Polyphenoloxidases immobilized in organic gels: Properties and applications in the detoxification of aromatic compounds

Gelatine gels originate from water in oil microemulsions in which the ternary system consists of isooctane/ sulfosuccinic acid bis [2-ethyl hexyl] ester/water; the solubilization of gelatin in the water pool of these microemulsions transforms them into viscous gels in which it is possible to cosolubilize various reactive molecules. These gels were used to immobilize two phenoloxidases, a laccase from Trametes versicolor and a tyrosinase from mushroom. The best balance between gel retention and catalytic activity was reached at a gelatine concentration of 2.5% (w/v) in the case of tyrosinase, while laccase immobilization was independent of gelatine concentration. Both enzymes kept the same optimum pH as the corresponding soluble controls, while a partial loss of activity was observed when they were immobilized. Immobilized enzymes showed an increased stability when incubated for several days at 4 degrees C with a very low release from the gels in the incubation solutions. The immobilization of tyrosinase and of laccase enhanced stability to thermal inactivation. Furthermore, gel-entrapped tyrosinase was almost completely preserved from proteolysis: more than 80% of the activity was maintained, while only 25% of the soluble control activity was detected after the same proteolytic treatments. A column packed with gel-immobilized tyrosinase was used to demonstrate that enzymes immobilized with this technique may be reused several times in the same reaction without loosing their efficiency. Finally, gel-entrapped tyrosinase and laccase were capable of removing naturally occurring and xeno-biotic aromatic compounds from aqueous suspensions with different degrees of efficiency. (c) 1995 John Wiley & Sons, Inc.     

Purification and characterization of a cold-adapted uracil-DNA glycosylase from Atlantic cod (Gadus morhua)

Uracil-DNA glycosylase (UDG; UNG) has been purified 17000-fold from Atlantic cod liver (Gadus morhua). The enzyme has an apparent molecular mass of 25 kDa, as determined by gel filtration, and an isoelectric point above 9.0. Atlantic cUNG is inhibited by the specific UNG inhibitor (Ugi) from the Bacillus subtilis bacteriophage (PBS2), and has a 2-fold higher activity for single-stranded DNA than for double-stranded DNA. cUNG has an optimum activity between pH 7.0-9.0 and 25-50 mM NaCl, and a temperature optimum of 41 degrees C. Cod UNG was compared with the recombinant human UNG (rhUNG), and was found to have slightly higher relative activity at low temperatures compared with their respective optimum temperatures. Cod UNG is also more pH- and temperature labile than rhUNG. At pH 10.0, the recombinant human UNG had 66% residual activity compared with only 0.4% for the Atlantic cUNG. At 50 degrees C, cUNG had a half-life of 0.5 min compared with 8 min for the rhUNG. These activity and stability experiments reveal cold-adapted features in cUNG.     

Optimization of gelatine extraction from grass carp (Catenopharyngodon idella) fish skin by response surface methodology

To establish the optimum gelatine extraction conditions from grass carp fish skin, response surface methodology (RSM) was adopted in this study. The effects of concentration of HCl (%, A), pre-treatment time (h, B), extraction temperature ( degrees C, C) and extraction time (h, D) were studied. The responses were yield (%) and gel strength (g). A=1.19%, B=24 h, C=52.61 degrees C and D=5.12h were determined as the optimum conditions while the predicted responses were 19.83% yield and 267 g gel strength. Gelling and melting points were 19.5 degrees C and 26.8 degrees C, respectively. Moreover, grass carp gelatine showed high contents of imino acids (proline and hydroxyproline) 19.47%. RSM provided a powerful tool to optimize the extraction parameters and the results may be adapted for industrial extraction of gelatine from grass carp fish skins.     

Utilisation of male Arctic capelin and Atlantic cod intestines for fish sauce production--evaluation of fermentation conditions

The present paper shows that the fish by-products male Arctic capelin and Atlantic cod intestines can be utilized as raw materials for the production of high value fish sauce for human consumption. By supplementing minced capelin with 5-10% enzyme-rich cod pyloric caeca, a good recovery of fish sauce protein (60%) was obtained after 6 months of storage. Although, the proteases present in cod pylorus caeca are cold adapted enzymes, a storage temperature of 26 degrees C gave a higher fish sauce recovery than storage at 21 degrees C. Initial alkalification of samples to pH 8 accelerated the tissue solubilisation and gave a better fish sauce recovery without adveresely affecting the pH of the final product. Addition of small amounts of calcium had no significant effects on either protease activity or protein recovery in the fish sauce fraction.     

Disposition of arsenobetaine in two marine fish species following administration of a single oral dose of [14C]arsenobetaine

The distribution and excretion of arsenobetaine in fish were investigated using whole body autoradiography and liquid scintillation counting. A single dose of synthesised [(14)C]arsenobetaine was orally administered to Atlantic salmon, Salmo salar L., and Atlantic cod, Gadus morhua L. Arsenobetaine was distributed to most organs within both species. Nevertheless, there were species differences in tissue distribution and excretory pattern. The highest level of arsenobetaine in Atlantic salmon was present in muscle tissue, while high levels of arsenobetaine were found in both muscle and liver (including gall bladder) from Atlantic cod. The results suggest that the major route of excretion was via urine, which seemed to be more important in Atlantic cod than in Atlantic salmon. Elimination of arsenobetaine via bile appeared to be negligible in both species.     

Gel strength of Ca-limited alginate gels made in situ

The gel strengths of Ca-alginate gels made in situ with different degree of cross-linking were determined by adapting three different methods: FIRA Jelly Tester, initial deformation (Youngs modulus, E) with a Stevens LFRA Texture Analyzer, and dynamic measurements with a Bohlin VOR Rheometer (dynamic storage modulus, G). The results showed that there were relatively large differences in absolute values, but that the deviations diminished when the results were expressed as relative gel strengths. The deviations of the Youngs modulus (E) from G increased with decreasing gel strength. Only dynamic measurements were suitable for quantifying low gel strengths.The gel strength and the breaking point were also measured as a function of the molecular weight of alginates isolated from stipes of Laminaria hyperborea. In the present Ca limited system, both the gel strength and the breaking point showed a marked increase with increasing molecular weight (Mw) up to 320–340 kD. This is considerably higher than with gels made by dialysis (Ca saturated), where the gel strength becomes independent on molecular weight around 100 kD. These results may have an impact on applications of alginate gels where the source of Ca crosslinking ions is limited.     

Characterization of kappa-carrageenan gels used for immobilization of Bacillus firmus.

In this study, aimed at a biochemical and physical characterization of kappa-carrageenan gels used for entrapment of Bacillus firmus NRS 783 (a superior producer of an alkaline protease), effects of carrageenan concentration, gelation temperature, initial cell loading, and strength of the curing agent (KCl) on the properties of cell-free and cell-laden gels were examined. The physical properties of the differently prepared gels that were examined included density, free volume fraction, mechanical strength, and change in gel volume during gel curing. The biochemical characteristics studied included viability of gel-entrapped cells, cell leakage from cell-laden gels, and cell penetration into cell-free gels. For the range of carrageenan contents investigated [between 2% and 5% (w/v)], the mechanical strength of the gels with/without KCl curing was observed to increase with an increase in carrageenan content of gels. The mechanical strength of each gel increased substantially upon extensive curing. Free volume fractions in excess of 0.8 were observed for all gels. Of cells that were viable prior to immobilization, 90-92% remained viable after formation and extensive curing of gels for cell-gel mixtures prepared at 45 degrees C. Attempts at prolonged storage of cell-laden gel beads at 0 degrees C as stock cultures of immobilized B. firmus were unsuccessful due to a significant decline in cell viability during such storage. On the basis of the cell leakage studies, the average pore sizes of 2%, 3%, 4%, and 5% gels were deduced to increase in the following order of carrageenan content (w/v): 4%, 3%, 2%, and 5%. Commensurate with the decrease in the average pore size (or the increased tightness of the gels) with increasing carrageenan content, both the extent of cell leakage and the extent of net cell penetration decreased with increasing carrageenan content for the first three gels. Owing to non-uniform distribution of free space and much larger pores, the extent of net cell penetration in 5% carrageenan gels was considerably low, while the extent of cell leakage in 5% carrageenan gels was an order of magnitude greater than the extents of cell leakage in the other three gels.     

Previously unrecognised division within Moritella viscosa isolated from fish farmed in the North Atlantic

Previously undocumented phenotypical and genetic variation was identified amongst isolates of Moritella viscosa collected from various geographical locations and from different fish species. The studied isolates could be split into 2 major phenotypically and genetically different clusters, one of which was consistent with the species type strain (NCIMB 13548). Isolates consistent with the type strain originated exclusively from Atlantic salmon farmed in Norway, Scotland and the Faroe Isles, although a single isolate from farmed Norwegian cod clustered closely with this group. The 'variant' cluster comprised isolates originating from Norwegian farmed rainbow trout, Icelandic farmed rainbow trout and salmon, Canadian farmed (Atlantic) salmon, Icelandic lumpsucker and only exceptionally from Norwegian salmon. With the exception of the single aforementioned cod isolate, all isolates from Norwegian farmed cod belonged to the variant cluster. Phenotypically, the clusters could be absolutely separated only by elevated haemolytic activity in the variant strain, although approximately half of these isolates also produced acid from mannose, in contrast to the typical (type) strain. While 16S rRNA gene sequencing was unable to separate the 2 clusters, Western blot analyses, plasmid profile analysis, pulsed field gel electrophoresis and gyrB gene sequence analysis produced clusters consistent with the phenotypic data. Macroscopically and histologically the disease in rainbow trout caused by the variant strain was consistent with that previously described in Atlantic salmon. The results of the present study may indicate a degree of host specificity of the typical strain for Atlantic salmon.     

Comparison of different extenders for the cryopreservation of Atlantic salmon spermatozoa

Fifteen extenders were produced by adding dimethyl sulfoxide (DMSO) at 8, 10 or 12% of diluent volume to 5 diluents. All extenders were cooled to 4 degrees C. Pooled Atlantic salmon (Salmo salar ) semen with greater than 90% progressive motility was kept at 4 degrees C and added to each extender so that the semen was diluted 1:3 (semen:extender). The equilibration time was less than 5 minutes at 4 degrees C. The extended semen was loaded into 0.5-ml straws and was cooled from 4 degrees C to -90 degrees C at a rate of 30 degrees C per minute. The straws were then plunged into liquid nitrogen for storage. Fluorometry was used to determine the viability of the semen in each of the extenders after freezing and thawing. Cryopreservation of Atlantic salmon semen in Extender 3 (0.137 M NaCl, 0.011 M KCl, 0.004 M Na(2)HPO(4).7H(2)O, 7.5 g/l L-alpha-lecithin and 12% dimethyl sulfoxide) and Extender 12 (0.100 M KHCO(3), 0.0065 M reduced glutathione, 0.125 M sucrose and 12% dimethyl sulfoxide) resulted in significantly (P<0.05) lower percentages of dead spermatozoa than for the other extenders. Furthermore, there was a significantly (P<0.05) lower percentage of dead cells in Extender 3 than in Extender 12.     

Histone-like proteins from Atlantic cod milt: stimulatory effect on Atlantic salmon leucocytes in vivo and in vitro

This study was carried out to reveal some characteristics of cationic proteins from Atlantic cod (Gadus morhua) milt chromatin and to investigate their ability to activate Atlantic salmon (Salmo salar) macrophages. Cationic proteins extracted from cod milt chromatin were fractionated on a cation exchange chromatography column. SDS-PAGE and amino acid analyses of the resulting fractions indicated that these proteins are similar to calf thymus histones. Two cationic protein fractions were used to stimulate leucocytes from Atlantic salmon in vitro and in vivo. Increased production of superoxide, measured as reduction of nitroblue tetrazolium (NBT), was used as indication of macrophage activation. Both fractions induced elevated superoxide anion production in the macrophages after 3 and 6 days of in vitro stimulation. Intraperitoneal injection of the cationic protein fractions in Atlantic salmon (100 mg kg(-1)) four days prior to slaughtering stimulated superoxide production when assayed after one and two days of cell cultivation. In macrophages from fish slaughtered two days after injection, activation could first be seen after two days of cell cultivation.     

Preparation and characterisation of gelatine from the skin of harp seal (Phoca groendlandica)

This paper describes a method for extraction of gelatine from the skin of harp seal at mild acid conditions and gives a chemical and physical comparison of this gelatine with other mammalian and cod-skin gelatines. As compared to the wet weight of unhaired skin, a recovery of 11% dry gelatine was achieved after acid treatment and a two step water extraction at 60 and 75 degrees C. The chemical and physical properties of the gelatine were similar to the properties of commercial gelatines made from bovine and porcine skin, but significantly different from the properties of Atlantic cod-skin gelatine. The results indicated that seal skin gelatine can be used as a substitute for standard commercial gelatines for food technology applications.     

Tissue astaxanthin and canthaxanthin distribution in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

A comparative investigation of tissue carotenoid distribution between rainbow trout, Oncorhynchus mykiss, and Atlantic salmon, Salmo salar, was undertaken to identify the relative efficiency of utilization of astaxanthin and canthaxanthin. Higher apparent digestibility coefficients (ADCs) (96% in trout vs. 28-31% in salmon; P<0.05), and pigment retention efficiencies (11.5-12.5% in trout vs. 5.5% in salmon; P<0.05), for both astaxanthin and canthaxanthin, were observed for rainbow trout. Astaxanthin deposition was higher than canthaxanthin in rainbow trout, while the reverse was true for Atlantic salmon, suggesting species-specificity in carotenoid utilization. The white muscle (95% in trout vs. 93% in salmon) and kidneys (0.5% in trout vs. 0.2% in salmon) represented higher proportions of the total body carotenoid pool in rainbow trout than in Atlantic salmon (P<0.05), whereas the liver was a more important storage organ in Atlantic salmon (2-6% in salmon vs. 0.2% in trout; P<0.05). The liver and kidney appeared to be important sites of carotenoid catabolism based on the relative proportion of the peak chromatogram of the fed carotenoid in both species, with the pyloric caecae and hind gut being more important in Atlantic salmon than in the rainbow trout. Liver catabolism is suspected to be a critical determinant in carotenoid clearance, with higher catabolism expected in Atlantic salmon than in rainbow trout.     

Flow cytometry assays of respiratory burst in Atlantic salmon (Salmo salar L.) and in Atlantic cod (Gadus morhua L.) leucocytes

The oxidation of dihydrorhodamine 123 (DHR) to the fluorescent rhodamine 123 (RHO) was detected using flow cytometry. This assay for detection of respiratory burst activity was established in peripheral blood leucocytes (PBL) and head kidney leucocytes (HKL) of Atlantic salmon and Atlantic cod. The leucocytes were stimulated by phorbol 12-myristate 13-acetate (PMA). For cod cells 10 times lower concentration of PMA had to be used compared to salmon cells, as higher concentrations were toxic and resulted in considerable cell death. The cells found to be RHO-positive were monocytes/macrophages and neutrophils based on the scatter dot plots, but for salmon also some small cells were found to have high fluorescence intensity both in the flow cytometry analyses and by fluorescence microscopy of cytospin preparations. The nature of these cells is not known. For cod leucocytes, such cells were not obvious. The instrument settings are a bit more demanding for cod, as cod cells die more easily compared to salmon cells. In both assays the limit between negative and positive cells has to be carefully considered. The presented flow cytometry protocols for measurements of respiratory burst in salmon and cod leucocytes can be applied in various studies where respiratory burst functions are involved, such as to verify if it is activated or suppressed in connection with infections and immunostimulation.     

Induction of Mx protein in Atlantic cod with poly I:C: immuno-cross reactive studies of antibodies to Atlantic salmon Mx with Atlantic cod

A polyclonal rabbit antiserum directed against the conserved region of the Atlantic salmon antiviral Mx1 protein was used to detect the putative Atlantic cod Mx protein using Western and dot blotting. A doublet band at about 75kDa and 65kDa was detected by Western blotting in kidney and spleen extracts of cod 3 and 4 days after i.p. injection with poly I:C but not in control fish injected with PBS. In blood leucocyte lysates, similar immunostaining could also be detected in Atlantic cod weakly after injection with PBS and more intensely after injection with poly I:C, suggesting some constitutive expression of Mx protein by leucocytes. Dot blot analysis showed that the Mx protein level was significantly higher in spleen, kidney, liver and gill of cod at least up to 4 days after injection with poly I:C when compared with the PBS-injected controls.     

Competition for shelter among over‐wintering signal crayfish and juvenile Atlantic salmon

Three separate effects on refuge use by signal crayfish Pacifastacus leniusculus and Atlantic salmon Salmo salar were examined: (1) the effect on Atlantic salmon of an addition of signal crayfish (doubling the total number of animals), (2) the effect on signal crayfish of an addition of Atlantic salmon and (3) intraspecific compared with interspecific competition, compared by holding total density of animals constant and varying the proportion of signal crayfish and Atlantic salmon in trials. Observations were made during winter, when both species are nocturnal. The proportion of Atlantic salmon sheltering was significantly lower in the presence than in the absence of signal crayfish when the interspecific treatment (Atlantic salmon plus signal crayfish) effected a doubling in density compared to the intraspecific treatment (Atlantic salmon alone). The proportion of signal crayfish sheltering was independent of the presence of Atlantic salmon. When total density was constant, the proportion of Atlantic salmon sheltering was significantly higher in intraspecific (52·8%) than interspecific trials (27·3%). Atlantic salmon out of shelter during the day in winter are believed to be very vulnerable to predators and the capacity for fish to share shelters with one another is known to be very low. Therefore, competition from crayfish for winter shelters may lead to detrimental effects on Atlantic salmon populations.     

Purification and characterization of trypsin from the poikilotherm Gadus morhua

A serine protease shown to be trypsin was purified from the pyloric caeca of Atlantic cod (Gadus morhua), and resolved into three differently charged species by chromatofocusing (pI 6.6, 6.2 and 5.5). All three trypsins had similar molecular mass of 24.2 kDa. N-terminal amino acid sequence analysis of cod trypsin showed considerable similarity with other known trypsins, particularly with dogfish and some mammalian trypsins. The apparent Km values determined at 25 degrees C for the predominant form of Atlantic cod trypsin towards p-tosyl-L-arginine methyl ester and N-benzoyl-L-arginine p-nitroanilide were 29 microM and 77 microM respectively, which are notably lower values than those determined for bovine trypsin (46 microM and 650 microM respectively). The difference was particularly striking when the amidase activity of the enzymes was compared. Furthermore, the kcat values determined for the Atlantic cold trypsins were consistently higher than the values determined for bovine trypsin. The higher catalytic efficiency (kcat/Km) of Atlantic cod trypsin as compared to bovine trypsin may reflect an evolutionary adaptation of the poikilothermic species to low environmental temperatures.     

Gelatinization and freeze-concentration effects on recrystallization in corn and potato starch gels

Freeze-concentration of starch gels was controlled by temperature and gelatinization with glucose and lactose. The aim of the study was to evaluate the effects of freezing temperature and gel composition on starch recrystallization behaviour of corn and potato starch gels (water content 70%, w/w) in water or glucose or lactose (10%, w/w) solutions. Starch gels were obtained by heating in differential scanning calorimetry (DSC). Samples of starch gels were frozen at -10 degrees C, -20 degrees C and -30 degrees C for 24h and, after thawing, stored at +2 degrees C for 0, 1, 2, 4 and 8 days. The extent of starch recrystallization was taken from the enthalpy of melting of the recrystallized starch by DSC. Freezing temperatures, glucose, lactose and the origin of the starch affected the recrystallization behaviour greatly. The recrystallization of amorphous starch during storage was enhanced by freeze-concentration of gels at temperatures above T'(m). Molecular mobility was enhanced by unfrozen water and consequently molecular rearrangements for nucleation could take place. Further storage at a higher temperature enhanced the growth and the maturation of crystals. In particular, glucose decreased the T'(m) of the gels and consequently lower freezing temperatures were needed to reduce enhanced recrystallization during storage. Freeze-concentration temperatures also showed a significant effect on the size and the perfection of crystals formed in starch recrystallization.     

Microbial spoilage and formation of biogenic amines in fresh and thawed modified atmosphere-packed salmon (Salmo salar) at 2 degrees C

AIMS: To evaluate the microbial spoilage, formation of biogenic amines and shelf life of chilled fresh and frozen/thawed salmon packed in a modified atmosphere and stored at 2 degrees C. METHODS AND RESULTS: The dominating microflora, formation of biogenic amines and shelf life were studied in two series of storage trials with naturally contaminated fresh and thawed modified atmosphere-packed (MAP) salmon at 2 degrees C. Photobacterium phosphoreum dominated the spoilage microflora of fresh MAP salmon at more than 10(6) cfu g(-1) and the activity of this specific spoilage organism (SSO) limited the shelf life of the product to ca 14 and 21 d in the two experiments. Despite the high levels of P. phosphoreum, less than 20 mg kg(-1) histamine was observed in fresh MAP salmon prior to sensory spoilage. Freezing eliminated P. phosphoreum and extended the shelf life of MAP salmon at 2 degrees C by 1-2 weeks. Carnobacterium piscicola dominated the spoilage microflora of thawed MAP salmon and probably produced the ca 40 mg kg(-1) tyramine detected in this product at the end of its shelf life. CONCLUSIONS: Photobacterium phosphoreum dominated the spoilage microflora of fresh MAP salmon but produced only small amounts of biogenic amines in this product. The elimination of P. phosphoreum by freezing allowed this bacteria to be identified as the SSO in fresh MAP salmon. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of P. phosphoreum as the SSO in fresh MAP salmon facilitates the development of methods to determine and predict the shelf life of this product, as previously shown with fresh MAP cod.