Research Journal of Biotechnology

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Vol. 13(9) September 2018

Toxicological and Histological Effects of Selected Bacterial Biopesticides on Aedes aegypti (L.), The Vector of Dengue in Saudi Arabia

Mahyoub Jazem A.*, Al-Ghamdi Khalid M. and Barnawi Abdulaziz B.M.

Page No: 1-7

Abstract: Mosquitoes are the carriers of severe well-known illnesses such as malaria, Japanese encephalitis, dengue fever, chikungunya fever, West Nile virus, yellow fever, lymphatic filariasis and zika virus. These diseases produce significant morbidity and mortalities in humans and livestock around the world. The present research investigated the toxicological and histological effects of three bacterial insecticide, Spinosad, Bacillus thuringiensis and B. sphaericus against the fourth-stage larvae of dengue and on zika virus vector, Aedes aegypti under laboratory as well as semi-field conditions. The lethal concentration to kill half (50%) of the population was found in Ae. aegypti fourth-instar larvae with LC50 0.005, 0.055, 0.414 ppm (laboratory conditions) and 0.007, 0.070, 0.563 ppm (semi-field conditions) respectively at 24 h.

In laboratory conditions, the tested effect of bacteria against larvae were 1.26, 1.27 and 1.35 folds compared with semi-field conditions. Also, these results indicate that mosquito larvae of Ae. aegypti were more susceptible to Spinosad than B. thuringiensi and B. sphaericus by about 9.59 and 77.12 folds respectively. The histological changes in mosquitoes targeted with bacterial insecticide were studied by light microscopy, evaluating the impact on mid-gut epithelial cells following treatment with LC50 of bacterial insecticide and the results showed detectable alteration in Gastric Epithelial Tissue (GEC) and fatty body (F). Overall, this study highlighted that bacterial insecticides can be proposed as effective tools for eco-friendly control of the dengue and Zika virus vectors.

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Antifungal activity of α-amylase inhibitor from the seeds of Fagopyrum esculentum against Aspergillus niger and Aspergillus flavus

Phirange Aparna Sudam and Sabharwal Sushma G.*

Page No: 8-14

Abstract: The bioefficacy of Fagopyrum esculentum α-amylase inhibitor (FEAI) as an antifungal agent was studied against the fungi Aspergillus niger and Aspergillus flavus. FEAI was purified using ion exchange chromatography on Poros-HS 50. The effects of FEAI on the growth of fungi were studied at varying concentrations of the inhibitor by evaluating the inhibition of growth by colony count method and reduction in mycelium dry weight in presence of the inhibitor. The changes in the morphology of FEAI treated mycelia were examined by light microcopy and by scanning electron microscopy.

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Synthesis of Zinc oxide nanoparticles using algal formulation (Padina tetrastromatica and Turbinaria conoides) and their antibacterial activity against fish pathogens

Rajeshkumar S.

Page No: 15-19

Abstract: The applications of zinc oxide nanoparticles are developing largely in the biomedical and another discipline. In this present study, we used the most applicable marine brown seaweeds such as Padina tetrastromatica and Turbinariae conoides based algal formulation for the green preparation of zinc oxide nanoparticles (ZnO NPs). The synthesized zinc oxide nanoparticles were preliminarily characterized by using a UV-visible spectrophotometer.

The morphology of zinc oxide nanoparticles was analyzed using Scanning Electron microscope (SEM). The clear images were observed in the figures. The crystalline nature of the zinc oxide nanoparticles was proved by X-ray Diffraction Assay (XRD pattern). The functional group responsible for the nanoparticles synthesis was analyzed by Fourier transform infrared spectroscopy (FTIR). Finally, the synthesized zinc oxide nanoparticles were tested against the fish pathogens.

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Improved Cry1Ac expression levels during Bt cotton cultivation

Srikanth Perumalla, Devasahayam Mercy, Singh Raghuvir and Masih Sam A.*

Page No: 20-25

Abstract: Decreasing resistance to insecticidal proteins from transgenic crops is a major strategy of insect management. Increased resistance is a major problem nowadays in insects’ pests which reduces efficiency of insecticidal proteins from Bacillus thuringiensis (Bt) produced by transgenic crops such as Bt cotton (Bollgard-II). Cultivation of Bt cotton with refuge nBt cotton is a strategy to delay resistance of lepidopteran insect pests. During this present study, field trails with four treatments with varying nBt refuge crops for quantification of Cry1Ac were done. Results showed that 50% Bt with 50% nBt treatment has highest Cry1Ac expression in lower leaves (8.249 µg/gm) followed by upper leaves (7.407 µg/gm), sepals (0.988 µg/gm) and boll bracts (0.987 µg/gm) respectively.

The Cry1Ac expression decreased consistently as the plant age increased in all the treatments. The morphological and yield parameters were recorded and highest plant height was found in 90% Bt with 10% nBt treatment such as 123.8669cm and 99.200 cm. It was also recorded that highest yield like boll size (gm/plant) and lint index were found in 75% Bt with 25% nBt treatment as 14.449, 9.979 (gm/plant) and 11.647, 9.426 respectively. We have concluded that highest morphology and yield depend upon number of Bt and nBt plants present in all treatments. This combination is a better option to farmers to increase their income and good strategy to defend the resistance in insect.

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Toxicity of biogenic gold nanoparticles fabricated by Hybanthus enneaspermus aqueous extract against Anopheles stephensi and Culex tritaeniorhynchus

Suman Thodhal Yoganandham*, Radhika Rajasree Santha Ravindranath, Gayathri Sathyamoorthy, Remya Rajan Renuka and Aranganathan Lakshminarayanan

Page No: 26-34

Abstract: In this study, rapid, simple approach was used for the biosynthesis of gold nanoparticles using Hybanthus enneaspermus aqueous extract and its toxicity studies on Anopheles stephensi and Culex tritaeniorhynchus. The biosynthesized gold nanoparticles were characterized for their size, morphology and crystallinity. Change in colour was observed to ruby red due to the formation of gold nanoparticles. The size and shape of the synthesized gold nanoparticles were confirmed by electron microscopy. The synthesized nanoparticles were mostly aggregated, oval and in the range of 19-27nm.

The infra-red spectroscopy studies revealed that hydroxyl group was present along with gold nanoparticles, which are responsible for their stabilization and reduction of gold nanoparticles. The biosynthesized gold nanoparticles showed larvicidal activity with the LC50 values of 28.15 and 26.05 mg/L against the fourth instar larvae of Anopheles stephensi and Culex tritaeniorhynchus.

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Impression of Missense Single Nucleotide Polymorphisms of Leptin Gene on the early onset of Obesity related Infertility in Female

Saranya G.M., Febin Prabhu Doss J. and Ramesh Pathy M.*

Page No: 35-47

Abstract: Missense mutations in LEPTIN (LEP) gene are more frequently linked to higher risk of acquiring complex metabolic disorders like obesity, type 2 diabetes and associated female infertility. Some nonsynonymous single nucleotide polymorphisms (nsSNP) in LEP gene affect both the function and structure of LEP protein. Thus, we have used various computational tools like nsSNP analyser, PolyPhen-2, Provean Protein, Fathmm, I-Mutant, SNPs and Go, ConSurf, Net Surf, PhD-SNP, PANTHER and SNP effect to identify the functional single nucleotide polymorphisms (SNPs) and indels in protein structure as listed in the dbSNP database.

In our in silico study, we have predicted seven potentially deleterious nsSNPs i.e., rs776443424 (S52F), rs886061972 (D61N), rs200915360 (D76Y), rs748408158 (L86F), rs724159998 (D100Y), rs28954113 (N103K), rs1051206328 (C117S). Using the Swiss PDB viewer, we have also analysed the free energy change and the structural visualization of wildtype and mutant protein. Among the seven nsSNPs, D100Y, D76Y and N103K have direct or indirect thwart on the amino acid interactions and hydrogen bonding networks of LEP. Of these, N103K functionally alters the LEP protein due to high free energy whereas D100Y and D76Y have structural impact. These findings may promote the selection of potential variants for future studies on onset of obesity related infertility in female.

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Designing self-ratiometric biogenic silica-nanoprobe for quantitative sensing of pH

Jeyanthi Palanivelu and Ramalingam C.*

Page No: 48-54

Abstract: Food sensors, sensitive to food properties like pH, water activity are trending issues in food processing and other food related field. Nanoparticles have inspired in the sensory studies owing to their high property of sensitivity and specificity. In this work we intended on the synthesis of silica nanoparticles from agricultural waste materials by acidic method. The synthesized nanoparticles were characterized by Particle size analyzer, Zeta potential, FTIR, XRD and EDX. The sensing property of biogenic SiO2 nanoparticles was achieved by covalently doping FITC dye.

The doped nanoparticles were analyzed using Fluorescent spectroscopy for its divergence in excitation intensity according to different pH standard solutions ranging from pH 2 to pH 9. Based on the standard excitation intensity, the pH sensitivity is measured in real time food samples. The FITC dye doped on biogenic SiO2 has a pKa value of 6.5 which makes it a prospective pH indicator.

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Phyllanthus fraternus a potent natural antioxidant as pharmaceutical supplement

Upadhyay Richa and Tiwari Kavindra Nath*

Page No: 55-62

Abstract: Oxidative stress is root cause of various degenerative diseases like cancer, atherosclerosis as well as diseases associated with ageing. Many antioxidant compounds naturally isolated from plants have been identified as free radical scavengers. Currently use of plant derived natural antioxidants in foods or medicinal products is increasing rapidly in comparison to synthetic antioxidants due to side effects of synthetics. In present study, antioxidant properties of whole plant extracts of Phyllanthus fraternus were evaluated by four different in vitro assays viz. radical scavenging activity by DPPH method, scavenging of superoxide by riboflavin-light-nitroblue tetrazolium assay, inhibition of lipid peroxidation using egg yolk homogenate and reducing power assay by Fe3+-Fe2+ transformation in presence of extract.

Total phenolic and flavonoid contents were measured by Folin-ciocalteu assay and AlCl3 colorimetric method respectively. The extracts were found to enrich with huge number of phenolic compounds and flavonoids. Ethanolic extract of P. fraternus possessed significant antioxidant activity as compared to aqueous extract. There was significant linear correlation between antioxidant property of extract and its reducing power as well as polyphenolic constituent. It has been concluded that P. fraternus extract can combat various chronic health problems caused by oxidative stress, thus it could be recommended as a potent pharmaceutical agent.

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Generation of a Monoclonal IgG Antibody to Polyethylene Glycol by Hybridoma Technology

Lal Annie Ankita* and Lall Rohit

Page No: 63-66

Abstract: An IgG mouse monoclonal antibody against polyethylene glycol has been generated. The antibody reacts with PEG and is able to recognize a PEGylated protein in plasma or serum at concentrations as low as 1 pg/mL. The antibody is readily purified in substantial quantities. The PEG IgG will find significant utility in the sensitive detection of PEG derivatives of biosimilars as well as free PEG molecules in plasma or serum during the pharmacokinetic characterization of PEGylated compounds.

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Production of Genetically Modified Grape (Vitis vinifera L.) Plants

Hemaid I. A. Soliman

Page No: 67-76

Abstract: Grape (Vitis vinifera L.) is one of the most economically important fruits in the world. High salinity stress adversely affects plant growth and limits agricultural production worldwide.

This study describes a successful method of somatic embryogenesis using in vitro-derived leaf explants and introduction of a vacuolar-type Na+/H+ antiporter gene from a halophytic plant, Atriplex gmelini (AgNHX1) confers salt tolerance to grape cv. Superior seedless using the Agrobacterium-mediated transformation. Callus embryogenic was induced on NN medium 2.0 mgL-1 2,4-D, 0.5 mgL-1 BAP and 0.5 mgL-1 NAA. Subsequent subculture of callus on NN medium containing 1.5 mgL-1 BAP, 0.5 mgL-1 kinetin and 0.5 mgL-1 NAA induced shoot organogenesis after eight weeks of culture. The leaf explants were co-cultivated with Agrobacterium strain LBA4404 harbouring the binary vector pBI121 which contained the AgNHX1 and nptII genes and putative transgenic plants were produced.

The presence and stable integration of AgNHX1 gene in transgenic plants was confirmed by PCR and northern blot hybridization. The transgenic grape plants overexpressing the AgNHX1 gene showed a strong tolerance to salt stress under 250 mM NaCl whereas non-transgenic plants died under the same conditions. Salt tolerance assays followed by salt treatments showed that the transgenic plants overexpressing AgNHX1 could survive under conditions of 250 mM NaCl for 4 weeks while the non-transgenic plants died under the same conditions. These results indicate that overexpression of the Na+/H+ antiporter gene in grape plants significantly improved their salt tolerance.

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Genetic association of geographic populations and species of lac insects of the genus Kerria (Hemiptera: Coccoidea) with EST-microsatellite markers

Kandasamy Thamilarasi*, Mishra Vinay, Toppo Neha, Ranjan Sanjeev, Mohanasundaram Arumugam, Sharma Kewal and Ramani Ranganathan

Page No: 77-89

Abstract: Lac insects belonging to the genus Kerria are commercially exploited for lac which has diversified industrial applications. India is the global leader in lac production; much of commercial lac is derived from the Indian lac insect, Kerria lacca (Kerr). Molecular marker studies of Kerria species are limited to RAPD and ISSR. We evaluated genetic diversity among 27 Kerria geographic populations belonging to thirteen species of Kerria, using 31 microsatellite markers developed from transcriptome of the Indian lac insect. The number of alleles per locus ranged between 2 and 6 with a mean of 2.74, showing polymorphism of 66.7-100%. UPGMA dendrogram based on Jaccard’s similarity coefficient grouped 27 Kerria accessions into three major clusters consisting of 2, 13 and 12 populations respectively and their similarity coefficient ranged between 0.61 and 0.95.

Based on similarity coefficients, the closest relationship was observed between Kerria pusana and Kerria pennyae and also between K. lacca and K. pennaye. K. chinensis appeared well separated from all other Kerria species studied. Geographically closer species were found more similar, in general. The present study reveals wide intra- and inter-specific genetic diversity among Kerria populations through EST-microsatellite markers and indicates the potential and usefulness of marker studies for lac insect improvement programmes.

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Biotic synthesis of silver nanoparticles from Momordica charantia (Cucurbitaceae) and its characterization studies

Gandhiraj V.*, Sathish Kumar K. and Narendrakumar G.

Page No: 90-99

Abstract: A collection of novel strategies was applied for the biosynthesis of silver nanoparticles by using plants. In the present study, green synthesis was carried using Momordica charantia and the optimization of silver nanoparticle synthesis was carried out by varying varieties, solvent, parts of fruit, plant extract concentration, metal ion concentration of silver nitrate solution, pH, temperature, reaction time and reaction condition. The characterization study was done using UV-Visible spectrophotometer, SEM, TEM, FTIR spectroscopy, EDX and XRD.

The results revealed that the shape of synthesized silver nanoparticle is spherical and size ranges from 75-150 nm in SEM and 50-200nm in TEM. FTIR identified nitro compounds, phenols, alcohols, aliphatic amines and alkenes. The chemical compound analysis is done using gas chromatography-mass spectroscopy (GC-MS).

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Alkaline Pectinase production by Bacillus thuringiensis strain KR Isolated from Vegetable Dumping Waste Soil

Dey Sujan, Roy Karabi, Barua Rasel, Uddin Md. Kamal and Hossain Md. Towhid*

Page No: 100-107

Abstract: Pectinase is one of the most important enzymes in present-day biotechnology. From the economical point of view, microbial pectinase has become increasingly significant. The aim of this study was the isolation and identification of potential pectinase producing microbes from soil, optimization of process parameters for maximum pectinase production and partial characterization of the pectinase enzyme. Primary screening was done on Yeast Extract-Pectin (YEP) agar medium and iodine-potassium iodide solution was used to detect the zone of clearance around the isolates. Among 40 isolates, the isolate (designated as K12) that showed potential pectinase activity was finally selected for detailed studies.

The selected isolate was identified as Bacillus thuringiensis strain KR by16S rRNA sequencing. The isolate was found to produce the maximum pectinase at 40°C with pH 8.0 during 72hrs incubation when grown into the medium containing citrus pectin and yeast extract as carbon and nitrogen sources respectively. The crude pectinase was partially characterized by optimizing temperature, pH, reaction time and substrate concentration. During enzyme-substrate reaction, the enzyme exhibited its maximum activity at 40°C with pH 8.0 for 30 minutes when 2% citrus pectin was used as a substrate. This alkaline crude pectinase showed a significant effect on fruit juice (apple/orange) clarification.

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Microbial Laccases in Food Processing Industry: Current Status and Future Perspectives

Yadav Mukesh*, Sehrawat Nirmala and Kumar Amit

Page No: 108-113

Abstract: Laccases (EC are copper-containing enzymes that catalyze the oxidation of organic and inorganic substrates including polyphenols, amino phenols, methoxy phenols, aromatic amines and ascorbate with concomitant four electron reduction of oxygen to water. Laccases are widely distributed in higher plants, fungi and bacteria. The ability of laccase to oxidize phenolic compounds and to reduce molecular oxygen to water has made it an enzyme of interest for various scientific and industry-based research groups. Microbial laccases are preferred for industrial and commercial processes due to easy and higher level of production in shorter time period, comparatively easy purification with respect to plants laccase.

It is generally used in textile industry, healthcare industry, pulp and paper industry, bioremediation and food industry. Food industry is growing rapidly particularly in developing countries and enzymes are now commonly used for processing of various food materials. Laccase is one the important industrial food enzyme commonly used for juice industry, baking and wine stabilization and bioremediation of food industry waste. An attempt has been made to collectively present the relevant information about microbial laccase used in food industry. Most of the review articles focus over various industrial applications but this review focuses over the applications of microbial laccases specifically in food industry for various processes.

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