Elucidation of protein aggregation in frozen cod and haddock by transmission electron microscopy/immunocytochemistry,light microscopy and atomic force microscopy

Polyclonal antibodies raised to both native cod myosin and actin as well as to aggregated proteins obtained from frozen cod stored for 11 months at ?10 °C were used to investigate disposition of muscle proteins in frozen cod and haddock fillets by transmission electron microscopy. Specimens from cod and haddock fillets, stored at ?10 °C, treated with anti‐aggregate antibody as the primary antibody, showed significantly more gold particles, especially around the protein aggregates and muscle fibres compared with fish stored at ?30 °C. Samples that were treated with anti‐myosin or anti‐actin antibody showed opposite results. Similar binding properties were observed in ELISA experiments involving the reaction of actin and myosin to both native and aggregate antibodies; thus immunological tests can be used for monitoring aggregate and texture changes in frozen stored fish. In addition, atomic force microscopy images obtained from cod muscle also indicated structural changes in frozen cod muscle proteins. The mica surface was covered with a continuous layer of muscle proteins comprising mainly small globular particles and a few large particles for the control cod sample stored at ?30 °C for 11 months. In contrast, cod fillets stored at ?10 °C showed a thin layer of proteins with small holes and an increased number of large particles denoting aggregates. Formation of ice crystals between the muscle fibres of frozen cod and haddock muscle was monitored without thawing by light microscopy at ?20 °C. The micrographs showed a greater proportion of large ice crystals and extensive protein fibre changes in fillets stored at ?10 °C compared with the control at ?30 °C. Copyright © 2004 Society of Chemical Industry     

A comparison of biochemical changes in cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) fillets during frozen storage

Changes in the muscle proteins of frozen cod fillets, which produce significant amounts of formaldehyde, and frozen haddock fillets, which produce negligible formaldehyde, were compared. Protein extractability and hydrophobicity and the amino acid contents of soluble and insoluble proteins, as well as formaldehyde formation, were investigated in matching pairs of cod and haddock fillets stored at ?10 and ?30 °C (control). Formaldehyde production in cod was much higher (845 and 1065 nmol g^?1 at 20 and 30 weeks respectively) than in haddock (93 and 101 nmol g^?1 after 20 and 30 weeks respectively) at ?10 °C. However, a rapid decrease in solubility of proteins, increase in hydrophobicity and decrease in the amino acid content of salt‐soluble proteins at ?10 compared with ?30 °C were observed in both species. The results showed that there were no significant differences in the nature of the protein changes between these two species, thus indicating that factors other than formaldehyde were involved in the denaturation of proteins and the formation of aggregates during frozen storage of cod and haddock fillets, especially at ?10 °C. © 2001 Society of Chemical Industry     

Changes in Electrophoretic Patterns of Gadoid and Non-gadoid Fish Muscle during Frozen Storage

Sodium dodecyl sulfate polyacrylamidc gel electrophoresis of fish muscle during frozen storage showed that a crosslinked protein of 280,000 daltons formed with gadoids, whereas this band did not occur with non-gadoid fish species. For cod, 20 days at -7°C are needed for the crosslink to appear, whereas for whiting only 3 days arc needed. Formaldehyde (FA) may be responsible for the crosslinking. Adding ascorbic acid to cod mince prior to freezing accelerated dimcthylamine and FA formation during frozen storage and accelerated the formation of the crosslinked protein, while leaving cod fillets on ice for 10 days prior to freezing, reduced the Instron hardness and the crosslinked protein did not appear with 50 days of frozen storage at -7°C.     

Development of Botulinal Toxin and Sensory Deterioration During Storage of Vacuum and Modified Atmosphere Packaged Fish Fillets

Toxin production by C. botulinum type E was studied in cod, whiting, and flounder filets packaged in air-permeable film, vacuum packages and packages flushed with N₂ or CO₂ during storage at 8°, 12° or 26°C. Cod and whiting filets were flushed with CO₂ and stored continuously at 4°C or cycled between 4° or 8° and 26°C. Cod and whiting fillets were flushed with gas mixtures and stored at 8°C or 26°C. Flounder deteriorated rapidly and was rejected by sensory evaluation prior to toxin detection during vacuum or modified atmosphere storage at 12°C and 8°C but after toxin detection at 26°C. Toxin was present either prior to or simultaneously with sensory rejection of cod and whiting fillets for all vacuum or modified atmosphere treatments and temperature regimens.     

Effect of lipid oxidation and frozen storage on muscle proteins of Atlantic mackerel (Scomber scombrus)

The effect of storage on the lipids and proteins in Atlantic mackerel stored for up to 24 months at ?20 and ?30 °C was studied. Traditional methods including the peroxide value, thiobarbituric acid‐reactive substances (TBARS) and a reverse phase HPLC method were used to determine the primary and secondary lipid oxidation products. All tests showed an increase in lipid oxidation products with storage time and at a higher storage temperature of ?20 °C compared with samples stored at ?30 °C. Antioxidants had a significant effect (P < 0.01) on the inhibition of lipid oxidation, as shown by the reduction in peroxide value and hydroxides, and malondialdehyde formation. Similarly, deterioration of protein structure and functionality in mackerel stored for 3, 6, 12 and 24 months was greater at ?20 than ?30 °C. ATPase activity in the myosin extract of Atlantic mackerel showed a significant decrease (P < 0.01) with progressive frozen storage. Protein solubility in high salt concentration (0.6 M NaCl) decreased (P < 0.01) during storage at both ?20 and ?30 °C but was greater at ?20 °C. Interestingly, antioxidants BHT, vitamin C and vitamin E protected the proteins against complete loss of ATPase activity and protein solubility to a significant level (P < 0.01) for up to 1 year at ?20 °C compared with samples stored without antioxidants. This study confirms the deleterious effect of lipid oxidation products on protein structure and function in frozen fatty fish. © 2002 Society of Chemical Industry     

Effects of Frozen Storage on the Ultrastructure of Bovine Muscle

Scanning electron microscopy (SEM) studies were carried out on bovine semitendinosus samples that were subjected both to long-term frozen storage and to repeated freeze-thaw cycles. Samples frozen at –18°C and stored up to 26 wk showed essentially no change in muscle ultrastructure. Samples frozen at liquid nitrogen temperature and stored at 2–3°C did show ice crystal damage within the muscle fiber. Repeated freeze-thaw produced essentially no change in muscle ultrastructure.     

Separation of Cod (Gadus morhua) Fillet Proteins by Electrophoresis and HPLC after Various Frozen Storage Treatments

Myofibrillar, sarcoplasmic (SAR) and total extractable proteins (TEP) were isolated from the same lot of frozen cod (Gadus morhua) fillets stored at -30°C, -22°C, -15°C, and -12°C and a simulated industrial fluctuating temperature (SIFT) program. HPLC and SDS-PAGE showed that myofibrillar and SAR of the fillets stored at -12°C and -15°C exhibited low and high molecular weight changes similar to the SIFT. A new indicator of myofibrillar frozen storage quality was developed using SDS-PAGE. TEP profiles by HPLC were time and temperature dependent. These observations by SDS-PAGE and HPLC were further evidence that changes in frozen stored cod muscle were partially due to covalent S-S crosslinking.     

Extractability of muscle protein as criterion for Technological evaluation of sea fish Part 1. Changes of protein fraction of Baltic herring and Baltic cod during storage in ice

Content of protein fractions dissolved in 0,5 – 5% NaCl solutions (total protein, myofibrillar protein and crude actomyosin) were investigated in meat tissue of Baltic cod and Baltic herring stored in ice at 1 °C (±1 °C). Cod was showing faster reduction in extractability of soluble proteins than herring during spoilage.     

Rheological Behavior of Frozen and Thawed Low-Moisture, Part-Skim Mozzarella Cheese

Stress relaxation and dynamic profiles of low-moisture, part-skim (LMPS) Mozzarella cheese cylinders refrigerated 14 days (control), frozen and thawed, and stored frozen and refrigerated up to 90 days were compared. Samples were frozen at ?30°C and stored at ?20°C. Thawing and refrigerated storage were at 5°C. Stress relaxation tests were conducted at 20°C and dynamic spectrometry at 20°C and 60°C. The frozen and thawed Mozzarella cheese tested at 20°C became harder and more elastic with storage time, while refrigerated stored samples became softer and more elasticoviscous with time. Upon melting, both go-day-frozen and go-day-refrigerated cheeses were less elastic and less viscous than 14-day-refrigerated samples.     

The effects of spoilage on the dielectric properties of frozen fish

Using a time-domain technique to obtain low frequency dielectric data the effects of spoilage on certain dielectric properties of frozen cod (Gadus morhua), haddock (Melanogrammus aeglefinus) and lemon sole (Microstomus kitt) have been measured at ? 78.5°C in the frequency range 0.025–10 kHz. The spoilage varied with the period of storage in ice prior to freezing. The measurements revealed a relaxation, the frequency of which in cod and haddock depended on pre-freezing quality of the fish. The relationship is discussed between this dependence and various other parameters and the relaxation is tentatively identified as that of a Maxwell-Wagner mechanism.     

Effect of Phosphate Blends on Stability of Cod Fillets in Frozen Storage

Atlantic cod fillets were dipped in commercial tripoly or metaphosphate solutions, frozen and stored at either –12 ° 0.5°C or –30°C (constant or with daily programmed fluctuations to ?26°C) for up to 26 wk. Phosphate treatments at both storage temperatures decreased thaw drip and cooked drip and yielded a product with higher raw and cooked moisture. Protein content of cooked drip from fillets stored at –12°C was reduced by phosphate treatment; no significant difference was found between treated and control samples at –30°C. Although salt extractable protein was lowered, phosphate treatment did not affect dimethylamine/formaldehyde formation. Sensory evaluation of treated fillets stored at –30°C (with daily fluctuation to –26°C) revealed phosphated fillets to be the most tender and, after 26 wk storage, the most highly acceptable. Tripolyphosphate treatment significantly retarded the increase of expressible fluid under abusive conditions of frozen storage.     

Cryoprotectants improve physical and chemical properties of frozen blue crab meat (Callinectes sapidus)

The storage stability of cryogenically frozen crab meat treated with cryoprotectants was compared with pasteurised and water-treated frozen samples to determine changes in physical and chemical properties. Commercially processed, hand-picked blue crab meat was treated with solutions of polydextrose, sucrose + sorbitol/sodium tripolyphosphate or water, vacuum packaged, and cryogenically frozen prior to storage at ?29°C. All treatments were compared with an untreated reference control stored at ?65°C and a pasteurised sample stored at 1.1°C. Samples were evaluated for physical and chemical changes at 4-week intervals over 32 weeks of storage. Cryoprotectants improved oxidative stability, drip loss, expressible moisture, texture, and colour compared to untreated reference and pasteurised samples.     

Effect of storage conditions on the quality of smoke-dried herring (Sardinella eba)

Samples of freshly smoked herring (Sardinella eba) at 140 g kg^?1 moisture were stored for a maximum period of 6 months using five different storage methods: in polyethylene bags at ambient temperature with and without desiccant, frozen ( ?20°), as well as the traditional and a modified oven storage technique. Their relative effectiveness in preserving the quality of the fish was evaluated in terms of storage losses, sensory changes and decomposition. Next to frozen storage, the most effective method was the modified procedure which gave storage yields of 97% and high sensory scores. Proteolytic and lipolytic deterioration was negligible. Although the traditional storage retained high sensory and chemical properties, over 30% storage losses were recorded. Storage in polyethylene bags at ambient temperature was ineffective, while inclusion of desiccant only delayed total decomposition beyond one month. Major organisms observed in stored smoked herring were Micrococcus, Bacillus, coryneform group, Aspergillus, Penicillium, Rhizopus and yeasts.     

Use of Cryoprotectants to Stabilize Functional Properties of Prerigor Salted Beef During Frozen Storage

Changes in functional properties (gelforming ability, water-holding capacity, and protein solubility) of salted prerigor beef and postrigor beef were measured over 6 months frozen storage at ?28°C, as affected by addition of 5.6% cryoprotectants [Polydextrose® or a mixture (1:1) of sucrose and sorbitol]. Addition of NaCl to comminuted muscle prior to freezing accelerated destabilization of muscle proteins with respect to functional properties. This effect was reduced by cryoprotectants; most effectively by sucrose/sorbitol and only slightly less effectively by polydextrose. As evidenced by the parameters investigated, quality of prerigor salted muscle treated with cryoprotectants and stored 6 months was approximately equal to that of untreated postrigor meat prior to freezing.     

Effect of Cryoprotectants on Frozen Burbot Fillets and a Comparison with Whitefish Fillets

Burbot fillets (Lota lota) were injected with sodium tripolyphosphate (STP), sodium glutamate (MSG), a high pH buffer or an antioxidant mixture (BHA, propyl gallate and citric acid) under high pressure, then frozen and stored at ?12°C. Control samples included untreated fillets stored at ?12°C and ?60°C, and STP dipped fillets. Samples treated with STP or the high-pH buffer had better textural properties than untreated control samples. Samples injected with STP exhibited no improvement as compared to the dipped STP samples and in some respects were worse. Burbot and whitefish have very different textural properties; however, the patterns of change during frozen storage are quite similar.     

Retention of Phytochemicals in Fresh and Processed Broccoli

Our objective was to determine whether steam blanching, storage and preparation affected concentrations of sulforaphane (SF), sulforaphane nitrile (SFN), cyanohydroxybutene (CHB), iberin (I) or iberin nitrile (IN) in fresh and frozen broccoli. Broccoli (var. “Arcadia”) was grown in St. Charles, IL over three seasons. Samples were steam blanched (2 min at 93 ± 5°C) within 24h of harvest, frozen and stored at ?20°C up to 90 days, and fresh broccoli was stored at 4°C up to 21 days. Samples were analyzed uncooked or microwave cooked. SF, SFN, I, IN and CHB were determined by GC in dichloromethane extracts from lyophilized samples. Rates of loss for CHB and SF were similar during storage of fresh broccoli. Blanching, storage, and microwave cooking decreased (p < 0.01) concentrations of each compound in fresh and frozen broccoli.     

Prediction of Quality in Frozen Cod (Gadus morhua) Fillets

Physical and chemical indices were determined on frozen cod (Gadus morhua) fillets stored for ca. 90 days at either - 12°C, - 15°C, - 22°C, - 30°C or under a set of simulated industrial fluctuating temperature conditions (SIFTC). Univariate and multivariate statistics on the quality indices gave a relationship between frozen storage textural deterioration and the chemical parameters as influenced by storage temperature. Results on the SIFTC resembled the - 12°C and - 15°C storage treatments. Chemical indices had lower activation energy values than those for the physical parameters. Ammonia, determined enzymatically, can be used as an index of frozen fish quality. The quadratic equations developed using the dependent variable of Instron raw peak force, independent of time and temperature, can predict the textural quality of frozen cod fillets.     

Foaming capacity of fish minces during frozen storage

This work covers a year's study of the foaming capacity (FC) of minced muscle of three fish species—blue whiting (Micromesistius poutassou R). horse mackerel (Trachurus trachurus L), and mackerel (Scomber scombrus L)—caught in two different seasons (January and July) and stored at – 18°C. Periodic assays were performed to determine protein solubility, FC and dimethylamine formation. The results show that FC is initially inhibited by the presence of the species own lipids; however, during frozen storage this effect is attenuated and thus FC increases. The results corroborate the view that protein solubility is not an indispensible requisite for FC, and that mere dispersion of proteins is sufficient. However, FC is not diminished by changes in protein during frozen storage when this deficiency is offset by sufficient protein concentration. On the other hand, FC is clearly affected by the formaldehyde generated during frozen storage, so that beyond a certain level the aggregates formed as a result of formaldehyde formation are too large to disperse, and hence FC decreases drastically.     

Thawing, Refreezing and Frozen Storage Effects on Muscle Functionality and Sensory Attributes of Frozen Cod (Gadus morhua)

Thawing and re-freezing of cod fillet blocks did not necessarily result in greater deterioration (relative to a ‘once frozen’ control) of cooked fish sensory attributes after 9 months frozen storage at –22°C. Thawed and refrozen fish muscle displayed a faster decline in myofibril protein solubility than once frozen controls and had reduced water-holding capacity but analysis of proton spin-spin relaxation times indicated no change in water location. The decline in protein solubility was not caused by complete protein unfolding. Long thawing times (30 hr) before re-freezing and storage resulted in cooked fish with more gray appearance and more stale flavor. Changes in fish muscle functionality did not enable direct inference of sensory attributes.     

Changes in the muscle of lemon sole (Pleuronectes microcephalus) after very long cold storage

The solubility of Lemon Sole muscle in 5% sodium chloride after storage at — 14 to — 15°C was shown to fall to the same minimum level as that of cod, but the reaction was very much slower. After storage for 5 years the protein had still been fairly soluble, and appeared to have reached the asymptote, but in the present experiment the fish were stored for over 17 years and the reaction was now evidently complete. It is complete in cod at this temperature in about 15 weeks.