Open Access Short Research Article

In Vivo Safety Assessment of the Plant Growth-Promoting Rhizobacterium Bacillus cereus RS87 and Rhizo-product

Ornrat Lohitnavy, Kanchalee Jetiyanon, Pinyupa Plianbangchang, Sakchai Wittaya-areekul

Biotechnology Journal International, Page 1141-1148
DOI: 10.9734/BBJ/2014/13507

In Vivo Safety Assessment of the Plant Growth-Promoting Rhizobacterium Bacillus cereus RS87 and Rhizo-product

Aims: To assess the acute toxicity and skin irritation potential of a rhizobacterium Bacillus cereus RS87 and the rhizo-product in rats and rabbits.

Study Design:  Adult Wistar rats were gavaged with a single dose of B. cereus RS87 in acute oral toxicity test and were applied with single doses of rhizo-product for 24 hours in acute dermal toxicity test.  New Zealand albino rabbits were applied with 0.5g rhizo-product in acute dermal irritation test.

Place and Duration of Study: Pharmaceutical and Natural Products Department, Thailand Institute of Scientific and Technological Research (TISTR), Pathum Thani, Thailand, between November 2013 and March 2014.

Methodology: Animal toxicity studies were carried out by the methods described in the Organization for Economic Co-operation and Development (OECD) test guidelines. Mortality data of animals were used to determine the median lethal dose (LD50) values after oral and dermal exposures to B. cereus RS87 and the rhizo-product along with distilled water as control. The skin irritation potential of the rhizo-product was evaluated in rabbits. Distilled water was used as a control. The average weight gains were calculated and gross examination at necropsy was performed.

Results: No mortality and no signs of toxicity were observed. The oral LD50 of B. cereus RS87 and dermal LD50 of rhizo-product in rats were greater than 9x108 CFU kg-1 and 15,000 mg kg-1(about 4.5x108 CFU kg-1), respectively. However, significant decrease in mean weight gain in the high-dose groups when compared to controls (21.40+/-1.47 versus 28.40 +/- 0.24 (male); 13.80+/-2.57 versus 20.20+/-0.58 (female)) were reported at day 8 after 24-hour dermal exposure to rhizo-product. No pathological changes in major organs were observed at necropsy.

Conclusion: B. cereus RS87 and the rhizo-product (about 3x107 CFU/g) have low acute toxicity and very low skin irritation potential, which was considered safe for humans. However, adverse effect needed to be further explored in the field experiment or in practical use.

Open Access Original Research Article

Studies on Immobilization of Cutinases from Thermobifida fusca on Glutaraldehyde Activated Chitosan Beads

Krishnamoorthy Hegde, Venkata Dasu Veeranki

Biotechnology Journal International, Page 1049-1063
DOI: 10.9734/BBJ/2014/12558


Studies on Immobilization of Cutinases from Thermobifida fusca on Glutaraldehyde Activated Chitosan Beads

Aims: To evaluate and optimize the activity and stability performance of two recombinant cutinases of Thermobifida fusca, Cut1 and Cut2 on glutaraldehyde activated chitosan beads.

Place and Duration of Study: Biochemical Engineering Laboratory, Department of Biotechnology, Indian Institute of Technology Guwahati, Assam India. Experiment conducted as a partial fulfillment to PhD degree from December, 2010 to January, 2014. 

Methodology: Purified cutinase were immobilized on chitosan beads by covalently coupling with glutaraldehyde. The biophysical properties of immobilized cutinase was analysed by FTIR, FESEM and the operational stability and activity of the immobilized cutinase was studied at different pH and temperature.  

Results: The optimal immobilization was achieved with 3% (v/v) glutaraldehyde activation and coupling pH of 8.5. Under this condition, 74% and 71% of immobilization was achieved for Cut1 and Cut2, respectively. Immobilized cutinase showed optimal activity at pH 8 with optimal functional range of pH 7.5 to 9 and 55ºC with operational stability in the range of 45ºC to 70ºC. The reusability and storage stability was found to be 80% after 10 reuse cycles and 50% after 13 days, respectively as compared to its initial activity. There was no loss in activity even after repeated freeze drying.

Conclusion: the cutinase immobilized on glutaraldehyde activated chitosan beads demonstrated better operational stability in comparison to free cutinase, showing chitosan as a potential support in the enzyme immobilization technology for industrial applications of T. fusca cutinases.

Open Access Original Research Article

Physicochemical, Trace Metal Analysis and Fatty Acid Profile of Python regius Fat

Osheke Shekins Okere, Bamidele Joseph Okoli, Moses Dele Adams, Samuel Omolabi Adeyemo

Biotechnology Journal International, Page 1064-1076
DOI: 10.9734/BBJ/2014/11677

Physicochemical, Trace Metal Analysis and Fatty Acid Profile of Python regius Fat

Physicochemical properties, metal analysis and fatty acid profile were carried out on Python regius fat with a view to ascertaining the immense therapeutic claim by traditional medical practitioners. Physicochemical analysis showed that the fat is lower than that of animal fats: cow fat, swine fat and goat fat. Trace metal analysis indicated that it contains 10000ppm Sodium, 620.00ppm Calcium, 94.26ppm Magnesium and 114.00ppm Iron. Python regius fat was found to contain 62% monounsaturated fatty acid, 10% polyunsaturated fatty acid and 28% saturated fatty acid. These results indicated that Python regius fat is a rich source of monounsaturated fatty acids; the healthy edible fat and can also be a good raw material for the soap and cosmetics industry. The results of the study showed that the fat has acid value and therefore less susceptible to rancidity. The saponification value of the fat is 164.09 mg KOH g-l showing its high triglyceride content, indicating its potential usefulness in the soap making industry. Its mineral content makes it a potentially useful source of electrolytes, which function in cellular activities such as enzyme action, muscle contraction, nerve action, blood clotting and water balance. The iodine value of 97.2 mg KOH g-l also indicated a fairly high amount of unsaturated fatty acids. The higher the iodine value, the greater the degree of unsaturation and the greater the susceptibility to oxidative rancidity. Overall, we therefore recommend the fat for use due to its diverse medicinal and industrial potentials.

Open Access Original Research Article

Searching for Algaecide or Algaestatic Effects of Several Plant Extracts on Phytoplankton: Preliminary Results

Conceição Fernandes, Sandra Barros, Victor Galhano, Ana Maria Geraldes

Biotechnology Journal International, Page 1077-1087
DOI: 10.9734/BBJ/2014/12898

Aims: Evaluate the in vitro effects of essential oils and water extracts of Laurus nobilis, Rosmarinus officinalis, Mentha suaveolens and Fraxinus angustifolia on the growth of Anabaena cylindrica and Chlorella vulgaris.

Study Design:  Experimental research.

Place and Duration of Study: The cyanobacterium Anabaena cylindrica and the green alga Chlorella vulgaris were used as test strains to evaluate the effects of plant extracts on algal growth. All experiments were undertaken in the Agricultural School of Bragança - Polytechnic Institute, from September 2010 to July 2011.

Methodology: Essential oils were obtained by means of hydrodistillation of the plants. The oils and the water that remained, after the hydrodistillation, were further used for the growth screening of Anabaena cylindrica and Chlorella vulgaris under axenic cultures. Both types of extracts were tested at different concentrations. The essential oil effects were evaluated by disc diffusion method and water extracts effects were evaluated in batch cultures.

Results: Essential oils had an algaecide effect in all tested concentrations (1:1; 1:3; 1:4 and 1:10) for both algal strains. Contrarily, none of the water extracts evidenced a complete algaecide effect. Nevertheless, promising results were obtained with      rosemary water extract since the highest concentrations (1:4) had an algaestatic effect on C. vulgaris. Conversely, the observed effects on A. cylindrica varied from cellular density decrease to an algaestatic effect. Therefore, the tested algal strains presented distinct responses to both extract types and concentrations.

Conclusions: Comparing the different extracts’ activity, it can be concluded that essential oils mostly influenced algal growth.

Open Access Original Research Article

In vitro and in silico Approach to Evaluate the Urease and Collagenase Inhibitory Activity of Embilica officinalis Gaertn Fruit

Sheema Bai, Anupma Malik, Leena Seasotiya, Pooja Bharti, Sunita Dalal

Biotechnology Journal International, Page 1088-1104
DOI: 10.9734/BBJ/2014/12339

In vitro and in silico Approach to Evaluate the Urease and Collagenase Inhibitory Activity of Embilica officinalis Gaertn Fruit

Aim: The key virulent factors of bacteria are enzymes. Urease and collagenase enzyme play a vital role in pathogenesis of wide array of bacterial strains and cause numerous diseases. So the aim of present study was to find out the potent drug candidate from Emblica officinalis Gaertn. fruit for these pathogenically important enzymes.

Study Design: A study was done to screen out the bacteria producing urease and collagenase from a stack of 19 bacterial strains and the positive strains were checked for their susceptibility to methanol and ethyl acetate extracts of Emblica officinalis Gaertn. fruit. Further extracts were investigated for their potential to antagonize these enzymes.

Place and duration of study: Department of Biotechnology KUK, Jwahar Lal University, Delhi between February 2012 and December 2013.

Methodology: Screening of bacteria and their susceptibility to methanol and ethyl acetate extracts of E. officinalis was done by using agar diffusion assay. Further investigation of extracts to antagonize urease and collagenase enzymes was checked by using phenol hypochlorite and gelatin diffusion assay respectively. GC-MS analysis, docking and ADME studies were conducted to screen for plant-based urease and collagenase inhibitors.

Results: Methanol extract inhibited Jack bean urease enzyme (IC50:0.74 mg/ml) more potently than collagenase Type 1 (IC50:1.13 mg/ml), while ethyl acetate extract inhibited collagenase completely (IC50:4.19 mg/ml) and was observed to be more effective than methanol extract (IC50:5.51 mg/ml). GC-MS analysis revealed an array of 28 and 30 compounds in methanol and ethyl acetate extract respectively. In silico study identified xylenol and erucylamide as active compounds of E. officinalis having good binding score with better ADME properties compared to standard compounds.

Conclusion: So our observations find application for the consideration of E. officinalis compounds for further validation towards development of effective drugs against these significant bacterial enzymes.

Open Access Original Research Article

Assessment of Roof Harvested Rainwater in Parts of Anambra State for Environmental Pollution Monitoring

J. N. Chukwuma, V. C. Nnodu, A. C. Okoye, E. C. Chukwuma

Biotechnology Journal International, Page 1105-1114
DOI: 10.9734/BBJ/2014/11876

Assessment of Roof Harvested Rainwater in Parts of Anambra State for Environmental Pollution Monitoring

Questions have been raised about the quality of roof harvested rainwater which has been shown to be temporally and spatially variable and at times not in compliance with drinking water guidelines. This study is therefore an investigation on the quality of roof harvested rainwater consumed by rural communities in parts of Anambra State Nigeria. The physico-chemical and microbiological parameters of the rainwater samples collected in the study area were analyzed with the view of determining the level of contamination as a result of anthropogenic activities in the study area. Roof harvested rainwater were collected from three stations, and a free-fall harvested rainwater sample was used as a control. The result shows that the physico-chemical parameters were all within permissible water quality standard as recommended by National Agency for Food and Drug Administration Control (NAFDAC) except for the presence of particles and for the micro-biological properties which were found quite unsatisfactory. Pearson Correlation Matrix of physico-chemical properties conducted indicated a strong positive correlation between Zinc and Iron which emphasizes common pathway and origin. The water samples were assessed using Water Quality Index (WQI), the WQI for the station 1, 2 and 3 were 71.68%, 60.19%, and 77.55% respectively. Low-cost microbial disinfection such as solar disinfection and pre-filtration or otherwise the proper maintenance of the entire Rain Water Harvesting (RWH) system could make the harvested roof rainwater potable for the study area.


Open Access Original Research Article

Antibacterial Activity of Ocimum gratissimum (Nchu-Anwu) and Vernonia amygdalina (Bitter-Leaf) Antibacterial Activity of Ocimum gratissimum (Nchu-Anwu) and Vernonia amygdalina (Bitter-Leaf)

A. U. Opara, R. C. Egbuobi, J. N. Dike Ndudim, C. E. Onyewuchi, J. K. Nnodim

Biotechnology Journal International, Page 1115-1122
DOI: 10.9734/BBJ/2014/11995

Antibacterial Activity of Ocimum gratissimum

(Nchu-Anwu) and Vernonia amygdalina

(Bitter-Leaf) Antibacterial Activity of

Ocimum gratissimum (Nchu-Anwu) and Vernonia amygdalina (Bitter-Leaf)

This study was carried out to evaluate the antibacterial activity of Ocimum gratissimum and Vernonia amygdalina on selected bacterial species, as well as comparing the antibacterial activities of their fresh and dried leaves. Fresh leave samples of the plants were collected from Okwu, Uratta in Owerri North, Imo State, Nigeria. The plants were identified and ethanolic and aqueous extractions of the dried and fresh leave samples were done using standard procedures. Antibacterial activity of the extracts was tested invitro against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyogenes using agar diffusion, punch method. The pattern of inhibition varied with plant extract, solvent used for extraction and test organisms. For the dried leave samples, all the extracts showed zones of inhibition above 5mm diameter against all the isolates, except hot water extract which had no activity for all. Ethanol extracts showed the highest zone of 20mm of O. gratissimum against Staphylococcus aureus and highest zone of 40mm by V. amygdalina against Pseudomonas aeruginosa. However, for the fresh leave samples, only Ethanolic extract of O. gratissimum was active against all the isolates. None of the fresh leave sample extracts of V. amygdalina was active against all the isolates, except the cold water extract which showed zones of inhibition of 10mm against E. coli and Streptococcus pyogenes each. It was also observed that Staphylococcus aureus had the lowest Minimum Inhibitory Concentration (MIC) of 0.48µg/ml and 0.82µg/ml against O. gratissimum and V. amygdalina respectively, and Minimum Bactericidal Concentration (MBC) of 0.28µg/ml and 0.61µg/ml respectively. From this work, it has been noted that the leaves of Ocimum gratissimum and Vernonia amygdalina have antibacterial properties. Government is therefore advised to invest money into more pharmacological researches on these extracts in order to make them better alternative to modern medicine for the treatment of infection caused by these pathogens.

Open Access Original Research Article

Antibacterial Effect of Pseudomonas aeruginosa Isolated from a Moroccan Hot Spring Discharge and Partial Purification of its Extract

Ilham Zahir, Abdellah Houari, Saad Ibnsouda

Biotechnology Journal International, Page 1123-1140
DOI: 10.9734/BBJ/2014/13406

Aims: The focus of this study was to isolate and to identify microorganisms possessing an antibacterial activity followed by a partial purification of their extracts.

Study Design: Screening and identification of bacteria with an antibacterial effect were performed and active substances responsible for the biological activity were localized and partially purified.

Place and Duration of Study: The study was carried out at laboratory of Microbial Biotechnology, Department of Biology, Faculty of Sciences and Technical, University Sidi Mohamed Ben Abdellah, BP 2202, Road of Immouzer, Fez, Morocco, during the period from February 2010 to August 2010.

Methodology: Samples of a hot spring discharge localized in the city Fez Morocco were explored to isolate compounds-producing microorganisms. The inhibitory spectrum of the isolate was evaluated against M. smegmatis, M. aurum, S. aureus, S. haemolyticus, B. subtilis, B. licheniformis, B. amyloliquefaciens, E. coli DH5α and Erwinia chrysanthemi by using agar well diffusion test and/or a modified spot-on-lawn assay. Identification of strain was executed on the basis of Gram stain, biochemical characteristics and PCR followed by DNA sequencing of 16S ribosomal RNA gene. Crude extract of the isolate was obtained by using ethyl acetate and was exposed to proteolytic enzymes (pepsin and proteinase K) and to heat treatment at 121°C (60 min), 100°C (20 min), 80°C (30 min), 37°C (3h) and kept at 4°C (six months). The antimycobacterial effect before and after every treatment was assessed by agar well diffusion method. Synthesis of antibacterial compounds was monitored during the isolate growth cycle. The extract was then fractionated by thin layer chromatography and the bioactivity was investigated with a bioautography technique followed by spots elution test.

Results: One bacterium was isolated having a broad antagonistic effect against all the tested bacteria. Based on biochemical characterization and 16S rDNA sequence analysis, the strain was identified as Pseudomonas aeruginosa. The antibacterial compounds were synthesized during the exponential growth phase and were not affected following heat treatment and proteases that indicated the non-proteinaceous nature of the active agents. The crude extract developed in chloroform: acetone (9:1) showed metabolite (s) at Rf = 0.68 which it may be pyocyanin, inhibiting the growth of M. smegamtis.

Conclusion: Metabolites of P. aeruginosa responsible for the sought effect were localized and characterized. These compounds might provide an alternative bio-resource for the bio-control of plant pathogens after their total purification in further investigation.