Open Access Original Research Article

Genetic Diversity and Molecular Characterization of Brazilian Wheat Varieties Obtained by Microsatellite Markers

André Luiz da Silva, Marcelo Berwanger de Oliveira, Elisa Serra Negra Vieira, Volmir Sérgio Marchioro, Francisco de Assis Franco, Ivan Schuster

Journal of Advances in Biology & Biotechnology, Page 1-10
DOI: 10.9734/JABB/2015/18628

Aims: Evaluating the genetic diversity and molecular characterization of wheat varieties in Brazil, using microsatellite markers.

Study Design: Random sampling of seeds from 32 varieties, was done.

Place and Duration of Study: Biotechnology lab, Coodetec, BR 467, km 98. Cascavel, PR, Brazil, between July 2011 to July 2012.

Methodology: Thirty-two varieties were evaluated with 23 markers, using capillary gel electrophoresis. After DNA extraction, and gels scoring, the genetic distances were obtained, the clustering by UPGMA method, the frequency of each allele and Probability of Random Identity (PRI).

Results: It was observed two to eight alleles by loci and genetic distances ranging from 0.31 to 0.90. The varieties were grouped into 11 groups. From the estimated PRI, 15 markers were identified that identify all 32 varieties with a maximum of 0.0001% PRI. High variability among wheat varieties was observed and also high efficiency in the identification of varieties with microsatellite markers.

Conclusion: This approach can be used in breeding programs and for the protection of intellectual property of wheat varieties breeders.

Open Access Original Research Article

Chemometric Assisted HPLC Method for the Simultaneous Estimation of Aspirin, Atorvastatin and Clopidogrel in Biological Matrix

Ramalingam Sathiyasundar, Kannappan Valliappan

Journal of Advances in Biology & Biotechnology, Page 1-10
DOI: 10.9734/JABB/2015/17269

Aim: A new efficient simple and robust liquid chromatographic method has been developed and validated for the simultaneous estimation of aspirin, atrovastatin and clopidogrel in human plasma sample.

Study Design: The investigation was focused on studying the influence of organic phase modifier, pH and flow rate, which modify the separation of chromatographic behavior of these compounds.

Methodology: To optimize the chromatographic factors that had a significant effect on separation attributes central composite design was applied. The global optimization of the chromatographic responses such as capacity factor, resolution and analysis time optimized derringer’s desirability function was employed.

Results: The optimized chromatographic condition was Acetonitrile: Methanol: 0.1% Triethylamine (pH 3.0 adjusted with Ortho phosphoric acid) (53.55: 05: 41.45 % v/v), 1.14 ml/min flow rate and measurement was performed PDA at 220 nm. The spiked plasma sample was extracted by a simple protein precipitation method and then the sample was extracted with cold methanol and solvents was evaporated by the dry air method.

Conclusion: This method can be useful for the computable determination of aspirin, atrovastatin and clopidogrel in human plasma suitable for bioequivalence and pharmacokinetic studies in healthy human volunteers.

Open Access Original Research Article

Optimised Production of Xylanase by Aspergillus brasiliensis under Submerged Fermentation (SmF) and Its Purification Using a Two-step Column Chromatography

Hooi Ling Ho, Zaki Iylia

Journal of Advances in Biology & Biotechnology, Page 1-22
DOI: 10.9734/JABB/2015/18129

Aims: Carbon and nitrogen source play a significant role in microorganisms growth especially for the production of enzymes. Expensive medium composition creates huge setbacks especially in the production of commercial enzymes such as xylanase. Hence, in this study, wheat bran as the agricultural residue supplemented with yeast extract was used as the sole carbon and nitrogen source to culture Aspergillus brasiliensis ATCC 16404. This cheap alternative carbon source was used comprehensively to replace the expensive medium especially xylan in the production of xylanase. In fact, one of the main factors of using wheat bran is due to its cost effectiveness and availability in current market. Therefore, the main objectives of this study were to elucidate the production of xylanase from A. brasiliensis under submerged fermentation (SmF) using wheat bran as the sole carbon source followed by purification of the xylanase to its homogeneity. SmF was chosen primarily as the mode of fermentation for the production of xylanase since it was easily operated in terms of controlling its parameters including the supply of oxygen and nutrients in a large culture volume for the growth of fungi using the optimised medium composition proposed. Subsequently, the purification of xylanase was performed in an easy two-step column chromatography after the precipitation with ammonium sulphate and dialysis, respectively. 

Methodology: In this study, the production of xylanase by A. brasiliensis was conducted under SmF using wheat bran as the prime carbon source followed by a two-step column chromatography of diethylaminoethanol (DEAE) Sepharose and Sephadex G-75 for the purification of xylanase before subjected to zymography analysis to detect the biological activity of the purified xylanase based on its substrates hydrolysation.

Results: From the results, high production of xylanase was exerted, producing 11.49 U/mL with protein concentration of 2.33 mg/mL at 72-hour of fermentation. The maximum spore count of 7.91 × 105 cells/mL with final pH of 6.9 was able to produce the optimum xylanase activity of 11.49 U/mL. Xylanase secreted from A. brasiliensis was precipitated using ammonium sulphate and purified via DEAE Sepharose and Sephadex G-75 column chromatography. The purified xylanase was illustrated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular weight of 36 kDa. Approximately a two and the half purification fold with a surprisingly high recovery of 38.15% was obtained. From our study, the purified extracellular xylanase was subjected to the zymography analysis using xylan supplemented potato dextrose agar (PDA) to analyze the biological application of xylanase in hydrolyzing its substrate of xylan. Since xylanase showed its activity only on xylan, therefore, the formation of visible halo ring in the zymography analysis was managed to indicate that this enzyme was an active xylanase after the two-step column chromatography.

Conclusion: The purified xylanase that obtained in this study possessed much greater potential in industrial applications since it had successfully demonstrated a visible convincing halo ring as a result of its active functional activity against its substrate of xylan.

Open Access Original Research Article

Evaluation of the Conventional Versus Two Rapid Microwave Processing Techniques Using the Masson Trichrome Histochemical Methtod

Tobias Peter Pwajok Choji, Samuel Ifedioranma Ogenyi, Anthony Ajuluchukwu Ngokere, Solomon Chuwang Chollom, Kizito Peter Jugu, Sati Dung Lokason

Journal of Advances in Biology & Biotechnology, Page 1-15
DOI: 10.9734/JABB/2015/19143

Aims: To evaluate the quality of paraffin blocks produced using the microwave and without the use of xylene. To evaluate the compatibility of the rapid microwave processing techniques on the histochemistry of collagen and muscle fibre as well as nuclear, cytoplasmic and extracellular components.

Study Design: Harvesting and fixation of tissues in 10% formalin. Grossing into triplicates, processing using three different techniques, checking for retraction of blocks, checking the quality of paraffin ribbon, Staining using the Masson Trichrome technique, grading of section quality and staining characteristics.

Place and Duration of Study: Anatomy Department, Nnamdi Azikiwe University, Nnewi Campus and National veterinary Research institute, Vom, Nigeria. The work was done between August and December, 2014.

Methodology: Liver, kidney and oesophagus were harvested from an apparently healthy rabbit. Human cervix, prostate, testis and prostate were obtained. All samples were fixed in 10% buffered formalin, cut into triplicates and processed using the three techniques, sectioned and stained simultaneously using the Masson Trichrome technique and evaluated.

Results: Processing cycles and reagent consumption per cycle were greatly reduced in the microwave techniques. Paraffin blocks as well as ribbons produced from the three techniques were of comparable quality. Though tissue integrity was comparable among the three techniques, the stroma of the cervix was better retained in the microwave with vacuum method. Dye uptake as well as section thickness were comparable among the three techniques. Nuclear, cytoplasmic and extracellular components presented with comparable histochemistry.

Conclusion: The rapid microwave techniques are cheap and fast with good ribonning. Reagent toxicity is greatly reduced as xylene is excluded. Muscle fibre and collagen fibre histochemistry for the rapid microwave methods are comparable to that of the conventional method. Cervical stroma is better retained in the rapid microwave methods than in the conventional method of paraffin wax processing. Microwave techniques should be encouraged since they can help to produce quality paraffin section within the shortest time possible at a cheaper rate than the conventional method without compromising section quality and tissue histochemistry.

Open Access Original Research Article

Application of Algae as a Biomass Feedstock Source at a Waste Water Treatment Facility

Klaus Dölle, Catherine Watkins

Journal of Advances in Biology & Biotechnology, Page 1-9
DOI: 10.9734/JABB/2015/19185

Algae species Cladophora glomerata and Vaucheria are growing in a trickling filter in the secondary treatment process at a Waste Water Treatment Facility. This study investigates the role of the algae and its potential application a as biomass feed source for renewable energy production with and without growth media. Utilizing different growth media, growth rates were found to be the highest on Styrofoam and plastic media. Phragmites australis bundles were found to have the most pragmatic application for both growth and harvest. C. glomerata exhibited the highest amounts of lipids, or total organic extracts (TOE).