Obesity-induced alterations in the gut microbiome in female mice fed a high-fat diet are antagonized by dietary supplementation with a novel, wax ester-rich, marine oil.

Dietary supplementation with calanus oil, a novel wax ester-rich marine oil, has been shown to reduce adiposity in high-fat diet (HFD)-induced obese mice. Current evidence suggests that obesity and its comorbidities are intrinsically linked with unfavorable changes in the intestinal microbiome. Thus, in line with its antiobesity effect, we hypothesized that dietary supplementation with calanus oil should counteract the obesity-related deleterious changes in the gut microbiota. Seven-week-old female C57bl/6J mice received an HFD for 12 weeks to induce obesity followed by 8-week supplementation with 2% calanus oil. For comparative reasons, another group of mice was treated with exenatide, an antiobesogenic glucagon-like peptide-1 receptor agonist. Mice fed normal chow diet or nonsupplemented HFD for 20 weeks served as lean and obese controls, respectively. 16S rRNA gene sequencing was performed on fecal samples from the colon. HFD increased the abundance of the Lactococcus and Leuconostoc genera relative to normal chow diet, whereas abundances of Allobaculum and Oscillospira were decreased. Supplementation with calanus oil led to an apparent overrepresentation of Lactobacillus and Streptococcus and underrepresentation of Bilophila. Exenatide prevented the HFD-induced increase in Lactococcus and caused a decrease in the abundance of Streptococcus compared to the HFD group. Thus, HFD altered the gut microbiota composition in an unhealthy direction by increasing the abundance of proinflammatory genera while reducing those considered health-promoting. These obesity-induced changes were antagonized by both calanus oil and exenatide.

Comparative study of muscle proteins in relation to the development of yake in three tropical tuna species yellowfin (Thunnus albacares), big eye (Thunnus obesus) and skipjack (Katsuwonus pelamis).

Burnt tuna (BT), or yake-niku, is a quality flaw of the muscle characterised by a pale colour and grainy and exudative texture. Cathepsin-L, water soluble and total protein components from normal and BT muscles, from three tropical tuna species - yellowfin (YFT, Thunnus albacares), bigeye (BET, Thunnus obesus) and skipjack (SKJ, Katsuwonus pelamis) - were compared by electrophoretic and western blot analyses to identify biomarkers for BT. As expected, SDS-PAGE patterns were species-specific but differences, due to BT, were observed only between some low ionic strength extracts of BET and YFT. Protein oxidation and cell proliferation analysed by immunoblotting did not show differences between BT and normal muscles. Gelatine zymography revealed different gelatinase activity patterns that, although not linked to BT, may affect the final texture of the muscle. A 43 kDa band, identified as creatine kinase by proteomic analysis, showed the potential to be a good indicator for BT in BET and YFT.

Comparison of food antioxidants and iron chelators in two cellular free radical assays: strong protection by luteolin.

Liver (HepG2) cells were incubated with 21 edible flavonoids, carotenoids, polyunsaturated fatty acid (PUFA) chromones, and metal chelators for 1 h, washed in PBS, and challenged in the cellular antioxidant activity (CAA) and the cellular lipid peroxidation antioxidant activity (CLPAA) assays. These microplate format assays assess the compounds' ability to protect against cytosolic peroxyl radicals (CAA) and induced membrane lipid peroxidation (CLPAA), respectively. Incubation encompassing a broad compound concentration range determined half-maximal inhibitory concentrations (IC(50)) by using sigmoidal curve fits. Overall, considering both assays, luteolin offered the greatest protection. The carotenoid astaxanthin offered only modest protection, whereas ß-carotene was ineffective. Subtle structural differences between flavonoids were found to have amplified effects on protective abilities, and mechanisms of flavonoid antioxidant action are discussed. Membrane-permeable iron chelators (deferasirox and SIH) offered strong protective effects in CLPAA, but not in CAA, suggesting that CLPAA is dependent on membrane-associated free iron ions.

Detection of tropomyosin and determination of proteins in crustacean oils.

Tropomyosin is known to be the main allergen in crustaceans and the objective of this study was to investigate if this protein could be detected in commercial crustacean oils from Antarctic krill (Euphausia superba) and the zooplankton Calanus finmarchicus. We also examined the possibility of determining the protein content in the oils by direct amino acid analysis. Western blotting showed that a commercial antibody against shrimp tropomyosin cross-reacted with a protein of similar size in Antarctic krill and C. finmarchicus. The protein tentatively identified as tropomyosin, was also detected in krill oil products, but not in oils from C. finmarchicus. The acetone-heptane method used for extracting proteins in the oils is however not optimal. Other extraction methods should therefore be considered when investigating the presence of allergenic proteins in oils. Direct amino acid analysis on oils should be further explored as a method for determining the total amount of proteins present.

Spectral changes of Atlantic salmon ( Salmo salar L.) muscle during cold storage as affected by the oxidation state of heme.

The spectra of fresh salmon fillets change due to storage and packaging atmospheres. The aim of this study was to demonstrate the effects of heme oxidation states on spectral development in salmon fillets and to investigate the origin of a shoulder peak representing important spectral variations during storage. Hyperspectral images of fresh salmon fillets and mince with various water contents were collected during storage under different atmospheres. In addition, the absorption spectra of extracted salmon hemoglobin and its derivatives (methemoglobin and deoxyhemoglobin) were obtained. Air storage resulted in an increased similarity between spectra of methemoglobin and salmon fillets in principal component analysis. Results from the mince storage demonstrated that absorption features at the shoulder peak could be related to water content in the salmon muscle. This study established that the formation of oxidized heme is the primary source of spectral variations that occur during air storage of fresh salmon. Changes in the status of heme due to storage and packaging can influence the appearance of the underlying water absorption at the shoulder peak and create variations in the salmon spectra.

Visible/near-infrared spectroscopy detects autolytic changes during storage of Atlantic salmon (Salmo salar L.).

UNLABELLED: Visible (VIS)/near-infrared (NIR) spectroscopy was used to investigate spectroscopic changes occurring during storage of Atlantic salmon fillets with and without bacterial growth. A storage experiment was conducted for 11 d postmortem. Bacterial growth was inhibited by soaking a group of salmon fillets in 3 mM NaN3 prior to storage, while a control group retained its normal bacterial growth. Spectra were obtained by directly applying the spectroscopic probe onto the loin part of each fillet stored under conditions accelerating degradation. Principal component analysis (PCA) was used to monitor and compare spectroscopic development of the 2 groups and the results showed that VIS/NIR spectral changes occurred in the control as well as the treated group of samples within a single day after filleting. After 2 d of storage, stored samples were distinguishable from those fresh in both groups and it was only after the microbial spoilage became pronounced (8 to 9 log colony forming unit [CFU]/g) that the spectra of the spoiled control samples could be differentiated from spectra of the treated samples with no bacterial growth. Microbial growth is therefore not the only explanation for the spectral variations prior to microbial spoilage. Nonmicrobial, autolytic changes including possible changes in the physical properties are also contributing. Our results show that VIS/NIR spectroscopy can detect autolytic changes occurring in salmon muscle during the early stage of storage, independent of microbial growth. PRACTICAL APPLICATION: Important spectroscopic changes occur even when microbial growth is not apparent. This indicates that VIS/NIR spectroscopy may be used to determine the degree of freshness before microbial spoilage becomes relevant.

Cathepsin D from Atlantic cod (Gadus morhua L.) liver. Isolation and comparative studies.

The isolated cathepsin D-like enzyme from Atlantic cod (Gadus morhua L.) liver was shown to be a monomer with a molecular mass of approximately 40 kDa. It was inhibited by Pepstatin A and had an optimum for degradation of haemoglobin at pH 3.0. The purified enzyme had lower temperature stability than bovine cathepsin D. Antibodies raised against the purified enzyme and against two C-terminal peptides of cod cathepsin D recognized a 40 kDa protein in immunoblotting of the samples from the purification process. Both antisera showed cross reactivity with a similar sized protein in liver from cod, saithe (Pollachius virens L.), Atlantic herring (Clupea harengus L.) and Atlantic salmon (Salmo salar L.). A protein of same size was detected in wolffish (Anarhichas lupus L.) liver with the antibody directed against the purified enzyme. This antibody also recognized the native enzyme and detected the presence of cathepsin D in muscle of cod, saithe, herring and salmon. These antibodies may be useful in understanding the mechanisms of post mortem muscle degradation in fish by comparing immunohistochemical localization and enzyme activity, in particular in cod with different rate of muscle degradation. They may also be used for comparing muscle degradation in different fish species.

An immunological study of glycosaminoglycans in the connective tissue of bovine and cod skeletal muscle.

The presence of sulfated glycosaminoglycans (GAGs) was demonstrated in the connective tissue of bovine and cod skeletal muscle by histochemical staining using Alcian blue added MgCl(2) (0.06 M and 0.4 M, respectively). For further identification of the sulfated GAGs, a panel of monoclonal antibodies, 1B5, 2B6, 3B3 and 5D4 was used that recognizes epitopes in chondroitin-0-sulfate (C0S), chondroitin-4-sulfate/dermatan sulfate (C4S/DS), chondroitin-6-sulfate (C6S) and keratan sulfate (KS), respectively. Light microscopy and Western blotting techniques showed that in bovine and cod muscle C0S and C6S were primarily localized pericellularly, whereas cod exhibited a more intermittent staining. C4S was expressed around the separate cells and also in the perimysium and myocommata. In contrast to bovine muscle, which hardly expressed highly sulfated KS, cod exhibited a very strong and consistent staining. Western blotting showed that C0S and C6S were mainly associated with proteoglycans (PGs) of high molecular sizes in both species. Contrary to bovine muscle, C4S in cod was associated with molecules of various sizes. Both cod and bovine muscle contained KSPGs of similar sizes as C4S. KSPGs of different sizes and buoyant densities, sensitive to keratanase I and II were found expressed in cod.

Stimulation of type I IFN activity in Atlantic salmon (Salmo salar L.) leukocytes: synergistic effects of cationic proteins and CpG ODN.

Unmethylated CpG motifs in DNA are recognised by vertebrate immune cells as pathogen signatures. Consequently, oligodeoxynucleotides containing CpG motifs (CpG ODNs) are able to enhance and direct immune responses. Recent studies have demonstrated that CpG ODNs activate antiviral immune responses in Atlantic salmon (Salmo salar L.) leukocytes, and are therefore promising agents as vaccine adjuvants or immunostimulants in fish. In this work, we report synergy of CpG ODN and cationic proteins in the stimulation of type I IFN activity in Atlantic salmon leukocytes. Different fractions of cationic histone proteins derived from cod milt or poly-l-arginine and poly-l-lysine were screened for their ability to enhance CpG ODN induced type I IFN activity in Atlantic salmon leukocytes. Optimal ratio of histones to CpG ODN was identified, and effects on transcription of type I IFN and antiviral Mx genes were studied. Delivery of CpG ODN with cationic proteins enhanced the production of type I IFN and succeeding Mx transcripts after two and five days of stimulation at substimulatory concentrations of CpG ODN. These results indicate that co-delivery of CpG ODN and cationic proteins enhance antiviral mechanisms in Atlantic salmon leukocytes as compared to CpG ODN alone.

Detection of TIMP-2-like protein in Atlantic cod (Gadus morhua) muscle using two-dimensional real-time reverse zymography.

Matrix metalloproteinases (MMPs) have been proposed to participate in postmortem degradation of fish muscle connective tissues during storage. In the extracellular matrix (ECM) of mammals, a group of specific tissue inhibitors of metalloproteinases (TIMPs) contributes in regulating the MMPs present. However, little information exists on the presence of TIMPs in fish. In this paper, the presence of TIMPs in the muscle of Atlantic cod (Gadus morhua) was investigated using gelatin affinity chromatography, real-time reverse zymography (RTRZ) and mass spectrometry (MS). Using RTRZ inhibitory action against cod muscle, proteinases binding to gelatin were detected in the muscle. The inhibitor had similar molecular weight (21 kDa) as a human recombinant TIMP-2 used as a reference sample. Because isoforms of TIMP-2 homologues with similar molecular weight have been suggested in fish, a two-dimensional RTRZ (2D RTRZ) method was designed. The new method showed the existence of only one form with inhibitory action against cod muscle proteinases. Finally, de novo sequencing of two peptides derived from the cod muscle inhibitor showed high homology to TIMP-2s both from human and other teleosts.

Tissue distribution, inhibition and activation of gelatinolytic activities in Atlantic cod (Gadus morhua).

Gelatinolytic activities in fish tissues with properties like matrix metalloproteinases (MMPs) have been paid little attention. However, they have been proposed to participate in post mortem degradation during storage and the disintegration of pericellular connective tissue during spawning. In this paper the distribution of gelatinolytic activities in liver, heart, muscle, gill, and male and female gonad of Atlantic cod (Gadus morhua) was studied by using gelatin SDS-PAGE, proteinase inhibitors, gelatin and lentil lectin Sepharose affinity chromatography. The amount of gelatin degrading enzymes varied from tissue to tissue. Most of the gelatin binding enzymes were found to be matrix metalloproteinases by adding galardin, a broad range MMP inhibitor, to the incubation buffer. A 72 kDa form of cod gelatin degrading enzyme had properties similar to human proMMP-2, as it could be activated by p-aminophenylmercuric acetate and trypsin. Like the human MMP-2 it did not bind to lentil lectin. An 83 kDa cod gelatin degrading enzyme had properties similar to the 92 kDa progelatinase B (proMMP-9). These properties were also similar to that of the 72 kDa form, except that the 83 kDa cod gelatinase was bound to lentil lectin, showing that it is a glycoprotein like MMP-9.

Origin and spoilage potential of the microbiota dominating genus Psychrobacter in sterile rehydrated salt-cured and dried salt-cured cod (Gadus morhua).

Salt-cured and dried salt-cured cod rehydrated using sterile water and equipment have a short shelf life at 4 degrees C due to high bacterial counts. The microbiota develops off-odours which partly can be described as musty, causing sensory rejection within 7-10 days of chilled storage. The microbiota composition was studied in a total of 38 samples obtained from 10 different, both commercial and laboratory produced, salt-cured and dried salt-cured cod products. The dominating bacterium, representing at least 90% of the total viable count in all products studied, was identified as belonging to the genus Psychrobacter; a Gram-negative, oxidase- and catalase-positive, nonpigmented, halotolerant, psychrotolerant, facultative aerobe and nonmotile bacterium. The morphology of the bacterium resembles coccobacilli and the cells occur most often in pairs. The bacterium was able to hydrolyze lipids, but not proteins. It did not produce H(2)S or TMA and the spoilage in rehydrated salt-cured and dried salt-cured cod is therefor different from what is observed in fresh cod. However, samples inoculated with Psychrobacter immobilis gave the same musty odour as spoiled control samples but earlier in the storage period and of a stronger intensity. In a field experiment, carried out to investigate the origin of the dominating bacterium, it was found that the microbiota in both sterile rehydrated commercially produced and laboratory (aseptically) produced salt-cured cod was dominated by this same bacterium. The bacterium was also isolated from cod skin mucus immediately after capture. The bacterium survived NaCl concentrations up to 25% (w/v) NaCl, stating its ability to survive during the salt-curing process. The dominating bacterium in rehydrated salt-cured and dried salt-cured cod seems to mainly originate from the fresh fish itself and not from contamination during processing.

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.