Purification and characterization of a thermostable alpha-galactosidase from Thermoanaerobacterium polysaccharolyticum

Food ingredients containing alpha-1,6-galactoside bonds elicit gastrointestinal disturbances in monogastric animals, including humans. Pretreatment of such ingredients with alpha-galactosidase (EC 3.2.1.22) has the potential to alleviate this condition. For this purpose, a thermostable alpha-galactosidase from Thermoanaerobacterium polysaccharolyticum was purified by a combination of anion exchange and size exclusion chromatographies. The enzyme has a monomeric molecular weight of approximately 80 kDa; however, it is active as a dimer. The optimum temperature for enzyme activity is 77.5 degrees C. Approximately 84 and 88% of enzyme activity remained after 36.5 h of incubation at 70 and 65 degrees C, respectively. Optimum activity was observed at pH 8.0, with a broad range of activity from pH 5.0 to 9.0. Different transition metals had weak to strong inhibitory effects on enzyme activity. The K(m) and V(max) of the enzyme are 0.29-0.345 mM and 200-232 micromol/min/mg of protein, respectively. Importantly, enzyme activity was only slightly inhibited by 75-100 mM galactose, an end product of hydrolysis. Enzyme activity was specific for the alpha-1,6-galactosyl bond, and activity was demonstrated on melibiose and soy molasses.     

Identification of fish species after cooking by SDS-PAGE and urea IEF: a collaborative study

A collaborative study, to validate the use of SDS-PAGE and urea IEF, for the identification of fish species after cooking has been performed by nine laboratories. By following optimized standard operation procedures, 10 commercially important species (Atlantic salmon, sea trout, rainbow trout, turbot, Alaska pollock, pollack, pink salmon, Arctic char, chum salmon, and New Zealand hake) had to be identified by comparison with 22 reference samples. Some differences in the recoveries of proteins from cooked fish flesh were noted between the urea and the SDS extraction procedures used. Generally, the urea extraction procedure appears to be less efficient than the SDS extraction for protein solubilization. Except for some species belonging to the Salmonidae family (Salmo, Oncorhynchus), both of the analytical techniques tested (urea IEF, SDS-PAGE) enabled identification of the species of the samples to be established. With urea IEF, two laboratories could not differentiate Salmo salar from Salmo trutta. The same difficulties were noted for differentiation between Oncorhynchus gorbuscha and Oncorhynchus keta samples. With SDS-PAGE, three laboratories had some difficulties in identifying the S. trutta samples. However, in the contrast with the previous technique, SDS-PAGE allows the characterization of most of the Oncorhynchus species tested. Only Oncorhynchus mykiss was not clearly recognized by one laboratory. Therefore, SDS-PAGE (Excel gel homogeneous 15%) appears to be better for the identification, after cooking, of fish such as the tuna and salmon species which are characterized by neutral and basic protein bands, and urea IEF (CleanGel) is better for the gadoid species, which are characterized by acid protein bands (parvalbumins). Nevertheless, in contentious cases it is preferable to use both analytical methods.     

Fecal microbiota of calves in the clinical setting: effect of penicillin treatment

The effect of parenteral penicillin treatment on the intestinal microbiota was determined by monitoring the phenotypic antimicrobial resistance among Escherichia coli in 19 calves (15 calves received treatment and four calves were healthy controls) and by examining changes in the fecal microbial community structure using molecular fingerprinting techniques in a subset of eight calves (five treated calves and three control calves). After five days of penicillin treatment an increased resistance to multiple unrelated antimicrobial agents, including non-β-lactams, was seen in E. coli from treated calves, and this was not seen in the controls. Automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphism (TRFLP) revealed that penicillin treatment causes a significant variation in the microbial structure within an individual calf. The study shows that parenteral administration of penicillin has an impact on the composition of the fecal microbiota in calves, and on the antimicrobial resistance pattern of their fecal E. coli.     

Investigations into the toxicity of Echium plantagineum in sheep: 1. Field grazing experiments

A field grazing trial was undertaken to monitor the health and production of crossbred sheep grazing pasture where Echium plantagineum constituted a considerable proportion of the available forage. The trial, conducted for 19 months over successive grazing seasons, demonstrated a significant difference in production, with sheep on the E. plantagineum pasture being lighter and growing less wool compared with sheep on Echium-free pasture. No mortalities involving pyrrolizidine alkaloid poisoning were recorded in sheep grazing E. plantagineum, although there was histological evidence of moderately severe liver damage associated with high liver copper concentrations in at least one sheep following the grazing of large quantities of the plant.     

Eye-drop DNA can induce IgA in the tears and bile of chickens

DNA vaccines protect chickens against lethal virus infections but whether they induce local antibody which is associated with preventing viral entry, is unknown. We were able to show how avian DNA vaccines can induce local IgA. 65 μg plasmid DNA encoding the reporter protein beta-galactosidase induced antigen-specific IgA in the tears of 6/10 birds, IgA in the bile of 4/10 birds and IgG in the serum of 2/10 birds. Giving the DNA by the intramuscular route, as is more usual, induced lacrimal IgA in 2/8 birds, biliary IgA in no birds and serum IgG in 4/8 birds. Eye-drop DNA therefore favoured local IgA whereas intramuscular DNA favoured serum IgG. Further to this preliminary work eye-drop DNA should be improved by adjuvants and cytokines as a way of inducing protective IgA at the mucosal surfaces of the alimentary and respiratory tracts.     

Degradation of wheat straw and alkaline hydrogen peroxide-treated wheat straw by Ruminococcus albus 8 and Ruminococcus flavefaciens FD-1

Degradation of wheat straw (WS) and alkaline hydrogen peroxide (AHP)-treated wheat straw (AHPWS) by Ruminococcus albus 8 and Ruminococcus flavefaciens FD-1 was determined by measuring the growth (OD600) of each bacterium and determining DM disappearance (DMD) of the substrate. Complex medium and defined medium with or without the addition of phenylpropanoic acid (PPA) and phenylacetic acid (PAA) were used. Tubes were incubated at 39 degrees C for 8 d. Both OD600 and DMD indicated that AHPWS was degraded to a much greater extent by either bacterium (R. flavefaciens FD-1, 60.8 +/- 1.8% and R. albus 8, 42.3 +/- 3.5%) vs untreated WS (R. flavefaciens FD-1, 16.5 +/- 1.8% and R. albus 8, 8.6 +/- 6%) in the complex medium. Most degradation occurred between d 1 and 4. With the complex medium, addition of PPA and PAA did not stimulate degradation by either bacterium. When the defined medium was used, the addition of PPA and PAA enhanced (P less than .05) degradation of AHPWS (39.6 +/- 2.6%) vs AHPWS with no added PPA and PAA (24.9 +/- 7.6%) by R. albus 8. There was no synergistic effect on degradation when the two species were co-cultured with either WS or AHPWS as the substrate. No effect of PPA and PAA on disappearance of AHPWS was observed for R. flavefaciens FD-1 or when the two bacteria were grown together. Dry matter disappearance analysis showed that R. flavefaciens FD-1 degraded AHPWS more rapidly (6.1 mg/d) than R. albus 8 did (4.2 mg/d) in complex medium.(ABSTRACT TRUNCATED AT 250 WORDS)