Analysis of protein structures and interactions in complex food by near-infrared spectroscopy. 2. Hydrated gluten

Hydrated gluten, treated with various salts, was analyzed by near-infrared (NIR) spectroscopy to assess the ability of this method to reveal protein structure and interaction changes in perturbed food systems. The spectra were pretreated with second-derivative transformation and extended multiplicative signal correction for improving the band resolution and removing physical and quantitative spectral variations. Principal component analysis of the preprocessed spectra showed spectral effects that depended on salt type and concentration. Although both gluten texture and the NIR spectra were little influenced by treatment with salt solutions of low concentrations (0.1-0.2 M), they were significantly and diversely affected by treatment with 1.0 M salt solutions. Compared to hydration in water, hydration in 1.0 M sulfate salts caused spectral effects similar to a drying-out effect, which could be explained by salting-out.     

Homogenization conditions affect the oxidative stability of fish oil enriched milk emulsions: oxidation linked to changes in protein composition at the oil-water interface

Fish oil was incorporated into milk under different homogenization temperatures (50 and 72 degrees C) and pressures (5, 15, and 22.5 MPa). Subsequently, the oxidative stability of the milk and changes in the protein composition of the milk fat globule membrane (MFGM) were examined. Results showed that high pressure and high temperature (72 degrees C and 22.5 MPa) resulted in less lipid oxidation, whereas low pressure and low temperature (50 degrees C and 5 MPa) resulted in faster lipid oxidation. Analysis of protein oxidation indicated that especially casein was prone to oxidation. The level of free thiol groups was increased by high temperature (72 degrees C) and with increasing pressure. Furthermore, SDS-PAGE and confocal laser scanning microscopy (CLSM) indicated that high temperature resulted in an increase in beta-lactoglobulin adsorbed at the oil-water interface. This was even more pronounced with higher pressure. Less casein seemed to be present at the oil-water interface with increasing pressure. Overall, the results indicated that a combination of more beta-lactoglobulin and less casein at the oil-water interface gave the most stable emulsions with respect to lipid oxidation.     

Analysis of moniliformin in maize plants using hydrophilic interaction chromatography

A novel HPLC method was developed for detection of the Fusarium mycotoxin, moniliformin in whole maize plants. The method is based on hydrophilic interaction chromatography (HILIC) on a ZIC zwitterion column combined with diode array detection and negative electrospray mass spectrometry (ESI(-)-MS). Samples were extracted using acetonitrile-water (85:15), and the extracts were cleaned up on strong anion exchange columns. By this procedure we obtained a recovery rate of 57-74% moniliformin with a limit of detection at 48 ng/g and a limit of quantification at 96 ng/g using UV detection at 229 nm, which is comparable to current methods used. Limit of detection and quantification using ESI(-)-MS detection was 1 and 12 ng/g, respectively. Screening of maize samples infected with the moniliformin producing fungi F. avenaceum, F. tricinctum, or F. subglutinans detected moniliformin levels of 1-12 ng/g in 15 of 28 samples using ESI(-)-MS detection. To our knowledge this is the first example of HILIC separation in mycotoxin analysis.     

Phylogenetic analysis of the SSU rRNA gene from the piscine diplomonad Spironucleus torosus (Diplomonadida: Hexamitinae)

Previous studies have recorded Spironucleus torosus Poynton et Morrison, 1990 from several species of gadoid fishes, including the only freshwater gadoid, the burbot Lota lota (L.). Two morphologically different isolates of S. torosus have been described (elongate and pyriform). Both have been found in saltwater, while only the elongate has been found in freshwater. To address the conspecificity of the two morphs of S. torosus, and to identify the source of S. torosus in burbot in Norway, we have sequenced the small subunit ribosomal RNA (SSU rRNA) gene from 43 isolates of S. torosus from six species of gadoid fishes sampled at 15 localities in Norway, Sweden and the Baltic Sea. Phylogenetic analyses of the SSU rRNA gene sequence data recovered two major clades, one containing mainly isolates from burbot, while the other contained isolates from marine gadoid fishes only. The genetic distance (based on 25 nucleotide substitutions in 789 base pairs) separating the two assemblages was not large enough to consider the two groups separate species. Spironucleus torosus isolated from burbot displayed limited genetic variation in the small subunit ribosomal RNA (SSU rRNA) gene along the post-Pleistocene migration route of its host. The present study is the first report of S. torosus in tusk Brosme brosme (Ascanius), whiting Merlangius merlangus (L.), and fourbeard rockling Enchelyopus cimbrius (L.).     

Cloning of two Atlantic salmon growth hormone receptor isoforms and <Emphasis Type="Italic">in vitro</Emphasis> ligand-binding response

Two isoforms of the full-length cDNA of the growth hormone receptor (GHR) of the Atlantic salmon (Salmo salar; ss) were cloned by a PCR approach using RACE. Respectively, the cDNA sequences of ssGHR isoforms 1 and 2 are 2654 and 2608 nucleotides long, with 1782 and 1773 nucleotide ORFs. The resulting coded proteins are 594 and 590 aa long, with 19 and 20 aa signal peptides. The two isoforms share 86% protein and 87% cDNA sequence similarity. Isoform 1 is most similar to other salmonid GHR isoforms 1 while isoform 2 is most similar to salmonid GHR isoforms 2 (93–95%). Similarity with other teleost species was lower (37–44%). The bioactivity of the cloned ssGHR was tested by transfecting the ssGHR isoform 1 cDNA into CHO-K1 hamster cells, incubating with recombinant salmon GH (sGH) or native ovine prolactin (oPRL), and measuring cell proliferation by the MTT assay. The ssGHR-transfected cells significantly increased proliferation when stimulated by sGH at all concentrations. oPRL stimulated ssGHR-transfected cells at higher concentrations due to receptor cross reaction. ssGHR isoforms 1 and 2 contain a single transmembrane domain and the typical conserved motifs found in other teleost GHRs, including four paired cysteine residues and five potential N-glycosylation sites in the extracellular domain, Box I and Box II, as well as seven potential tyrosine phosphorylation sites in the intracellular domain. However, in salmonids, these motifs differ from those of other teleosts, and could be responsible for differentiated hormone binding, signal transduction and response.     

Adoption of milk cooling technology among smallholder dairy farmers in Kenya

Factors influencing adoption of milk cooling technology were studied with data for 90 smallholder dairy farmers who were randomly selected from seven dairy cooperative societies in Kiambu County, Kenya. Logistic regression identified the age of the household head, daily household milk consumption, freehold land ownership, fodder production area, number of female calves, cooperative membership and cooperative services as significant factors influencing farmers’ willingness to invest in milk cooling technology. These findings offer an entry point for increased interventions by policy makers and various dairy sector stakeholders in promoting milk cooling technology with the aim of significantly reducing post-harvest losses and increasing the sector’s competitiveness.     

Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), hatcheries: studies on broodstock, eggs, fry and environment

The occurrence of Flavobacterium psychrophilum at four rainbow trout hatcheries was investigated to provide more knowledge about the reservoirs and transmission of this bacterium. Broodstock were sampled at stripping (including both unfertilized and fertilized eggs), and the offspring were then sampled at the eyed egg and fry stages. Water and surface samples (e.g. hatchery trays) were also sampled. Flavobacterium psychrophilum was found in ovarian fluid and milt, indicating that broodstock may serve as a reservoir and are latent carriers of the pathogen. Flavobacterium psychrophilum was not found on or inside eggs, but further egg studies will be necessary to elucidate the possibility of vertical transmission of the pathogen. Flavobacterium psychrophilum was isolated from water samples, but only from water that had been in close contact with farmed rainbow trout or eggs. Flavobacterium psychrophilum isolates were characterized and compared with well-characterized strains, using degradation of elastin, serotype and ribotype profiles. Different ribotypes of F. psychrophilum were found between hatcheries, but a common ribotype A was found at all four hatcheries. Different ribotypes were found in broodstock without clinical disease, whereas only a few ribotypes (mostly ribotype A) were found in diseased fry. The same ribotype A was found in broodstock, in water samples from hatchery trays and in fry, which suggests the possibility of transmission of F. psychrophilum between broodstock and offspring.     

Analysis of exotoxins produced by atypical isolates of Aeromonas salmonicida,by enzymatic and serological methods

In this study, exotoxins produced by 62 Aeromonas salmonicida strains and the bacterium Haemophilus piscium were analysed. Enzymatic assays, zymograms and serological detection were used to monitor secretion by bacterial strains of the previously described exotoxins P1, GCAT and AsaP1 and also the extracellular P2 metallo-gelatinase and a serine caseinase, which is different from the P1 protease and has not yet been characterized. Based on the results, the strains were divided into five groups. One comprised the type strains for A. salmonicida ssp. masoucida, H. piscium and 36% of the atypical isolates, and another, a type strain for A. salmonicida ssp. smithia together with 14% of the atypical isolates. A second type strain of A. salmonicida ssp. smithia was grouped with 8% of the atypical isolates. The largest group contained the type strains for A. salmonicida ssp. achromogenes and 38% of the atypical isolates. The type strains for A. salmonicida ssp. salmonicida were in the last group with all the four typical strains and 4% of the atypical isolates. The combination of zymogram and serological detection used is recommended as the most reliable method for characterizing A. salmonicida strains according to their exotoxin secretion.     

Estradiol-17β-induced calcium uptake and resorption in juvenile rainbow trout,Oncorhynchus mykiss

Juvenile rainbow trout, Oncorhynchus mykiss, were injected with estradiol-17β (E₂) in order to study the source of extra calcium needed during vitellogenesis. E₂-treatment increased the calcium uptake from the external medium as well as calcium mobilization from muscle and scale. Judged by the increase in plasma protein-bound calcium levels, the E₂-induced increase in calcium uptake is an apparent over-mobilization of calcium, i.e., the calcium uptake of the fish is in excess of what is found bound to plasma proteins. As the calcium excretion and calcium space (calculated from free plasma calcium levels) were unaffected, the excess calcium is suggested to be incorporated into internal calcium stores. This implies that the systems regulating vitellogenesis and calcium balance are integrated on the mechanistic or endocrine level, and that E₂ causes calcium mobilization of a magnitude geared to the needs of the sexually maturing female.