Effect of antioxidants, citrate, and cryoprotectants on protein denaturation and texture of frozen cod (Gadus morhua)

To investigate the role of antioxidants and cryoprotectants in minimizing protein denaturation in frozen lean fish, cod fillets were treated with either antioxidants (vitamin C (500 mg kg(-1)) or vitamin C (250 mg kg(-1)) + vitamin E (250 mg kg(-1))), antioxidants (vitamins C + E 250 mg kg(-1)each) with citrate (100 mg kg(-1)), cryoprotectants (4% (w/w) sucrose + 4% (w/w) sorbitol), or a mixture of antioxidants (vitamins C + E 250 mg kg(1)), citrate (100 mg kg(-1)), and cryoprotectants (sucrose 40 g kg(-1) + sorbitol 40 g kg(-1)). Untreated and treated fish samples were stored at -10 degrees C; cod fillets stored at -30 degrees C were used as a control. Stored frozen samples were analyzed at intervals for up to 210 days for changes in protein extractability, thermodynamic parameters (transition temperature T(m) and enthalpy DeltaH), structure by FT-Raman spectroscopy, and rheological properties by large and small deformation tests. Results indicated that protein denaturation and texture changes were minimized in the presence of cryoprotectants, as well as in the presence of antioxidants with citrate, antioxidants alone, or the mixture of antioxidants, citrate, and cryoprotectants. In the presence of increased formaldehyde levels in fish treated with vitamin C, toughening was still lower compared to that of the -10 degrees C control due to the antioxidant property of vitamin C. Thus, ice crystal formation and lipid oxidation products are the major factors that cause protein denaturation in lean frozen fish, and antioxidants in addition to cryoprotectants can be used to minimize toughness.     

Raman spectroscopic study of structural changes in Hake (Merluccius merluccius L.) muscle proteins during frozen storage

This paper examines changes in the structure and functionality of fish muscle proteins at frozen storage temperatures known to render very different practical storage lives (-10 and -30 degrees C). Apparent viscosity and dimethylamine (DMA) content showed drastic temperature-related differences during storage. Raman spectroscopy revealed the occurrence of some structural changes involving secondary and tertiary protein structures. The changes in secondary structure were quantified, showing an increase of beta-sheet at the expense of alpha-helix structure. The nuC-H stretching band near 2935 cm(-)(1) increased in intensity, indicating denaturation of the muscle proteins through the exposure of aliphatic hydrophobic groups to the solvent. These structural changes were more pronounced at -10 degrees C but occurred at both storage temperatures, whereas changes in apparent viscosity and DMA only occurred in storage at -10 degrees C. The possible utility of these structural changes for quality assessment is discussed.     

Structural changes of hake (Merluccius merluccius L.) fillets: effects of freezing and frozen storage.

Structural changes in hake (Merluccius merluccius L.) fillets as affected by freezing method and frozen storage temperature have been studied through Raman spectroscopy and related to changes in texture and functionality. Changes in protein secondary structure were observed due to storage temperature, accompanied by changes in apparent viscosity and shear resistance. Samples at -10 degrees C showed greater structural alteration than at -30 degrees C in terms of increase of beta-sheets at the expense of alpha-helices. An increase of unordered protein structure was found only in samples stored at -10 degrees C. Exposure of buried tryptophan residues was observed at both storage temperatures. The decrease of the deltaCH(2) band upon storage suggested an increase of hydrophobic interactions of aliphatic residues. Except for liquid air frozen fillets, all samples showed a decrease of the nuO-H/nuC-H band ratio compared to the fresh ones, this decrease being higher the harsher the conditions.     

Collagen content in farmed Atlantic salmon (Salmo salar L.)

To clarify fish flesh quality problems and softening of fish muscle tissue during chilled storage, the collagen content, types I and V, and its changes in solubility during storage on ice in muscle of farmed Atlantic salmon (Salmo salar L.) were analyzed. The contents of acid-soluble, pepsin-soluble, and insoluble collagen in white muscle were determined in fresh fish muscle and after 3 days of storage in ice. The total collagen content was 2.9 g kg(-)(1) fresh weight. During storage on ice, a progressive change in solubility of muscle collagen was found. For acid- and pepsin-soluble collagen fractions, significantly higher and lower values, respectively, were detected. The presence and quantification of types I and V collagen in the different collagen fractions was determined also, but no significant difference in solubility during storage was found. The result suggested that collagen fibers of Atlantic salmon have a high solubility in acid solutions and contain few cross-links.     

Effect of various cryostabilizers on the production and reactivity of formaldehyde in frozen-stored minced blue whiting muscle

The production of formaldehyde in frozen-stored minced blue whiting muscle was described by a rectangular hyperbolic model, and the effectiveness of each cryostabilizer is discussed in terms of its parameters. The maltodextrins assayed noticeably inhibited formaldehyde production, this effect being greater at -20 degrees C than at -10 degrees C. Sucrose was only effective at -20 degrees C. It seems that these compounds act by restricting molecular diffusion. The effect of each cryostabilizer on formaldehyde binding was closely regulated by its effect on production. This is discussed in terms of the binding equation parameters. The binding of formaldehyde during frozen storage was dependent on protein rearrangements leading to reactive groups becoming available. The constraints of cryostabilizers on molecular diffusion reduced the exposure of these groups. Consequently, the interpretation of formaldehyde reactivity was biased, leading to conclusions different from those that would be obtained from a study done under standard conditions.     

Cytochrome oxidase as an indicator of ice storage and frozen storage

The potential of cytochrome oxidase as an indicator of ice storage and frozen storage of fish was investigated. Optimal assay conditions for cytochrome oxidase in a crude homogenate from cod muscle were studied. Maximal cytochrome oxidase activity was found at pH 6.5-7.5 and an assay temperature of 30 degrees C. Maximal activation by Triton X-100 was obtained in a range of 0.62-1.25 mM Triton X-100. The specificity of the assay was high, as cytochrome oxidase was inhibited 98% by 33 microM of the specific inhibitor sodium azide. The coefficient of variation of cytochrome oxidase activity in different cods was 21%, and the coefficient of variation of different analyses on the same homogenate was 5%. It was shown that ice storage of muscle samples before they were frozen and thawed resulted in a major freezing-induced activation of cytochrome oxidase activity. The enzyme may therefore be used as an indicator of frozen fish to determine if the fish has been stored on ice before freezing. Cytochrome oxidase activity showed also potential as an indicator of frozen storage, as it was possible to distinguish between the frozen storage temperatures -9, -20, and -40 degrees C.     

Protein and lipid oxidation during frozen storage of rainbow trout (Oncorhynchus mykiss)

This study aimed at investigating protein and lipid oxidation during frozen storage of rainbow trout. Rainbow trout fillets were stored for 13 months at -20, -30, or -80 degrees C, and samples were analyzed at regular intervals for lipid and protein oxidation markers. Lipid oxidation was followed by measuring lipid hydroperoxides (PV), as well as secondary oxidation products (volatiles) using dynamic headspace GC-MS. Free fatty acids (FFA) were measured as an estimation of lipolysis. Protein oxidation was followed using the spectrophotometric determination of protein carbonyls and immunoblotting. Significant oxidation was observed in samples stored at -20 degrees C, and at this temperature lipid and protein oxidation seemed to develop simultaneously. FFA, PV, and carbonyls increased significantly for the fish stored at -20 degrees C, whereas the fish stored at -30 and -80 degrees C did not show any increase in oxidation during the entire storage period when these methods were used. In contrast, the more sensitive GC-MS method used for measurement of the volatiles showed that the fish stored at -30 degrees C oxidized more quickly than those stored at -80 degrees C. Detection of protein oxidation using immunoblotting revealed that high molecular weight proteins were oxidized already at t = 0 and that no new protein oxidized during storage irrespective of the storage time and temperature. The results emphasize the need for the development of more sensitive and reliable methods to study protein oxidation in order to gain more explicit knowledge about the significance of protein oxidation for food quality and, especially, to correlate protein oxidation with physical and functional properties of foods.     

Physicochemical changes and mechanism of heat-induced gelation of arrowtooth flounder myosin

Physicochemical changes of myosin during heating were investigated to elucidate the mechanism of heat-induced gelation of arrowtooth flounder (ATF) myosin at high ionic strength. Changes in dynamic properties indicated ATF myosin formed a gel in three different stages as shown by the first increase in G' (storage modulus) at 28 degrees C, followed by the decrease at 35 degrees C and the second increase at 42 degrees C. DSC thermogram showed the onset of myosin denaturation at 25 degrees C with two maximum transition temperatures at 30 and 36 degrees C. The decrease in alpha-helical content indicated ATF myosin began to unfold at 15 degrees C and the unfolding continued until it reached 65 degrees C. Turbidity measurement showed myosin began to aggregate at 23 degrees C and the aggregation was complete at 40 degrees C. Surface hydrophobicity increased consistently in the temperature range studied, 20-65 degrees C. Sulfhydryl contents decreased significantly at 20-30 degrees C due to the formation of disulfide linkages but remained constant at temperatures >30 degrees C. ATF myosin was shown to be extremely sensitive to heat, resulting in denaturation at lower temperature than other fish myosin. Denaturation was initiated by unfolding of the alpha-helical region in myosin followed by exposure of hydrophobic and sulfhydryl residues, which are subsequently involved in aggregation and gelation processes.     

Antioxidant protection of bulk fish oils by dispersed sugars and polyhydric alcohols

Selected sugars (fructose, sucrose, or raffinose) and polyhydric alcohols (sorbitol or mannitol) were equilibrated directly with bulk fish oil (10% by weight, excess) and exposed to fluorescent lighting (2550 Lx) for 24 h at 5 degrees C. Data for room temperature-equilibrated samples revealed that polyols functioned as antioxidants in fish oil. Increased times and temperatures of equilibration (to 90-110 degrees C, 1-2 mmHg, to 2 h) greatly enhanced the antioxidant activity of polyols in fish oil exposed to light. Under accelerated oxidation conditions (60 degrees C) in the dark, dispersed sorbitol in bulk fish oil greatly suppressed the peroxide value, primarily by chelating transition metals, while fructose showed a limited antioxidant activity. Sugars with a lower molecular weight and smaller numbers of equatorial OH groups exhibited a higher rate of permeation of sugars into fish oil triacylglycerols and hence rendered greater antioxidant activities. The treatment of bulk fish oils with polyols and then using the oils in the preparation of emulsions greatly reduced their antioxidant activities as compared to those observed for treated bulk oils. The introduction of polyols dissolved in propylene glycol into bulk fish oils at 90 degrees C (0.025% polyol, 0.25 h of equilibration) provided a similar antioxidant activity to that imparted by the introduction of polyols into the oil by equilibrating excess polyols (10% by weight) with them at 90-110 degrees C for 2 h. However, regardless of the method of the introduction of polyols to bulk fish oil, an elevated temperature (90 degrees C) exposure during fish oil treatment was required to induce a notable antioxidant activity.     

Differential scanning calorimetry and circular dichroism spectrometry of walleye pollack myosin and light meromyosin

The thermodynamic properties of myosin and its C-terminal fragment, light meromyosin (LMM), from walleye pollack, a typical cold-water fish efficiently utilized on an industrial scale, were analyzed by using differential scanning calorimetry (DSC) and circular dichroism (CD) spectrometry. Recombinant walleye pollack LMM expressed in Escherichia coli was also subjected to DSC and CD measurements for reference. The two proteins prepared from frozen surimi showed three endothermic peaks, the transition temperatures (T(m)) of which were quite similar, although overall DSC patterns differed considerably from one another. Their alpha-helical contents determined by CD were low compared to values reported before for other species. On the other hand, recombinant LMM gave four endothermic peaks at 27.4, 30.8, 36.5, and 43.4 degrees C in DSC and showed an alpha-helical content of approximately 80%. The peak at 27.4 degrees C could not be observed in walleye pollack LMM prepared from frozen surimi and thus was possibly attributed to its C terminus, because this extreme C-terminal region is supposedly truncated during preparation of LMM by tryptic digestion.     

Free polyunsaturated fatty acids cause taste deterioration of salmon during frozen storage

Increased intensity of train oil taste, bitterness, and metal taste are the most pronounced sensory changes during frozen storage of salmon (Refsgaard, H. H. F.; Brockhoff, P. B.; Jensen, B. Sensory and Chemical Changes in Farmed Atlantic Salmon (Salmo salar) during Frozen Storage. J. Agric. Food Chem. 1998a, 46, 3473-3479). Addition of each of the unsaturated fatty acids: palmitoleic acid (16:1, n - 7), linoleic acid (C18:2, n - 6), eicosapentaenoic acid (EPA; C20:5, n - 3) and docosahexaenoic acid (DHA; C22:6, n - 3) to fresh minced salmon changed the sensory perception and increased the intensity of train oil taste, bitterness, and metal taste. The added level of each fatty acid ( approximately 1 mg/g salmon meat) was equivalent to the concentration of the fatty acids determined in salmon stored as fillet at -10 degrees C for 6 months. The effect of addition of the fatty acids on the intensity of train oil taste, bitterness and metal taste was in the order: DHA > palmitoleic acid > linoleic acid > EPA. Formation of free fatty acids was inhibited by cooking the salmon meat before storage. Furthermore, no changes in phospholipid level were observed during frozen storage. The results suggest that enzymatic hydrolysis of neutral lipids plays a major role in the sensory deterioration of salmon during frozen storage.     

Antioxidant activity of dietary oregano essential oil and alpha-tocopheryl acetate supplementation in long-term frozen stored turkey meat

The effects of dietary oregano essential oil and alpha-tocopheryl acetate supplementation on the oxidative stability of long-term frozen stored turkey meat were investigated. Thirty 12-week-old turkeys, randomly divided into five groups, were given a basal diet or a basal diet supplemented with 200 mg of alpha-tocopheryl acetate kg(-1), or 100 or 200 mg of oregano oil kg(-1), or 100 mg of oregano oil plus 100 mg of alpha-tocopheryl acetate kg(-1) for 4 weeks prior to slaughter. Lipid oxidation in breast and thigh meat was assessed after 1, 3, 6, and 9 months of frozen storage at -20 degrees C prior to or following 7 days of refrigerated storage at 4 degrees C. Results showed that oregano oil increased the oxidative stability of breast and thigh meat during the frozen storage. Dietary oregano oil at the inclusion level of 200 mg kg(-1) feed was significantly (p < 0.05) more effective in delaying lipid oxidation compared to the level of 100 mg kg(-1), but equivalent to dietary alpha-tocopheryl acetate supplementation at 200 mg kg(-1), which in turn was inferior to dietary supplementation of 100 mg kg(-1) oregano essential oil plus 100 mg kg(-1) alpha-tocopheryl acetate that was significantly (p < 0.05) superior to all other treatments. Thigh meat was more susceptible to oxidation than breast meat, although the former contained alpha-tocopherol at markedly higher levels. Mean alpha-tocopherol levels in breast and thigh meat from all treatments decreased during the frozen storage, the decrease being sharper between 1 and 3 months of frozen storage for breast and between 3 and 6 months for thigh meat. Oregano oil supplementation increased (p < 0.05) the retention of alpha-tocopherol in meat, the increase being positively correlated with the supplementation level. However, the retention of alpha-tocopherol in meat could only partly elucidate the antioxidant activity exhibited by dietary oregano oil supplementation.     

Chemical composition, antioxidant properties, and thermal stability of a phytochemical enriched oil from Acai (Euterpe oleracea Mart.)

Phenolic compounds present in crude oil extracts from acai fruit ( Euterpe oleracea) were identified for the first time. The stability of acai oil that contained three concentrations of phenolics was evaluated under short- and long-term storage for lipid oxidation and phenolic retention impacting antioxidant capacity. Similar to acai fruit itself, acai oil isolates contained phenolic acids such as vanillic acid (1,616 +/- 94 mg/kg), syringic acid (1,073 +/- 62 mg/kg), p-hydroxybenzoic acid (892 +/- 52 mg/kg), protocatechuic acid (630 +/- 36 mg/kg), and ferulic acid (101 +/- 5.9 mg/kg) at highly enriched concentrations in relation to acai pulp as well as (+)-catechin (66.7 +/- 4.8 mg/kg) and numerous procyanidin oligomers (3,102 +/- 130 mg/kg). Phenolic acids experienced up to 16% loss after 10 weeks of storage at 20 or 30 degrees C and up to 33% loss at 40 degrees C. Procyanidin oligomers degraded more extensively (23% at 20 degrees C, 39% at 30 degrees C, and 74% at 40 degrees C), in both high- and low-phenolic acai oils. The hydrophilic antioxidant capacity of acai oil isolates with the highest phenolic concentration was 21.5 +/- 1.7 micromol Trolox equivalents/g, and the total soluble phenolic content was 1252 +/- 11 mg gallic acid equivalents/kg, and each decreased by up to 30 and 40%, respectively, during long-term storage. The short-term heating stability at 150 and 170 degrees C for up to 20 min exhibited only minor losses (<10%) in phenolics and antioxidant capacity. Because of its high phenolic content, the phytochemical-enriched acai oil from acai fruit offers a promising alternative to traditional tropical oils for food, supplements, and cosmetic applications.     

Biochemical and functional properties of Atlantic salmon (Salmo salar) muscle proteins hydrolyzed with various alkaline proteases

Protein hydrolysates (5, 10, and 15% degrees of hydrolysis) were made from minced salmon muscle treated with one of four alkaline proteases (Alcalase 2.4L, Flavourzyme 1000L, Corolase PN-L, and Corolase 7089) or endogenous digestive proteases. Reaction conditions were controlled at pH 7.5, 40 degrees C, and 7.5% protein content, and enzymes were added on the basis of standardized activity units (Azocoll units). Proteases were heat inactivated, insoluble and unhydrolyzed material was centrifuged out, and soluble protein fractions were recovered and lyophilized. Substrate specificities for the proteases was clearly different. Protein content for the hydrolysates ranged from 71.7 to 88.4%, and lipid content was very low. Nitrogen recovery ranged from 40.6 to 79.9%. The nitrogen solubility index was comparable to that of egg albumin and ranged from 92.4 to 99.7%. Solubility was high over a wide range of pH. The water-holding capacity of fish protein hydrolysates added at 1.5% in a model food system of frozen minced salmon patties was tested. Drip loss was on average lower for the fish protein hydrolysates than for egg albumin and soy protein concentrate, especially for Alcalase hydrolysates. Emulsification capacity for fish protein hydrolysates ranged quite a bit (75-299 mL of oil emulsified per 200 mg of protein), and some were better than soy protein concentrate (180 mL of oil emulsified per 200 mg of protein), but egg albumin had the highest emulsifying capacity (417 mL of oil emulsified per 200 mg of protein). Emulsification stability for fish protein hydrolysates (50-70%) was similar to or lower than those of egg albumin (73%) or soy protein concentrate (68%). Fat absorption was greater for 5 and 10% degrees of hydrolysis fish protein hydrolysates (3.22-5.90 mL of oil/g of protein) than for 15% hydrolysates, and all had greater fat absorption than egg albumin (2. 36 mL of oil/g of protein) or soy protein concentrate (2.90 mL of oil/g of protein).     

Rheological and physicochemical properties of starches from moist- and dry-type sweetpotatoes

Although starch makes up from 50 to 70% of sweetpotato (SP) dry matter, its role in cooked texture is unknown. The purpose of this research was to characterize raw starches isolated from SP cultivars and experimental selections (C/S) with a wide range of textural properties when cooked and to investigate the relationship between textural properties of the cooked roots and characteristics of the isolated starches. Shear stress measured by uniaxial compression of cooked SP cylinders served as an objective measure of SP texture. Starches were isolated from C/S representing three SP texture types: moist (Jewel and Beauregard); intermediate (NC10-28 and NC2-26); and dry (NC6-30 and NC8-22). The following parameters of isolated starches were measured: amylose content by colorimetric and differential scanning calorimetric (DSC) methods; swelling power, solubility, gelatinization enthalpy (DeltaH), and pasting properties by Brabender amylograph (BA) and rapid viscoanalyzer (RVA). Pasting temperatures for SP C/S measured by BA and RVA were significantly correlated. Due to high shear degradation in RVA, RVA viscosities of starch suspensions decreased as much as 40% during cooking at 95 degrees C, whereas the BA viscosities changed little at this temperature. There were no statistically significant differences among the C/S for amylose or DeltaH. However, significant C/S differences in swelling power, solubility, and pasting properties were observed. Although differences in some rheological and physical properties were observed for C/S starches, shear stress was statistically correlated only with DSC onset temperature (r = 0.78), indicating that factors other than the properties measured on isolated starches are mainly responsible for the texture of cooked SP C/S.     

Differential Scanning Calorimetric Study on the Effects of Frozen Storage on Gluten and Dough

Water transport from the gluten to the starch paste during frozen storage of a dough was studied by analysis of the differential scanning calorimetry (DSC) peak shape for gluten and dough. The results indicate that in gluten stored at ‐15°C, the water content in the gluten phase decreased by ≈1% over the first three weeks. An apparent steady state was attained over this period and the amount of ice did not increase further. Such changes were not observed for samples stored at ‐25°C. Qualitatively similar observations were made for dough stored at either ‐15 or ‐25°C. The greater sensitivity of dough to frozen storage is tentatively attributed to a slightly lower glass transition temperature in dough.     

The effects of frozen storage conditions on lycopene stability in watermelon tissue

The purpose of this investigation was to evaluate the rate of deterioration of lycopene in watermelon tissue during frozen storage, because little is known about the stability of watermelon tissue lycopene under cold storage conditions. Heart tissue from each of nine individual watermelons was stored at -20 or -80 degrees C as either small chunks or puree and periodically sampled over a year's time. Initial freeze-thaw experiments indicated that a small percentage of lycopene, approximately 4-6%, degraded during an initial freeze-thaw. Analyses of the samples showed a loss of approximately 30-40% lycopene over a year's storage at -20 degrees C and a loss of approximately 5-10% over the same period at -80 degrees C. Lycopene was slightly more stable in pureed compared with diced watermelon tissue at -20 degrees C, but not at -80 degrees C. The kinetic data were best fitted by application of two simultaneous, first-order decay processes. HPLC analysis of the samples after a year's storage suggested that beta-carotene was more stable during storage at -20 degrees C than was lycopene.     

Determinant parameters and components in the storage of virgin olive oil. Prediction of storage time beyond which the oil is no longer of "extra" quality.

This work studies the changes in the quality indices standardized by the European Union, together with the evolution of the oxidative stability and sterols, polyphenols, alpha-tocopherol, pigments, and fatty acids contents throughout the storage of Picual and Hojiblanca olive cultivar "extra" virgin olive oils at 2 degrees C + darkness and 30 degrees C + illumination. Only two quality indices (K(270) and sensory evaluation) indicate the loss of the extra quality of the oil during storage, and there is an excellent correlation between initial stability and the time to reach the limit of K(270) > 0.25 (after which the oil quality is no longer of "extra" quality). This time can be predicted with an error of <10%, which is of great commercial interest and previously unknown. Also unknown until now is that the changes in polyphenols, pigments, and alpha-tocopherol with storage time follow first-order kinetics.     

Phase transitions, solubility, and crystallization kinetics of phytosterols and phytosterol-oil blends

The thermal properties, solubility characteristics, and crystallization kinetics of four commercial phytosterol preparations (soy and wood sterols and stanols) and their blends with corn oil were examined. Differential scanning calorimetry (DSC) revealed narrow melting peaks between 138 and 145 degrees C for all phytosterol samples, reversible on rescan. Broader and less symmetrical melting transitions at lower temperatures with increasing oil content were observed for two samples of phytosterol-oil admixtures. The estimated, from the solubility law, deltaH values (34.7 and 70.7 mJ/mg for wood sterols and stanols, respectively), were similar to the DSC experimental data. Fatty acid esters of soy stanols differing in the chain length of the acyl groups (C2-C12) exhibited suppression of the melting point and increase of the fusion enthalpy with increasing chain length of the acyl group; the propionate ester exhibited the highest melting point (Tm: 151 degrees C) among all stanol-fatty acid esters. Solubility of phytosterols in corn oil was low (2-3% w/w at 25 degrees C) and increased slightly with a temperature rise. Plant sterols appeared more soluble than stanols with higher critical concentrations at saturation. The induction time for recrystallization of sterol-oil liquid blends, as determined by spectrophotometry, depended on the supersaturation ratio. The calculated interfacial free energies between crystalline sediments and oil were smaller for sterol samples (3.80 and 3.85 mJ/m2) than stanol mixtures (5.95 and 6.07 mJ/m2), in accord with the higher solubility of the sterol crystals in corn oil. The XRD patterns and light microscopy revealed some differences in the characteristics among the native and recrystallized in oil phytosterol preparations.