Objective
- To investigate the biochemical changes and mechanisms causing the aggregation of proteins in frozen fish in order to predict and control fish quality in terms of texture, nutrition and food safety.
- To bridge gaps in our knowledge of the interaction of myofibrillar and collagen proteins from selected fish with formaldehyde and lipid oxidation products using advanced and less empirical techniques such as spectroscopy, radiolabelling and microscopy with a view to inhibiting toughening of frozen fish.
- To relate the physiological and nutritional state of the fish on formaldehyde production, lipid changes, water-protein ratio and muscle toughening.
- To identify, from the body of knowledge obtained, the mechanisms of interaction of fish proteins during frozen storage in order to establish ways of inhibiting aggregation and toughening of fish and to enhance the nutritional value.
SUMMARY
This study is concerned with the investigation of the nature of aggregation including myofibrillar protein-protein; protein-formaldehyde and protein-lipid interactions in gadoid fish namely cod (Gadus morhua) and hake (Merluccius merluccius), with an under-utilised species blue whiting (Micromesistius poutassou) and a non-formaldehyde producing species haddock (Melanogrammus aeglefinus) used for comparison. The different species are being used as appropriate to the countries (UK, Spain and Portugal) involved. The studies are being undertaken on fresh fish and over a period of time in frozen storage at - 10oC to accelerate changes and compared to controls stored at-30oC. A storage trial has commenced and systematic studies in model systems, soluble and insoluble aggregates and intact tissue are being undertaken using a powerful range of non-empirical techniques namely spectroscopy (NMR, Raman and fluorescence); 14C labelled TMAO to monitor the formation of formaldehyde in hake, and immunocytochemistry combined with electrophoretic, differential scanning calorimetry (DSC), chromatographic, spectroscopic and rheological techniques to clearly define the interaction of proteins with formaldehyde or lipid oxidation products. Findings from the above will be related to differences in red and white muscle tissue and to the formaldehyde and lipid levels in fish before and after spawning to determine the effect of physiological and nutritional parameters on formaldehyde formation and changes in lipids and proteins.
INTRODUCTION
One of the major and intractable problems in the field of fish technology which requires to be resolved is the variation in the stability of commercially important lean gadoid species like cod, hake, pollack, haddock and whiting. The instability resulting from the denaturation and aggregation of proteins especially during frozen storage, results in texture changes such as toughening of the muscle which makes the fish unpalatable.
Previous studies and reviews (Mackie, 1993; Shenouda,1980; Sikorski et al ,1976), have not provided a coherent explanation for the development of toughness of fish flesh during frozen storage. During the past four years, however, the present Consortium undertook major systematic studies on the changes occurring in proteins on frozen storage of cod, hake and haddock at -20 and -30oC and reported key findings on the changes in proteins and causes of aggregation in fish flesh include the effect of freezing, presence of formaldehyde and lipid oxidation products.
The overall conclusion of the study (Project UP3.647 EC Final Report 1995) is that cod, haddock and to a greater extent hake, stored at both -20 and -30oC, increased in toughness with a significantly greater change at -20oC due to protein aggregation. The aggregates formed in the initial periods of storage are non-covalently linked mainly by hydrophobic bonds, hydrogen bonds and electrostatic linkages. After about 8 months at -20oC and 12 months at -30oC in cod and earlier in hake, covalently linked ring-shaped aggregates were formed causing changes in texture. Fluorescence was detected in aggregates from cod and haddock and fluorescent peptides were isolated from the SDS-insoluble precipitate from cod which indicated the presence of lipid oxidation compounds in preliminary studies. Model studies incorporating eicosapentanoic acid produced a similar blue-white fluorescence and SDS-insoluble aggregates with cod proteins. During storage of cod and hake, denaturation of myosin heavy chain occurred first followed by actin, and then myosin light chains, tropomyosin and troponin. Since no differences in the amino acid profiles of cod and haddock were detected after a year of storage at -20 or -30oC, it is proposed that during frozen storage formaldehyde forms non-covalent and non acid-resistant covalent linkages such as those evident in the initial stages of frozen storage. This stage is followed by the formation of disulphide bonds and finally the acid resistant non-disulphide covalent bonds, which were detected in the latter stages of storage. The covalently linked precipitates appear to be mainly due to lipid oxidation products, which may play a predominant role in the aggregation of proteins thereby affecting the organoleptic properties of frozen stored gadoid fish. Further studies are therefore being undertaken on the role of lipid oxidation products as well as formaldehyde on the aggregation of fish proteins, using the knowledge gained with the novel applications of non-empirical methods and protocols developed in the last study.
RESEARCH TASKS
TASK 1 INTERACTIONS OF PROTEINS IN FROZEN STORAGE
Sub-task 1.1. Preparation and characterisation of soluble and insoluble aggregates in hake and blue whiting
Sub-task 1.2. Microstructural characterisation of soluble and insoluble aggregates
Sub-task 1.3. Preparation of salt-soluble aggregates in cod and their characterisation
Sub-task 1.4. Preparation and characterisation of insoluble aggregates from stored frozen cod
Sub-task 1.5. The disposition of proteins in aggregates by immunocytochemistry
Sub-task 1.6. Association of structures in whole tissue during frozen storage of cod and haddock
TASK 2 INTERACTIONS OF FORMALDEHYDE WITH MYOFIBRILLAR PROTEINS AND COLLAGEN IN INTACT MUSCLE
Sub-task 2.1. Systematic studies on the effect of formaldehyde on muscle proteins
Sub-task 2.2. Effect of formaldehyde on the myosin subfragments by NMR
Sub-task 2.3. Effect of formaldehyde on the microstructure of fish proteins
Sub-task 2.4. Atomic force microscopy of fish proteins treated with formaldehyde
Sub-task 2.5. The ability of formaldehyde to produce fluorescent compounds during frozen storage of cod and hake
Sub-task 2.6. The presence and type of oxidised lipid on the ability of formaldehyde to produce fluorescent compounds during frozen storage of cod and hake
TASK 3. FORMATION OF FORMALDEHYDE AND ITS INTERACTION WITH PROTEINS IN HAKE USING RADIOLABELLED TRACERS.
Sub-task 3.1. Preparation of radiolabelled 14C TMAO and its breakdown in hake flesh
Sub-task 3.2. The SDS-insoluble residue from 14 C- treated frozen hake will be examined by autoradiography
Sub-task 3.3. The interaction of radiolabelled formaldehyde with amino acids and peptides
TASK 4. INTERACTION OF LIPID OXIDATION PRODUCTS WITH FISH PROTEINS
Sub-task 4.1. Lipid oxidation product formation during frozen storage.
Sub-task 4.2. Investigation of fluorescent peptides formed in cod and haddock by fluorescence spectroscopy.
Sub-task 4.3. The association of long chain fatty acid oxidation products with protein aggregation
Sub-task4.4. Characterisation of SDS -insoluble residue from 3H-EPA-treated frozen hake by autoradiography.
Sub-task 4.5. Identification of cross-linking peptides
Sub-task 4.6. Effect of antioxidants on lipid-protein interactions
TASK 5. EFFECT OF PHYSIOLOGICAL CHANGES ON THE MAGNITUDE OF AGGREGATION IN HAKE AND BLUE WHITING
Sub-task 5.1. Investigation of changes in lipids and lipid oxidation products
Sub-task 5.2. Investigation of changes in proteins by electrophoresis, FPLC and amino acid analysis
Sub-task 5.3. Measurement of formaldehyde production frozen storage
Sub-task 5.4. Rheological and microstructural analysis of the extent of aggregation.
REFERENCES
Final Report (1995). FAR UP3.647.
Careche, M and Tejada, M. (1994). Hake natural actomyosin interaction with free fatty acids during frozen storage. J. Sci. Food Agric. 64, 501-507.
Careche, M and Tejada, M (1990). The effect of neutral and oxidised lipids on functionallity in hake (Merluccius merluccius L.) a dimethylamine and formaldehyde-forming species during frozen storage. Food Chem, 36, 113-128.
Del Mazo, M.L. Huidobro, A., Torrejon, P., Tejada, M., and Careche, M (1994). Role of formaldehyde in the formation of natural actomyosin aggregates in hake during frozen storage. Z.Lebensm. Unters. Forsch., 198, 549-464.
Howell, N.K. Shavila Y., Grutveld, M and Williams, S. (1996). High-Resolution NMR and Magnetic Resonance Imaging (MRI) studies on fresh and frozen Cod (Gadus morhua) and Haddock (Melanogrammus aeglefinus). J. Sci Food and Agric.72 49-56.
Howell, N.K. (1995). Interaction of proteins with small molecules. In Ingredient Interactions -Effects on Food Quality. Ed. A. Gaonkar. Marcel Dekker, N.Y. Pp.269-289.
Iyambo, A. (1994). The structure and physicochemical properties of frozen Atlantic cod (Gadus morhua) and Namibian Hake (Merluccius capensis) PhD Thesis. University of Surrey. Guildford.
Mackie, I (1993). The effect of freezing on flesh proteins. Food Reviews International 9. 575-610.
Tejada,M., Careche, M., Torrejon, P., Del Mazo, M.L. Solas, M.T. Garcia, M.L. and barba, C.(1996). Protein extracts and aggregates formed in minced cod (Gadus morhua) during frozen storage. J.Agric. Food Chem. 44, 3308-3314.
Fields of science (EuroSciVoc)
- natural sciences chemical sciences organic chemistry aldehydes
- natural sciences chemical sciences electrochemistry electrolysis
- natural sciences biological sciences biochemistry biomolecules proteins
- natural sciences biological sciences biochemistry biomolecules lipids
- natural sciences chemical sciences organic chemistry amines
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GU2 7XH GUILDFORD
United Kingdom
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