In Vitro Studies on the Adhesion of Fibrobacter Succinogenes Strain D3 to Macrocrystalline Cellulose

In the present study, the factors affecting adhesion of Fibrobacter succinogenes strain D3 to microcrystalline cellulose avicel were investigated. Fibrobacter succinogenes showed the highest percentage of adhesion (85%) during late exponential phase of growth. During lag phase, 50 - 55% of the bacterial cells were adherent and during death phase, 60 - 70% of the cells were adherent. Adhesion of bacterial cells to avicel was significantly (P<0.05) affected by pH and temperature and significant (P<0.05) interaction between these two factors was also observed. The optimum pH for cell adhesion was 6.5 and the optimum temperature was 39°C. At pH 6 . 5 , the adhering ability of the cells was reduced when the temperature was raised to 5 0°C and 60°C or lowered to 4°C and 22°C. At this pH, the effect of temperature on adhesion was greater at high temperature than at low temperature. At 50°C and 60°C, only 20 - 27% adhesion was observed but at 4°C and 22°C, 48 - 5 8% adhesion was obtained. At other combinations of condition (PH 4.0, 5.6, 7.0, 8.0 and temperature 4, 22, 39, 5 0, 60°C), less than 20% adhesion was observed. The adhering ability of the bacterial cells was also reduced after the cells were treated with proteolytic enzymes such as thermolysin and pronase. Lipase and dextranase did not affect the adhesion of the cells. The study usmg scannmg electron microscopy and transmission electron microscopy showed that the adhesion of F. succinogenes was first mediated by fine structures radiating from the outer layer of the cell and then by the glycocalyx. Initial adhesion by these fine structures was observed after 1 0 min of incubation with avicel. After 1 8 h of incubation, the bacteria had digested away the cellulose at the point of contact and penetrated into the substrate. Pits of digestion surrounding the bacteria were particularly evident after 30 h of incubation and larger digestion pits were observed after 56 h of incubation. Studies were also carried out to detect the cellulose-binding proteins (CBPs) of the cells. In this study, Buffer A supplemented with 1% (w/v) carboxymethylcellulose (CMC), 1 0% (w/v) cellobiose or 5% (w/v) sodium dodecyl sulphate (SDS) were used to elute CBPs from avicel incubated with cell lysate of F. succinogenes. Buffer A supplemented with CMC was found to elute two maj or proteins ( 1 20 kDa and 1 00 kDa) and a few minor proteins ranging from 35 kDa to 60 kDa. Buffer A supplemented with cellobiose or SDS eluted proteins with approximate weights of 240, 1 20 and 1 00 kDa. These three CBPs (240, 1 20 and 1 00 kDa) were involved in the adhesion process of the cells as cells with reduced adhering ability after being treated with proteolytic enzymes such as thermo lysin and pronase did not show these CBPs. Other than possessing the ability to bind, the 240 kDa CBP showed xylanase activity and the 1 20 kDa protein showed carboxymethylcellulase (CMCase) activity in the cell lysate. The 1 00 kDa CBP did not show any of the two enzyme activities shown by 240 kDa and 1 2 0 kDa CBP

Manipulation of Lactobacillus Probiotic Strains to Produce Heterologous Beta-Glucanase for Chickens

Application of enzymes as feed additives is common in the livestock industry, especially in poultry, to eliminate the antinutritional factors present in the diets of chickens. However, the efficiency of enzymes seldom achieves their desired effects because of destruction during feed processing and unsuitable conditions in the gastrointestinal tract. Thus, in the present study, investigations were carried out to evaluate the potential of 12 Lactobacillus strains as delivery vehicles for a heterologous β-glucanase enzyme in poultry. The 12 Lactobacillus strains used were L. crispatus I12, L. acidophilus I16 and I26, L. fermentum I24, I25, C16 and C17, and L. brevis I23, I211, I218, C1 and C10. The strains were found to exhibit resistance to chloromphenicol, erythromycin and tetracycline in varying degrees. The erythromycin resistance of L. acidophilus I16 and I26, and L. fermentum I24 and C17 could be cured by using novobiocin, and L. brevis C10 cured by using acriflavin. The chloromphenicol and tetracycline resistances of all the resistant strains were not eliminated even after prolonged curing in sublethal concentrations of individual or mixtures of curing agents such as novobiocin, ethidium bromide, acriflavin or SDS. Electrotransformation efficiency of the Lactobacillus strains was affected by growth phase, growth and recovery medium, cell density, electroporation buffer, buffer strength, plasmid concentration and electrical pulse. At optimized conditions, the strains were transformed at 103-104 transformants/μg plasmid DNA. The erythromycin susceptible wild-type strains (L. crispatus I12, L. brevis I23, I211 and I218, and L. fermentum I25) and cured derivatives (L. acidophilus I16C and I26C, L. brevis v C10C, and L. fermentum I24C and C17C) were then transformed at optimized conditions with plasmid pSA3b6, which carried a β-glucanase gene from Bacillus amyloliquefaciens. Five wild-type Lactobacillus strains, namely, L. crispatus I12, L. fermentum I25, L. brevis I23, I211 and I218 and a cured derivative, L. brevis C10C, which could retain the plasmid at a comparatively higher rate, were used for subsequent studies. The Lactobacillus transformants were found to secrete 32-52 U/ml of β- glucanase. Optimum activity of the enzyme was at 39 oC and pH 5-6. A loss of 0.4-1.6 U/generation of β-glucanase was observed when the strains were grown under nonselective pressure. PCR analyses of gastrointestinal samples of chickens fed transformed Lactobacillus strains revealed that the strains could not persist for more than 24 h in the gut. The β- glucanase activity detected in the jejunum and ileum of chickens fed transformed Lactobacillus strains was found to be 2-9.4 folds higher than those obtained from other intestinal sites. In the feeding trial, supplementation of transformed Lactobacillus strains to chickens significantly (P<0.05) improved the body weight by 2.5 %, and the feed conversion ratio by 1.0-2.6 %. In addition, the apparent metabolizable energy, digestibilities of crude protein and dry matter of feed were improved by 3.4 %, 5.9 % and 3.5 %, respectively. The intestinal fluid viscosity was reduced by 21-46 %. The relative weights of organs and intestinal segments (pancrease, liver, duodenum, jejunum, ileum, cecum and colon) were also reduced by 6-27 %, and the relative length of intestinal segments (duodenum, jejunum, ileum and cecum) was reduced by 8-15 %. Histological examination of the intestinal tissues showed that the jejunal villus height of chickens fed diet supplemented with transformed Lactobacillus strains was significantly (P<0.05) higher than those of chickens fed other dietary treatments. The transformed Lactobacillus strains were also found to reduce the time of feed passage rate by 2.2 h. vi The results of the present study showed that the Lactobacillus strains have the potential to be used as delivery vehicles for a heterologous β-glucanase enzyme in poultry

Electrophoresis : what does a century old technology hold for the future of separation science?

Electrophoretic separation was first demonstrated in the year of 1807 and has since been a staple tool used by biologists and chemists for more than a century since its inception. From the initial crude paper electrophoresis system to today’s modern automated electrophoresis system, the development of electrophoresis systems have been driven by the advancement of technology such as miniaturization, precision engineering, biochemistry, electrical and electronics. These advancements were introduced to meet the requirement for faster and better resolution of results. This paper reviews the evolution of the electrophoresis technology over one century and provides an insight into the possible future development of electrophoresis.Various aspects of the electrophoresis system such as the performances, designs, usages, separation phases, and biochemistry were analyzed. The technological advancements for this field have been evidenced by the increasing complexity of the electrophoresis system. A peek into the possible future for the world of electrophoresis has been provided by drawing insights from the missing links of current technologies. It is both exciting and equally perplexing to explore the promises that this seeming simple separation technology holds for the future

Display of the VP1 epitope of foot-and-mouth disease virus on bacteriophage T7 and its application in diagnosis

Foot-and-mouth disease (FMD) is a highly contagious epidemic disease threatening the cattle industry since the sixteenth century. In recent years, the development of diagnostic assays for FMD has benefited considerably from the advances of recombinant DNA technology. In this study, the immunodominant region of the capsid protein VP1 of the foot-and-mouth disease virus (FMDV) was fused to the T7 bacteriophage and expressed on the surface of the bacteriophage capsid protein. The recombinant protein of about 42 kDa was detected by the anti-T7 tag monoclonal antibody in Western blot analysis. Phage ELISA showed that both the vaccinated and positive infected bovine sera reacted significantly with the recombinant T7 particle. This study demonstrated the potential of the T7 phage displaying the VP1 epitope as a diagnostic reagent

Estimation of 16S RNA gene copy number in several probiotic Lactobacillus strains isolated from the gastrointestinal tract of chicken.

The copy numbers of 16S rRNA genes in 12 probiotic Lactobacillus strains of poultry origin were analyzed. Genomic DNA of the strains was digested with restriction endonucleases that do not cut within the 16S rRNA gene of the strains. This was followed by Southern hybridization with a biotinylated probe complementary to the 16S rRNA gene. The copy number of the 16S rRNA gene within a Lactobacillus species was found to be conserved. From the hybridization results, Lactobacillus salivarius I 24 was estimated to have seven copies of the 16S rRNA gene, Lactobacillus panis C 17 to have five copies and Lactobacillus gallinarum strains I 16 and I 26 four copies. The 16S rRNA gene copy numbers of L. gallinarum and L. panis reported in the present study are the first record. Lactobacillus brevis strains I 12, I 23, I 25, I 211, I 218 and Lactobacillus reuteri strains C 1, C 10, C 16 were estimated to have at least four copies of the 16S rRNA gene. In addition, distinct rRNA restriction patterns which could discriminate the strains of L. reuteri and L. gallinarum were also detected. Information on 16S rRNA gene copy number is important for physiological, evolutionary and population studies of the bacteria

Purification of bacteriophage M13 by anion exchange chromatography

M13 is a non-lytic filamentous bacteriophage (phage). It has been used widely in phage display technology for displaying foreign peptides, and also for studying macromolecule structures and interactions. Traditionally, this phage has been purified by cesium chloride (CsCl) density gradient ultracentrifugation which is highly laborious and time consuming. In the present study, a simple, rapid and efficient method for the purification of M13 based on anion exchange chromatography was established. A pre-packed SepFast™ Super Q column connected to a fast protein liquid chromatography (FPLC) system was employed to capture released phages in clarified Escherichia coli fermented broth. An average yield of 74% was obtained from a packed bed mode elution using citrate buffer (pH 4), containing 1.5 M NaCl at 1 ml/min flow rate. The purification process was shortened substantially to less than 2 h from 18 h in the conventional ultracentrifugation method. SDS-PAGE revealed that the purity of particles was comparable to that of CsCl gradient density ultracentrifugation method. Plaque forming assay showed that the purified phages were still infectious

Phytochemical compounds and antibacterial activity of Jatropha curcas Linn. extracts

The present study was conducted to determine the phytochemical compounds in different solvent extracts of Jatropha curcas Linn. plant and antibacterial activity of crude extracts. Aqueous, methanolic and hexane extracts of various plant parts were analysed for phytochemical compounds by spectrophotometry, high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry analysis (GC-MS). Antibacterial activity was studied by paper disc diffusion assay against Gram positive and Gram negative bacteria. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by micro-broth dilution. The root bark methanolic extract contained high phenolics (11.51 mg gallic acid equivalents/g DW) and flavonoids (0.94 mg rutin equivalents/g DW). Kernel meal aqueous extract contained high saponins (0.65 mg diosgenin equivalents/g DW) and the methanolic extract contained 1.13 mg/g DW phorbol esters. Phytochemicals detected by RP-HPLC were pyrogallol, gallic acid, naringin, rutin and vanillic acid. The main compounds detected by GC-MS were oxalic acid (root bark), acetic acid and oleic acid (stem bark). Inhibition zones ranged from 8.0 to 17.7 mm. Low MIC (1.2 to 2.3 mg/ml) and MBC (0.4 to 6.3 mg/ml) values were observed in methanolic extract of all plant parts. The present study showed that stem bark, root bark and kernel meal of J. curcas contained compounds with antibacterial activities. The results indicate the potential of J. curcas as a source of antibacterial compounds

A low cost, simplified and battery-powered mobile electrophoresis system suitable as edutainment tool.

Electrophoresis originated since 1807 and has been a staple tool used by biologist and chemist over the centuries since its inception. From paper electrophoresis system to today’s modern automated electrophoresis system, the development of electrophoresis systems have been driven by the advancement of technology and also by the requirement of better and faster resolution of results. This paper reviews the progress of electrophoresis over the decades and into possible future development of electrophoresis

Effects of dietary prebiotics, probiotic and synbiotics on performance, caecal bacterial populations and caecal fermentation concentrations of broiler chickens

Background: In view of a worldwide attempt to restrict or ban the use of antibiotics as growth promoters in animal production, probiotics, prebiotics and combinations of both, as synbiotics, have been suggested as potential alternatives. In this study, the effects of a prebiotic (isomalto-oligosaccharides, IMO), a multi-strain probiotic (consisting of 11 Lactobacillus strains), and a combination of these dietary additives as a synbiotic on the performance, caecal bacterial populations and concentrations of caecal volatile fatty acids and non-volatile fatty acids of broiler chickens were evaluated. Results: Supplementation of 1g kg−1 probiotic (PRO); 5 g kg−1 prebiotic IMO (PRE05); 10 g kg−1 prebiotic IMO (PRE10); synbiotic consisting of 1g kg−1 probiotic + 5 g kg−1 prebiotic IMO (SYN05); or synbiotic consisting of 1g kg−1 probiotic + 10 g kg−1 prebiotic IMO (SYN10) significantly (P < 0.05) improved weight gain of broiler chickens at 22–42 and 1–42 days of age, and feed conversion rate from 1 to 21, 22–42 and 1–42 days of age. The supplementation of probiotic (PRO), prebiotics (PRE05 and PRE10) or synbiotics (SYN05 and SYN10) also significantly (P < 0.05) increased the caecal populations of lactobacilli and bifidobacteria, and decreased the caecal Escherichia coli at 21 days of age, and increased the caecal VFA at 21 and 42 days of age. In all parameters studied, synbiotics did not show a two-fold synergistic effect, when compared to those of probiotic or prebiotic alone. Conclusion: The results of the study indicated that prebiotic IMO (5 g kg−1 or 10 g kg−1), probiotic and their combinations as synbiotics were effective in improving the performance of broiler chickens and in increasing the caecal beneficial bacteria and fatty acids