Proteome analysis elucidating post-mortem changes in cod (Gadus morhua) muscle proteins

Proteome analysis was successfully applied to study the alterations in fish muscle proteins during ice storage. The processes occurring during post-mortem metabolism are known to lead to characteristic changes in the texture and taste of fish muscle. Endogenous proteases are anticipated to play the major role in these processes, although the exact mechanisms during fish meat tenderization have yet to be depicted. Protein changes in cod (Gadus morhua) muscle were followed during 8 days of storage. Within the partial proteome (pI 3.5-8.0, MW 13-35 kDa) significant changes were found in 11 protein spots. In nine protein spots the intensity increased, and for eight of these the increases were significant (p < 0.05) within the first 2 h post-mortem. In contrast, two protein spots decreasing in intensity showed significant (p < 0.03) changes after 8 days, thereby indicating that in the fish muscle different biochemical processes are involved in the protein changes observed post-mortem.     

pH-induced shift in hemoglobin spectra: a spectrophotometeric comparison of atlantic cod ( Gadus morhua ) and mammalian hemoglobin

Due to a pH-sensitive effect in many fish hemoglobins (Hb), analytical errors may occur when mammalian Hb is used as a standard in quantitative spectrophotometric multicomponent analysis of fish blood. The aim of this work was to examine differences in the optical spectra of mammalian (human) and fish (farmed Atlantic cod) Hb subjected to pH 7.4 and 6.5. The absorption spectra of the common derivatives, deoxy- (HHb), oxy- (OHb), carboxy- (COHb), and methemoglobin (metHb), were determined in the spectral range of 450-700 nm. The metHb spectra of fish differed considerably from the corresponding human Hb spectra, whereas only minor differences in OHb, HHb, and COHb were found. Cod Hb was significantly (P < 0.05) influenced by a drop in pH compared to mammalian Hb. This resulted in deoxygenation of the Hb and increased autoxidation. For human Hb, a pH-independent isosbestic point in the spectra of OHb, HHb, and metHb at 523 nm was found. This isosbestic point was not found in the absorption spectra of cod Hb. In conclusion, spectra of cod metHb and human metHb behave differently. This must thus be taken into account in spectrophotometric multicomponent analysis. Ideally, Hb in muscle or blood should be determined by comparison to a standard made from the same species.     

Water distribution in brine salted cod (Gadus morhua) and salmon (Salmo salar): a low-field 1H NMR study

Low-field (LF) (1)H NMR T 2 relaxation measurements were used to study changes in water distribution in lean (Atlantic cod) and fatty (Atlantic salmon) fish during salting in 15% NaCl and 25% NaCl brines. The NMR data were treated by PCA, continuous distribution analysis, and biexponential fitting and compared with physicochemical data. Two main water pools were observed in unsalted fish, T 21, with relaxation times in the range 20-100 ms, and T 22, with relaxation times in the range 100-300 ms. Pronounced changes in T 2 relaxation data were observed during salting, revealing changes in the water properties. Salting in 15% brine lead to a shift toward longer relaxation times, reflecting increased water mobility, whereas, salting in saturated brines had the opposite effect. Water mobility changes were observed earlier in the salting process for cod compared to salmon. Good linear correlations ( F 相似文献   

Flavor and quality characteristics of salted and desalted cod (Gadus morhua) produced by different salting methods

Flavor characterization and quality of salt-cured and desalted cod (Gadus morhua) products was studied using sensory analysis and gas chromatography techniques. The products were produced in Iceland using two different processing methods (filleting and splitting) and three different salting procedures, i.e., the old single-step kench salting or a multistep procedure, and presalting (injection and brine salting or only brine salting), which was followed by kench salting. The main difference observed was between fillets and split fish, where the split fish was darker and had stronger flavor characteristics. Comparison of different salting procedures showed that the use of presalting improved the appearance of the salted products, which can be described as increased lightness and reduced yellowness of the products. In the same products, the intensity of curing flavors was milder, as described by sensory analysis and key aroma compounds. Derivatives from lipid and protein degradation contribute to the characteristic flavor of the salted products.     

Effects of fish meal replacement with full-fat soy meal on growth and tissue fatty acid composition in Atlantic cod (Gadus morhua)

Atlantic cod of initial mean weight approximately 220 g were fed a control diet and three diets in which fish meal (FM) was replaced with increasing levels of full-fat soybean meal (FFS) supplied at 12, 24, and 36% of dry diet, for 12 weeks. There were no significant differences in final weights, but the specific growth rate (SGR) was significantly higher in fish fed the control (FFS0) diet compared to fish fed the FFS12 and FFS36 diets, and the feed conversion ratio (FCR) was significantly lower in fish fed the FFS0 diet compared to the other three treatments. The fatty acid (FA) compositions of the cod muscle and liver were highly affected by dietary treatment, and linear relationships between dietary and tissue FA concentrations were shown for some of these. Moreover, selective utilization or accumulation in the tissues of specific FA was suggested by the results.     

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.     

Effect of chitosan and chitin on the separation of membranes from proteins solubilized by pH shifts using cod (Gadus morhua)

Studies with isolated membranes and isolated membranes suspended in muscle proteins solubilized at pH 3 showed that mixing chitosan and membranes at this low pH followed by a pH adjustment to 10.5 could sediment membranes effectively at 4000 g. In the solubilized muscle homogenate, the effectiveness of membrane removal by chitosan at 4000 g for 15 min was molecular weight dependent. About 80% of the phospholipids and 28% of proteins were sedimented from solubilized muscle homogenate by mixing muscle homogenate (10 g of muscle tissue homogenized with 90 mL of distilled water) with 10 mL of MW 310-375 k chitosan (10 mg/mL in 0.1 N HCl) before solubilizing it at pH 10.5, whereas 55% of the phospholipids and 12% of proteins were sedimented by mixing muscle homogenate with the MW 310-375 k chitosan before solubilizing the homogenate at pH 3. Low molecular weight chitosans (at MW 1k or 33k) showed little effect on membrane sedimentation under the same conditions. Chitin was not useful for removing membranes at either pH 3 or 10.5, whether added before or after pH adjustment.     

Characterization of volatile compounds in chilled cod (Gadus morhua) fillets by gas chromatography and detection of quality indicators by an electronic nose

Volatile compounds in cod fillets packed in Styrofoam boxes were analyzed during chilled storage (0.5 degrees C) by gas chromatography (GC)-mass spectrometry and GC-olfactometry to screen potential quality indicators present in concentrations high enough for detection by an electronic nose. Photobacterium phosphoreum dominated the spoilage bacteria on day 12 when the fillets were rejected by sensory analysis. Ketones, mainly 3-hydroxy-2-butanone, were detected in the highest level (33%) at sensory rejection, followed by amines (TMA) (29%), alcohols (15%), acids (4%), aldehydes (3%), and a low level of esters (<1%). The electronic nose's CO sensor showed an increasing response with storage time coinciding with the production of ethanol and 2-methyl-1-propanol that were produced early in the storage, followed by the production of 3-methyl-1-butanol, 3-methyl-butanal, 2,3-butandiol, and ethyl acetate. Lipid-derived aldehydes, like hexanal and decanal, were detected in similar levels throughout the storage time and contributed to the overall sweet odors of cod fillets in combination with other carbonyls (3-hydroxy-2-butanone, acetaldehyde, 2-butanone, 3-pentanone, and 6-methyl-5-heptene-2-one).     

Bioactive compounds in cod (Gadus morhua) products and suitability of 1H NMR metabolite profiling for classification of the products using multivariate data analyses

This work investigates the suitability of (1)H NMR spectroscopy to identify the fate of some bioactive compounds in seafood submitted to several processing conditions and examines the possibility of using (1)H NMR spectroscopy profiling to classify such products. Perchloric acid extracts of cod white muscle from newly killed and (i) unprocessed, (ii) boiled, and (iii) fried fillets and from (iv) frozen fillets, (v) the frozen fillets after thawing, and (vi) their drip loss and from (vii) rehydrated cod klippfish (n = 5) were analyzed by 500 MHz (1)H NMR spectroscopy. It was possible to identify taurine, betaine, anserine, creatine, and trimethylamine oxide (TMAO) in all extracts examined, and frozen fish was recognizable by the presence of dimethylamine (DMA). None of the heating procedures seemed to induce the loss of bioactive compounds from the fillet, but freezing and thawing did: the compounds were lost in what is known as drip loss. About 80% of the samples were correctly classified using a probabilistic neural network procedure having as inputs the scores of the first 20 principal components of the principal component analysis of a selected region of the NMR spectra.     

Effects of oxidized dietary oil and vitamin E supplementation on lipid profile and oxidation of muscle and liver of juvenile atlantic cod (Gadus morhua)

The effects of oxidized dietary lipid and the role of vitamin E on lipid profile, retained tocopherol levels, and lipid oxidation of juvenile Atlantic cod (Gadus morhua) were evaluated following a 9-week feeding trial. Four isonitrogenous experimental diets containing fresh or oxidized (peroxide value of 94 mequiv/kg) fish oil with or without added vitamin E (alpha-tocopherol or mixed tocopherols) were fed to juvenile cod in duplicate tanks. There was no significant (P > 0.05) influence on major lipid classes of cod liver and muscle by diet with the exception of sterols. Sterols content was increased in liver but decreased in muscle by oxidized dietary oil in the absence of vitamin E. Dietary vitamin E supplementation decreased the sterols level in cod liver but with no significant (P > 0.05) effect on their level in the muscle. Fatty acid composition varied between lipid fractions in muscle tissue and was affected by the diet. Oxidized oil significantly (P < 0.05) decreased the deposition of alpha-tocopherol in liver but not in muscle. gamma- and delta-Tocopherols from dietary tocopherol mixtures were retained at very low levels in liver, but higher retention was observed in muscle tissue. The oxidative state of both liver and muscle, as measured by the 2-thiobarbituric acid reactive substances (TBARS) and headspace propanal, negatively correlated with tissue vitamin E levels. It is suggested that oxidized oil affected juvenile Atlantic cod by causing vitamin E deficiency in certain tissues and that these effects could be alleviated by supplementation of a sufficient amount of dietary vitamin E. The results also indicate that mixed tocopherols were good antioxidants for Atlantic cod, although less effective than alpha-tocopherol alone in many tissues with the exception of muscle, where gamma- and delta-tocopherols were deposited at relatively high levels.     

Hemoglobin-mediated lipid oxidation and compositional characteristics of washed fish mince model systems made from cod (Gadus morhua), herring (Clupea harengus), and salmon (Salmo salar) muscle

The use of washed cod light muscle minces in mechanistic studies of hemoglobin (Hb)-mediated fish lipid oxidation has largely increased in the past 5 years. Although cod light muscle has a low level of intrinsic lipid oxidation catalysts, a prerequisite for a good oxidation model system, we believe it cannot fully mimic the oxidation kinetics taking place in other fish species being more susceptible to lipid oxidation. The aim of this study was to systematically investigate whether washed mince model systems useful in Hb-mediated oxidation studies could be prepared also from herring (Clupea harengus) and salmon (Salmo salar) light muscles. The kinetics of oxidation in the washed models was measured during ice storage (+/-Hb), and the results were related to compositional differences. Minces from cod, herring, and salmon light muscles were washed 3 times with 3 volumes of water and buffer. A 20 microM portion of Hb and 200 ppm streptomycin was then added, followed by adjustment of pH and moisture to 6.3 and 86%, respectively. Samples with or without Hb were then stored on ice, and oxidation was followed as peroxide value (PV), rancid odor, redness (a*) loss and yellowness (b*). Prior to storage, all minces and models were also analyzed for total lipids, fatty acids, alpha-tocopherol, proteins, Hb, Fe, Cu, and Zn. Hb-mediated lipid oxidation appeared within 2 days on ice in all models. Small differences in the oxidation rates ranked the models as herring > cod > salmon. These differences were ascribed to more preformed peroxides and trace elements in the herring model, and more antioxidants in the salmon model. Controls, without Hb, stayed stable in all cases except herring, where a very slight oxidation appeared, especially if the herring raw material had been prefrozen. In conclusion, fattier fish like dark muscle species and salmonoids are useful for making washed mince model systems and would be a better choice than cod if there is an interest in the oxidation kinetics of such species.     

Ultrastructure of the myofibrillar component in cod (Gadus morhua L.) and hake (Merluccius merluccius L.) stored at -20 degrees C as a function of time.

Transmission electron microscopy and image analysis techniques were used to study the ultrastructure of the myofibrillar component in cod and hake muscle stored at -20 degrees C for varying periods of time. Cod muscle showed a deformation of the hexagonal array of thick filaments with the storage time, reflected in an increase in the eccentricity value, a parameter defined to measure changes in the ratio of maximum to minimum hexagon diameter, and an increase in the cross-linkings between the filaments. Degradation of cod thick filaments leading to detachment was also visible upon prolonged storage. In hake muscle significant changes were not found in the arrangement and morphology of thick filaments during frozen storage, suggesting a high incidence of intrafilament aggregation. The ultrastructural differences in the array of thick filaments between species were accompanied by a difference in the textural measurements.     

In vivo bioinsecticidal activity toward Ceratitis capitata (fruit fly) and Callosobruchus maculatus (cowpea weevil) and in vitro bioinsecticidal activity toward different orders of insect pests of a trypsin inhibitor purified from tamarind tree (Tamarindus indica) seeds

A proteinaceous inhibitor with high activity against trypsin-like serine proteinases was purified from seeds of the tamarind tree (Tamarindus indica) by gel filtration on Shephacryl S-200 followed by a reverse-phase HPLC Vidac C18 TP. The inhibitor, called the tamarind trypsin inhibitor (TTI), showed a Mr of 21.42 kDa by mass spectrometry analysis. TTI was a noncompetitive inhibitor with a Ki value of 1.7 x 10(-9) M. In vitro bioinsecticidal activity against insect digestive enzymes from different orders showed that TTI had remarkable activity against enzymes from coleopteran, Anthonomus grandis (29.6%), Zabrotes subfasciatus (51.6%), Callosobruchus maculatus (86.7%), Rhyzopertha dominica(88.2%), and lepidopteron, Plodia interpuncptella (26.7%), Alabama argillacea (53.8%), and Spodoptera frugiperda (75.5%). Also, digestive enzymes from Diptera, Ceratitis capitata (fruit fly), were inhibited (52.9%). In vivo bioinsecticidal assays toward C. capitata and C. maculatus larvae were developed. The concentration of TTI (w/w) in the artificial seed necessary to cause 50% mortality (LD50) of larvae was 3.6%, and that to reduce mass larvae by 50.0% (ED50) was 3.2%. Furthermore, the mass C. capitata larvae were affected at 53.2% and produced approximately 34% mortality at a level of 4.0% (w/w) of TTI incorporated in artificial diets.     

Dynamics of humic fractions and microbial activity under no-tillage or reduced tillage,as compared with native pasture (Pampa Argentina)

Humic fractions, arginine ammonification and soil respiration were monitored in spring, summer and autumn 1999 in natural pasture soil and in no-tillage or reduced-tillage soil under maize. The Typic Argiudoll soils, typical of the Argentine rolling pampa, can be structurally unstable, particularly when conventionally tilled, a form of soil management affecting the humification process. The no-tillage soil had a lower content of fulvic acids than the reduced-tillage soil in spring and summer, probably because the humification process was favored by residue management in no-tillage soil, with a significant increase in the most stable fraction. Both arginine ammonification and CO₂ were significantly correlated with the humic acids and humin contents. No significant correlation was found with fulvic acids,probably due to the lability and high variability of this fraction. A high correlation was found between arginine ammonification and CO₂. The highest index values were generally observed in natural pasture soil, whereas no-tillage soils showed a higher index value than reduced-tillage soils throughout, confirming the hypothesis that humification is more intense in the presence of organic residues.     

再生稻根干物质量及根系活力与产量的相关性研究

对不同施N水平、不同品种再生稻根干物质量及根系活力与产量的相关性研究结果表明 ,再生稻头季稻产量与根系干物质量及根系活力密切相关 ;再生稻再生分蘖的生长发育依赖于头季稻残留的根系 ,再生季稻穗数及产量与头季稻成熟期和再生季稻齐穗期的根系机能呈极显著线性正相关 ,并与芽肥施用量呈抛物线形相关     

Procyanidin fractions from pine (Pinus pinaster) bark: radical scavenging power in solution, antioxidant activity in emulsion, and antiproliferative effect in melanoma cells

Pine (Pinus pinaster) bark is a rich source of procyanidin oligomers. From a total polyphenolic extract, we have generated fractions of different procyanidin composition. The mixtures, devoid of gallate esters, were active as free radical scavengers against ABTS(*+), DPPH, and HNTTM. Pine bark fractions were tested for antioxidant activity in solution (hydrogen donation and electron transfer) and emulsion (inhibition of lipid peroxidation) and compared with their galloylated counterparts from grape origin. While galloylation clearly influenced the free radical scavenging efficiency in solution, it did not seem to play a determinant role in protection against lipid peroxidation in emulsion. The fractions were very mild inhibitors of cell proliferation. Because gallate esters appear to interfere with crucial cell functions, gallate free pine procyanidins may be the innocuous chemopreventative agents of choice for many applications in food and skin protection.     

Glutathione S-transferases of italian ryegrass (Lolium multiflorum): activity toward some chemicals, safener modulation and persistence of atrazine and fluorodifen in the shoots

Many varieties of Italian ryegrass (Lolium multiflorum) show resistance to herbicides; while this ability was frequently attributed to alterations in the target sites of the herbicide's action of the plant or to an efficient oxidative metabolism, little attention has been paid to glutathione S-transferases (GSTs), which are a family of detoxifying enzymes involved in the inactivation of many toxic compounds. To investigate the role of GSTs, seedlings of Italian ryegrass were treated with four herbicides (atrazine, fenoxaprop-ethyl, fluorodifen, metolachlor) and a safener (fenchlorazol-ethyl). All the treatments were well tolerated by the plant, with very low decreases in terms of fresh weight and length of shoots. Regarding GST activity, the chemicals generally determined significant increases in the above enzyme activity toward the model-substrate CDNB. Therefore, the herbicides most GST inducing and the safener were tested themselves as enzyme substrates: constitutive GST activities toward atrazine, fluorodifen and fenchlorazol-ethyl were found, and, in addition, these activities were significantly induced by the safener. Following these results, a HPLC procedure was standardized in order to investigate the persistence of atrazine and fluorodifen in the seedlings of Italian ryegrass and the effect on this of the safener. It was found that the residual amounts of the two herbicides in the shoots were significantly reduced following the safener treatments.     

Surface runoff phosphorus (P) loss in relation to phosphatase activity and soil P fractions in Florida sandy soils under citrus production

Phosphorus losses by surface runoff from agricultural lands have been of public concern due to increasing P contamination to surface waters. Five representative commercial citrus groves (C1-C5) located in South Florida were studied to evaluate the relationships between P fractions in soils, surface runoff P, and soil phosphatase activity. A modified Hedley P sequential fractionation procedure was employed to fractionate soil P. Soil P consisted of mainly organically- and Ca/Mg-bound P fractions. The organically-bound P (biological P, sum of organic P in the water, NaHCO₃ and NaOH extracts) was dominant in the acidic sandy soils from the C2 and C3 sites (18% and 24% of total soil P), whereas the Ca/Mg-bound P (HCl-extractable P) accounted for 45-60% of soil total P in the neutral and alkaline soils (C1, C4 and C5 soils). Plant-available P (sum of water and NaHCO₃ extractable P fractions) ranged from 27 to 61 mg P kg⁻¹ and decreased in the order of C3>C4>C1>C2>C5. The mean total P concentrations (TP) in surface runoff water samples ranged from 0.51 to 2.64 mg L⁻¹. Total P, total dissolved P (TDP), and PO₄³⁻-P in surface runoff were significantly correlated with soil biological P and plant-available P forms (p<0.01), suggesting that surface runoff P was directly derived from soil available P pools, including H₂O- and NaHCO₃- extractable inorganic P, water-soluble organic P, and NaHCO₃- and NaOH-extractable organic P fractions, which are readily mineralized by soil microorganisms and/or enzyme mediated processes. Soil neutral (55-190 mg phenol kg⁻¹ 3 h⁻¹) and natural (measured at soil pH) phosphatase activities (77-295 mg phenol kg⁻¹ 3 h⁻¹) were related to TP, TDP, and PO₄³⁻-P in surface runoff, and plant-available P and biological P forms in soils. These results indicate that there is a potential relationship between soil P availability and phosphatase activities, relating to P loss by surface runoff. Therefore, the neutral and natural phosphatase activities, especially the natural phosphatase activity, may serve as an index of surface runoff P loss potential and soil P availability.     

Quantitation of volatiles and nonvolatile acids in an extract from coffee beverages: correlation with antioxidant activity

The antioxidant activities of a commercial brewed coffee were investigated by measuring malonaldehyde (MA) formation from oxidized cod liver oil using a gas chromatographic method (MA-GC assay) and a thiobarbituric acid method (TBA assay). The highest antioxidant activity obtained by the MA-GC assay was from regular whole brewed coffee (97.8%) at a level of 20%, and the highest antioxidant activity obtained by the TBA assay was from decaffeinated whole brewed coffee (96.6%) at a level of 5%. Among 31 chemicals identified in a dichloromethane extract, guaiacol, ethylguaiacol, and vinylguaiacol exhibited antioxidant activities, which were comparable to that of alpha-tocopherol. Among nine chlorogenic acids (three caffeoylquinic acids, three feruloylquinic acids, and three dicaffeoylquinic acids) identified, 5-caffeoylquinic acid contained the greatest amount both in regular (883.5 microg/mL) and in decaffeinated (1032.6 microg/mL) coffees; it exhibited 24.5% activity by the MA-GC assay and 45.3% activity by the TBA assay at a level of 10 microg/mL. Caffeic and ferulic acids showed moderate antioxidant activities in both assays.     

Pre-extraction preparation (fresh, frozen, freeze-dried, or acetone powdered) and long-term storage of fruit and vegetable tissues: effects on antioxidant enzyme activity

Activities of the antioxidant enzymes ascorbate peroxidase, catalase, dehydroascorbate reductase, glutathione reductase, guaiacol peroxidase, monodehydroascorbate reductase, and superoxide dismutase were assayed in honeydew (Cucumis melo L.) fruit and spinach (Spinacia oleracea L.) leaves either as fresh, frozen to -80 degrees C, frozen in liquid nitrogen, freeze-dried, or acetone powder, representing the various ways tissues are treated prior to enzyme extraction. Treated tissues were analyzed following treatment or stored for up to 8 weeks at -80 degrees C. Enzyme activities in fruit frozen with or without liquid nitrogen and leaves frozen with or without liquid nitrogen or freeze-dried were equal to those of fresh tissue. Enzyme activities in freeze-dried or acetone-powdered fruit and leaves and in acetone-powdered tissues were significantly higher or lower than those in fresh tissue. Enzyme activities in both tissues frozen with or without liquid nitrogen and stored for 8 weeks at -80 degrees C changed little; those in freeze-dried and acetone-powdered tissues, however, significantly increased/decreased over the same period. Fresh tissue should be used in antioxidant enzyme assays, but if storage is necessary, tissues should be placed directly into a -80 degrees C freezer.