Research Journal of Biotechnology

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Research Journal of Biotechnology





Random Mutagenesis on Wautersia Eutropha for Improving Poly-β-Hydroxybutyrate Production from Inexpensive Carbon Sources

ADas Santanu, Sikdar Munmun and Veerasamy Muralikrishnan

Wautersia eutropha, a source of homopolymer and heteropolymers of biodegradable poly-β-hydroxybutyrate [P(3HB)], a potential alternative to recalcitrant plastics, was subjected to random mutagenesis using ultraviolet irradiation. Five mutant isolates, namely Wautersia eutropha M1, Wautersia eutropha M2, Wautersia eutropha M4, Wautersia eutropha M5, Wautersia eutropha M6 were obtained on screening for poly-β-hydroxubutyrate yield. All five mutant strains and wild-type Wautersia eutropha rendered better P(3HB) yield in minimal salt broth supplemented with surplus fructose than nutrient broth. Wautersia eutropha M2 and Wautersia eutropha M5 were screened for improved P (3HB) yield. Wautersia eutropha M5 efficiently utilized cane molasses rendering a highest P(3HB) content of 57.42% at 20g.L-1 concentration whereas the other two strains showed insignificant P(3HB) yield from molasses. P(3HB) content increased with increasing concentration of sago industry liquid waste by the screened strains with a highest P(3HB) yield of 56.67% at 30g.L-1 concentration by Wautersia eutropha M5. Arrack gave least growth and poor P(3HB) yield by the screened strains.

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Valuable Antioxidant and Antimicrobial Extracts from Rhizophora Mucronata of Asiatic Mangrove Forests

Haq Imdadul, Sani Wirakarnain, Philip Koshy , Rafat Arash, Hossain A.B.M. Shariff and Taha Rosna Mat

Abstract: The antioxidant and antimicrobial properties of ethanol, methanol and chloroform extracts of Rhizophora mucronata leaves were examined in this study. The antioxidant activities of the samples were evaluated using a combination of enzymatic and non-enzymatic methods namely superoxide dismutase deter­­m­ination, erythrocyte haemolysis protection and 2,2-diphenyl-1-picrylhydrazil free radical scavenging assays. Folin-Ciocalteu reagent method was used to estimate the amount of total phenolic compounds of the extracts. Ethanol, chloroform and methanol extr­acts of the leaves showed the highest antioxidant pote­n­tial in superoxide dismutase, erythrocyte haemo­lysis and free radical scavenging assays respectively. The highest total phenolic content was found in ethanol extract followed by methanol extract. Paper disk diffusion method was applied to determine the antimi­cr­obial activities of ethanol and methanol extracts of the leaves. Both ethanol and methanol extracts could inhibit the growth of Escherichia coli, Staphylococcus aureus and Bacillus cereus while no inhibition was detected against Pseudomonas aeruginosa.

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Effect of Corn Oil as a Vehicle on Kidney of Female Swiss Albino Mice Mus Musculus by Gavages Method

Anjali S.,Tanuja, Nath A., Singh J. K. and Ravish K.

Abstract: The purpose of this study was to investigate the effects of corn oil on histological and biochemical parameter of kidney of Female Swiss albino mice. Mi­ce were administered corn oil at the dose of 10 ml /kg body weight for the period of six weeks. No morp­hological alterations were observed. But fur soiling was observed among few mice of treated groups during the experimental period. Histological exa­min­ation of kidney of female mice revealed mild alte­rations like tubular disarrangement. Few foci of hem­orrhage and fatty degeneration were observed only in four weeks and six weeks corn oil treated groups. Mice of one week and two weeks group sho­w­ed neither abnormal condition nor any severe lesio­ns in the kidney. Comparative study of treated groups of mice with control group of mice was done. The serum biochemical test or kidney function test (Urea and creatinine) were estimated by Fully Autom­ated Biochemistry Analyzer (Model No.-Selectra- E” (Vita lab) by Merck. These data showed that corn oil at the rate of 10 ml/kg body weight can be used as a vehicle for toxicological studies but may cause adve­rse effects on the renal tubules if used for prolonged periods.

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The Extraction Efficiency of Apigenin from Scutellaria barbata comparing pressurized Liquid extraction and Soxhlet extraction < /p>

Lee Hanna and Shim Sang Hee

Abstract: Pressurized liquid extraction of apigenin from Scutellaria barbata was compared to the traditional Soxhlet extraction using methanol. The conditions for pressurized liquid extraction were as follows: the extraction solvent was 100% ethanol, 50% aq ethanol or water; the extraction temperature was 120˚C; static extraction time was 5 min. For the traditional Soxhlet extraction, the sample was boiled at 50˚C for three hours. According to calculations by regression parameters using HPLC analysis, the extraction yields of apigenin were 1.25 mg/g by pressurized liquid extraction (extraction solvent, 100% ethanol; temperature, 120˚C; static extraction time, 5 min), 0.32 mg/g by pressurized liquid extraction (extraction solvent, 50% aq ethanol; temperature, 120˚C; static extraction time, 20 min) and 0.88 mg/g by Soxhlet extraction using methanol. Pressurized liquid extraction using 100% ethanol yielded 74.4 % higher apigenin than using 50% aq ethanol and using 100% ethanol yielded 29.6% higher apigenin than using the Soxhlet extraction technique.

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Antibacterial and Antifungal Activity of different Lichens Extracts and Lichen Acid

Kosanić Marijana and Ranković Branislav

Antibacterial and antifungal activity of the acetone, methanol and aqueous extracts of the lichens Lecanora atra, Parmelia reticulata, Parmelia oomph­alodes and salazinic acid from Parmelia reticulata lichen were explored in vitro against six species of bacteria and eight species of fungi. The antimicrobal activity was estimated by the disc-difusion method and determination of the minimal inhibitory concentration (MIC) by the Broth tube dilution method. The aqueous extracts of all of the tested lichens were inactive, whereas the acetone and methanol extracts showed antimicrobial activity against the majority of the tested organisms. Among the extracts, the strongest activity was found in the acetone extract of the lichen Parmelia omphalodes where the least measu­red MIC value was 0.78 mg/ml. Salazinic acid also showed strong activity against bacteria and fungi. The lowest measured MIC value was 0.39 mg/mL related to the Klebsiella pneumonie species. The bacteria were more sensitive related to the tested fungi. Generally, Bacillus subtilis and Bacillus mycoides were the most sensitive of the tested bacterial species while Botrytis cinerea and Candida albicans were the most sensitive fungal species. There was no antimicrobial activity against Escerichia coli species. The results obtained show that the tested lichen and their compound demostrated a strong antimicr­obal effect against the tested microorganisms.

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In Vivo Immunomodulating Activities of Ethanolic Leaf Extract of Stevia rebaudiana in Albino Rats

Shukla Shruti , Mehta Archana, Kim Myunghee and Ayyadurai N.

Abstract The present study was undertaken to explore the in vivo immunomodulating activities of ethanolic leaf extract of Stevia rebaudiana (Bertoni) in albino rats. The immunomodulatory activities were determined by neutrophil adhesion test, haemagglutinating anti­body (HA) titre, delayed type hypersensitivity (DTH) response and phagocytic activity. The response pro­duced by oral administration of ethanolic leaf ext­ract showed a significant increase in neutrophil adhe­sion and delayed type hypersensitivity (DTH) response. With 300, 400 and 500 mg/kg/day doses of the extract, the DTH responses of 24 h (mean ± SD % increase in paw volume) were found to be 0.41 ± 0.01, 0.50 ± 0.01 and 0.58 ± 0.01 respectively as compared to the corres­ponding value of 0.18 ± 0.01 for the control group. These differences in DTH response were statistically significant (P<0.05). The augmentation of humoral immune response to sheep red blood cells by ethanolic extract (200 - 500 mg/kg) was evident by increase in antibody titres in rats. A dose-related increase in antibody titres was observed. The ethanolic leaf extract also evoked potent response towards phagocytosis in carbon clearence assay. This study demonstrates potent immunomodulatory activities and therapeutic potential of Stevia rebaudiana ethanolic leaf extract for the prevention of immunological disorders.

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In Vitro Regeneration of Chlorophytum Borivilianum using Different Explants – A Rare Medicinal Herb

Dwivedi Rashmi

Abstract: Chlorophytum borivilianum Sant et Fernard, commonly known as safed musli belonging to the Lilliaceae family is an important medicinal herb of commercial importance. Chlorophytum borivilianum with about 17 species found in India, is a very popular herb in traditional Indian medicine and constitutes a group of herbs used as ‘Rasayan’ or adaptogen. In the present study, shoot growth and multiplication respon­ses using different explants were compared on solid medium. Crown and fruit were used as an explant and cultured on MS medium with different concentrations of BAP (6-benzyl amino purine) at 1.0, 2.0, 3.0, 4.0, 5.0 mg/l and IAA (Indole acetic acid) at 1.0 and 1.5 mg/l. Shoot proliferation was maximum (75%) with 5.0 mg/l BAP when compared with other combinations of phytohormones. Fruit explants also showed response in the treatment using 2 mg/l BAP and 5 mg/l BAP without further dedifferentiation. In vitro grown shoot buds produce good profuse roots with 75% rooting in half MS medium with 500 mg/l activated charcoal. 90% surv­ival rate of the plantlets was observed during accl­im­atization. This protocol can be successfully utilized for the large scale clonal multiplication and germplasm conservation of rare valuable medicinal herb.

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PCR-Based Differentiation of Phenotypically similar Gram-Negative Bacilli recovered from CSF Samples

Nagaveni S., Rajeshwari H., Oli Ajay Kumar, Patil S. A. and Kelmani Chandrakanth R.

Broad-range bacterial PCR combined with DNA sequencing was used as a means of identifying clinically relevant isolates of gram-negative bacilli recovered from cerebrospinal fluid (CSF). Genes coding for bacterial 16S rRNA genes were amplified. The study was designed to compare phenotypic with molecular identification. These results indicated that preliminary phenotypic testing had misidentified several isolates due to the morphological changes induced by the antibiotics. This invites efforts to be focused on the application of PCR with broad-range bacterial primers to improve the etiologic diagnosis of bacterial infections.

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Efficacy of Probiotic Toothpaste on subjects with Periodontitis - A Randomised Double-Blind Study

Philip Koshy , Swaminathan Dasan, Muniandy Sekaran and Yaacob Hashim

Abstract: Probiotics have been successful in the treatment of digestive related diseases. However, its use to overcome dental disorders is an innovative approach in Oral Biology. Probiotics has been shown to possess antibacterial activity and it thus can control pathogens from causing dental diseases like gingivitis. Gingivitis can lead to periodontitis when not treated and eventually to loss of teeth. Past studies indicate that probiotics also lowers the pH so that bacterial dental plaque cannot form mature dental plaque and calculus that can lead to periodontal disease. In this clinical study, Probiotic toothpaste is assessed in this randomized parallel, double blind study to evaluate its efficacy, in comparison to a placebo, on patients with chronic gingivitis or early periodontitis. The toothpaste contains bacteriocin extracted specifically from lactic acid bacteria found in Probiotics. This randomized, parallel, double blind study, in which 60 adult patients diagnosed with chronic gingivitis or early periodontitis, 30 of whom were subjected to the test Probiotic toothpaste and the remaining 30 used a control toothpaste evaluated the healing of gingival tissues using clinical indices. This parallel study was carried out over a period of 2 months. The clinical indices used in this study were Plaque Index (PI), Gingival Index (GI), Papillary Bleeding Index (PBI) and Stain Index. The results of this study indicated that the Probiotic toothpaste containing bacteriocin was marginally significant in its efficacy to control plaque and gingivitis when compared to the placebo toothpaste and both the test and control toothpastes had no effect on dental staining.

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Spectrometric Analysis of Quinazoline Alkaloids from Methanolic Leaf Extract of Adhatoda vasica Nees.

Khadiker S. and Tenguria R.K.

Abstract: Methanolic extract of leaves of Adhatoda vasica Nees. were prepared using Soxhletion method. Obtained leaf extract were purified with the help of column and thin layer chromatography followed by acid hydrolysis and methylation. These isolated and purified compounds were subjected to spectral analysis. On the basis of Mass spectrum, IR spectrum and NMR spectrum analysis, it may be concluded that leaf extract of Adhatoda vasica Nees. had the compounds namely Phenoxy Vasicolinone with molecular weight 413 and Phenyl Adhatodine with molecular weight 397. Both obtained compounds are the quinazoline alkaloids in which two fused benzene rings are present.

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Genetic Structure of an Endangered Cycas Revoluta using RAPD Markers

Sarethy Indira P., Gulati Natasha, Bansal Akanksha, Gupta Vibhuti, Malhotra Kunal and Gabrani Reema

Abstract: Cycads are one of the most primitive species and represent a key point in evolution between ferns and angiosperms. Cycas revoluta is an introduced species in India, widely exploited for its ornamental and medicinal features, facing extensive habitat destruction. Eight accessions were collected from three regions. These were considered to belong to three populations assuming each population to be homogeneous. Genetic diversity within and amongst accessions was studied using fifteen RAPD primers. Multiple bands of scorable intensity were reprodu­cibly generated from 10 decamers. A total of 51 loci (82.26%) were found to be polymorphic. Co-efficient of gene differentiation (GST) was 0.4260, indicating that 57.4% of variation existed within populations. To remove the upward bias of GST, AMOVA analysis was used to confirm that a non-significant amount of genetic diversity (25%) was attributable to variance between populations while a larger share (75%) of the diversity occurred within populations. The principal co-ordinates analysis also confirmed AMOVA results. Since our sampling was from locations widely separated (2000 km), this level of genetic diversity could be because of genetic drift or ancestral polymorphisms. Based on our results and the high level of genetic diversity observed, conserv­ation strategies should be formulated focusing on collection of samples from as many locations as possible.

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Molecular Identification of ‘Candidatus Phytoplasma Cynodontis’ associated with Bermuda Grass Disease in Rajasthan, India

Gaur R.K., Raizada Richa and Gupta Vijai K.

Abstract: Bermuda grass showing symptoms of white leaf disease has been observed in the area of Rajasthan, India. The diseased plants showed typical white leaf symptoms, proliferation of auxiliary shoots, bushy growing habit, small leaves and shortened stolons. Using polymerase chain reaction (PCR) amplification with P1/P6 universal primer followed by R16F2n/R16R2 primers, all symptomatic plants tested positively; whereas no amplification product was obtained from non-symptomatic plants. RFLP analysis of PCR products with HaeIII, RsaI or AluI endonuclease generated fragment profiles that were identical for all white leaf samples. The phytoplasma 16/23S intergenic region was sequenced directly with primers R16F2n/R16R2 and compared by BLAST analysis with those of other phytoplasma rDNA sequences archived in GenBank. The highest sequence homology (95%) obtained was with that of ‘Candidatus Phytoplasma cynodontis’ (GenBank Accession No.Y14645), the type member of the BGWL (16SrXIV) group. This is the first molecular identification of ‘Ca. Phytoplasma cynodontis’ (16SrXIV group) associated with Bermuda grass white leaf disease in West India (Rajasthan).

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Isolation, Identification and Media Optimization of Thermostable Alpha Galactosidase producing Geobacillus Sp.

Chavan Sanjay and Sabharwal Sushma

Abstract: Thermophilic alpha galactosidase producing bacteria have been isolated by enrichment of the soil samples with soybean meal for 24 hrs at 60 °C. The isolated strain is aerobic and gram positive growing, optimally at pH 8.0.This bacterial strain was identified as a Geobacillus sp. by its colony morphology and biochemical tests. The growth pattern of the bacteria was studied at various stress conditions. The alpha galatosidase activity was detected in the culture filtrate after 15 hours of incubation period. The bacterial strain used different carbohydrates as the carbon source; however the maximum production of alpha galactosidase occurred when 1.5% (w/v) soybean meal was used. The use of organic nitrogen favored the production of alpha galactosidase in the isolated strain compared to inorganic nitrogen sources.

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Studies on Establishment of Primary Cell Cultures from Hepatopancreatic Tissue of Penaeus Vannamei Post Larvae using Different Biological Fluid Extracts

Rao Jaganmohan P. and Rao Sambasiva K.R.S.

Abstract: A primary cell culture system was developed for the cells of hepatopancreatic tissue of Penaeus vannamei post larvae. Minced tissue fragments of hepa­topancreas were disaggregated with 0.1% trypsin. The cells were seeded at different densities in three different media viz. minimum essential medium (MEM), Hanks balanced salt solution (HBSS) and L-15 medium. Epitheliod like cells were developed in all media. The cultured conditions like time of disaggre­gation, seeding density, temperature, CO2 concentr­ations were optimized. The primary cell cultures were also established by supplementing all the three media with different biological fluid extracts like Fetal bovine serum (FBS), Prawn muscle extract (PME) and Fish fry extract (FFE). Maximum number of viable cells were obtained with 30 minutes disaggregation and when cells were seeded at a density of 106 cells/ml, primary cell cultures were best established in L-15 medium among the three media used with different biological fluids like FBS (20%), prawn muscle extract (PME) and fish fry extract (25%). They were survived for a period of 50 days. Cell cultures were best survived with 5% CO2 and at 300C temperature.

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Rapid and Reproducible ‘Green’ Synthesis of Silver Nanoparticles of consistent shape and size using Azadirachta Indica

Anuradha J., Abbasi Tasneem and Abbasi S.A.

Abstract: Rapid and reproducible biomimetic synthesis of silver nanoparticles was achieved with the aqueous extract of neem (Azadirachta indica). The extract served as reducing as well as stabilizing agent in extracellularly synthesizing silver nanoparticles at ambient temperatures. At stochiometric ratios differing in a fairly wide band, the nanoparticle formation was complete within 4-6 hours and exhibited characteristic plasmon vibration peak at 433±3 nm. It is brought out that reproducible synthesis of nanoparticles of consistent shapes and sizes is achievable in an environment-friendly manner.

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Full Length cDNA derived Novel Peptides belonging to Esculentin Family from Skin of Indian Bronzed Frog Hylarana Temporalis

Reshmy Vimaladevi, Kumar Kesavakurup Santhosh and George Sanil

Abstract: Esculentins constitute the most potent antimicrobial peptides from frog skin. The present study describes the successful isolation of 3 novel esculentin -2 peptides from the skin secretion of Indian bronzed frog Hylarana temporalis. The deduced open reading frames encoding the biosyn­thetic precursors of each esculentin consisted of 76 amino acid residues. Precursors of these novel pept­ides, named esculentin-2TEa, esculentin-2TEb and esculentin-2TEc, from H.temporalis showed 12 amino acid differences than the nearest homologous peptide reported so far. This forms the first report of escue­lentin peptides from Hylarana temporalis, endemic to India and Srilanka.

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Arbuscular Mycorrhizal Symbiosis: An Overview

Jaya Kumari T.R.

Abstract: Arbuscular mycorrhizas formed by obligately symbiotic soil fungi of the Phylum Glomeromycota occurs in the roots of more than 95% of plants belonging to diverse ecological groups in natural as well as disturbed/contaminated habitats. They offer mu­ltit­udes of benefits to host plants including enhanced nut­ri­ent and water uptake, tolerance/ resistance to biotic and abiotic stresses etc. They have recently been identified as the most viable strategy to remediate environmental degradations and contaminations em­an­a­ting from anthropogenic activities. Their taxon­omy is undergoing massive modifications due to molec­ular methods of elucidation of the phylogenetic relationships. The phylum is currently known to comprise approximately 200 species distributed am­ong ten genera. By far the largest AM fungal genus known is Glomus, with more than 100 species. Reports on the incidence of arbuscular mycorrhizas in crops of diverse categories add to the growing body of knowledge that they are ubiquitos, universal and ecologically significant components of the ecosystems.

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