity, apparent viscosity, etc.). The extracted amn was also analyzed by electrophoresis (sds-page) and gel filtration chromatography, to study possible changes in composition, presence of soluble aggregates and bonds that form them. These extracts were studied by transmission electron microscopy in minced cod muscle. On the other hand, the aggregate not extracted in saline was treated with different agents to study the types of bonds involved and their changes throughout conservation. The fractions extracted by this treatment were analyzed by electrophoresis (sds-page) to know the participation of the different myofibrillar proteins in the formation of the aggregate not extracted in saline. To study the effect of fa, both alone and in combination with that of freezing and freezing, two model systems were prepared with cod muscle amn, hake and blue whiting. In the first one, the effect of different concentrations of fa was studied, and in the second, the extractability in different agents of the insoluble amn aggregates formed during freezing and preservation in freezing in the presence or not of fa. Soluble aggregates, formed by secondary and / or covalent bonds, were detected in the extracted fractions, the size of which increased throughout conservation. Secondary bonds, disulfide and covalent participated in the formation of the non-extracted aggregate in 0.6 m nac1. The proportion of the same changes throughout the conservation time, depending on the species and the degree of integrity. A relationship was observed between these changes and the amount of fa formed. A greater participation of myosin was also observed in the formation of non-extracted aggregates in 0.6 m nac1 and its greater involvement in covalent bonds. The fa modified the aggregation mechanism of the conserved amn in freezing and caused changes in the proportion of the different bonds involved in the formation of insoluble aggregates depending on the functionality of the amn studied.
The main objective of the present work was to study the denaturation and protein aggregation during freezing preservation, giving special interest to the study of the types of bonds and proteins involved in this aggregation. Chopped cod, hake and blue whiting muscle and cod fillets were used as a sample to observe the effect of the degree of muscle integrity in this species. Changes in muscle functionality (protein solubility, apparent viscosity, texture) and in formation of free dimethylamine (dma) and free formaldehyde (fa) were measured periodically throughout preservation. In parallel, in each of the controls, the natural actomyosin (amn) was isolated from the muscle in saline solution, determining some functional and biochemical parameters of the same (atpasic activity, surface hydrophobicity, apparent viscosity, etc.). The extracted amn was also analyzed by electrophoresis (sds-page) and gel filtration chromatography, to study possible changes in composition, presence of soluble aggregates and bonds that form them. These extracts were studied by transmission electron microscopy in minced cod muscle. On the other hand, the aggregate not extracted in saline was treated with different agents to study the types of bonds involved and their changes throughout conservation. The fractions extracted by this treatment were analyzed by electrophoresis (sds-page) to know the participation of the different myofibrillar proteins in the formation of the aggregate not extracted in saline. To study the effect of fa, both alone and in combination with that of freezing and freezing, two model systems were prepared with cod muscle amn, hake and blue whiting. In the first one, the effect of different concentrations of fa was studied, and in the second, the extractability in different agents of the insoluble amn aggregates formed during freezing and preservation in freezing in the presence or not of fa. Soluble aggregates, formed by secondary and / or covalent bonds, were detected in the extracted fractions, the size of which increased throughout conservation. Secondary bonds, disulfide and covalent participated in the formation of the non-extracted aggregate in 0.6 m nac1. The proportion of the same changes throughout the conservation time, depending on the species and the degree of integrity. A relationship was observed between these changes and the amount of fa formed. A greater participation of myosin was also observed in the formation of non-extracted aggregates in 0.6 m nac1 and its greater involvement in covalent bonds. The fa modified the aggregation mechanism of the conserved amn in freezing and caused changes in the proportion of the different bonds involved in the formation of insoluble aggregates depending on the functionality of the amn studied.
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Tesis de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Nutrición y Bromatología II, leída el 04-06-1996