Research Journal of Biotechnology

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Vol. 14(9) September 2019

Isolation, screening, identification and optimization of growth conditions of glucose oxidase producing microbes from various sources

Singh Sujeet Pratap, Modi Dinesh Raj and Tiwari Rajesh Kumar

Page No: 1-17

Abstract: This work aimed to isolate and screen glucose oxidase producing microorganisms from samples collected from seven various sources and determining the ability of the screened organisms to produce glucose oxidase. The isolated and screened organisms were further identified as Pseudomonas Spp. and Actinomycetes Spp. by biochemical characteristics and phylogenetic analysis of 16 sRNA genes extracted from the isolates.

A total of 52 isolates were isolated from sources like plant sap, spoiled plant remains, cane molasses, soil from rice field and poultry farm, spoiled onion sap and spoiled sweet potato. Two strains showed glucose oxidase presence when tested. Molecular identification was established by 16 sRNA analyses. Optimal growth conditions of the two strains were taken to experimental setup and confirmed by the maximum amount of growth obtained in presence of several sources of carbon, nitrogen, various pH levels, temperature and numerous substrates required for growth.

Agrobacterium-mediated β-glucuronidase gene transformation in Espand

Hiva Mohammad Nejhad, Hedayat Bagheri and Aso Nobakht Vakili

Page No: 18-23

Abstract:Espand (Peganum harmala) is a valuable medicinal plant with antioxidant, antimutagenic and anticancer properties. To increase certain substances in cells, metabolic engineering is a powerful tool which needs an optimized regeneration and transformation protocol. The aim of current study was to set up a regeneration and transformation protocol for Espand. Hypocotyls of three Iranian Espand cultivars (Yazd, Amol and Uremia) were cultured on MS medium supplemented with different concentration of BA (0, 1, 2 and 3 mg l-1) and NAA (0, 0.1 mg l-1). The highest frequency of regeneration was achieved by Amol ecotype with 93% shoot regeneration on 0.01 mg l-1 NAA and 2 mg l-1 BAP medium.

For transformation, Amol hypocotyls were inoculated with Agrobacterium tumefaciens strain LBA4404 (pBI121) which contained neomycin phosphotransferase (nptII) and an intron-containing β-glucuronidase (GUS) genes. Kanamycin-resistant shoots were successfully regenerated on selection medium. An overall transformation frequency of 37% was achieved and confirmed by polymerase chain reaction. T1 segregation confirmed the genotypic ratio of 1:2:1 based on NPTII selectable marker gene. Also, GUS expression of randomly green selected T1 transgenic plants confirmed stability of transgene.

Targeted Metabolic Profiling of Black Turmeric (Curcuma caesia Roxb.) Accessions for Industrially Important Compounds

Tamang Deeki L., Manivannan S., Upadhyay Sujata and Roy Biswajit G.

Page No: 24-28

Abstract: Black turmeric accessions collected from two regions of Himalayas showed a high difference in volatile oil content as well as the metabolite content. Highest amount of volatile oil 24.91% was obtained in accessions from Arunachal Pradesh. A positive correlation was also observed between the rhizome colour and volatile oil content. Analysis of the volatile oil using High Performance Liquid Chromatography (HPLC) revealed that in majority of the accessions, Linalool (up to 52.12%) followed by Ocimin (up to 32.45%) and Camphor (up to 16.95%) contents were higher than the other components. Our targeted profiling approach grouped the accessions into four different chemo types viz. high camphor, high 1, 8 cineole and curcumin, high ocimin and high linalool groups.

Two-way Hierarchical Cluster Analysis (HCA) revealed the relationship among the accessions as well as metabolites analyzed. From the study it emerged that the accessions viz. BTAR-05 and BTAR-04 rich in linalool and BTAR-06 and BTAR-05 rich in ocimin are the promising chemo types which could be industrially utilized.

Erythromycin and penicillin toxic post-products management using Pleurotus ostreatus

Sośnicka Agata, Lubiński Olgierd and Turło Jadwiga

Page No: 29-36

Abstract: The purpose of this research was to investigate the possibility of using post-products of antibiotic fermentation process as components of Pleurotus ostreatus fungus culturing media in order to find a suitable way of their utilization. Some analytical methods were applied to determinate the post-product contents of: a dry substance (drying in the moisture analyser), ash (roasting in a quartz crucible), calcium (ASA method), nitrogen (Dumas-Unterzaucher method), protein (Kiejdahl method), fat (Soxhlet method), amino acids (the volume method) and sugar (Schoorl method). Also, the antibiotic activity was evaluated (agar disk-diffusion method).

The research compares the effects of different concentrations of antibiotic fermentation post-products used to breed the fungus in shake-flasks cultures and in bioreactors on the fungus growth and ability to degrade the waste material.

Analysis of the Co-expression of genes and Gene Expression Pattern of co-expressed genes in Sinorhizobium meliloti strain 1021

Mathur Manisha

Page No: 37-42

Abstract: In the present study, standard statistical algorithms and bioinformatics approaches are being used to do clustering of gene expression data groups in the Sinorhizobium meliloti strain 1021. Comprehensive phylogenetic analysis using clustered data has also been included. This investigation of gene expression of Sinorhizobium meliloti strain 1021 demonstrates that genes with important functions can vary in their expression levels. Output, which is displayed graphically, conveys the clustering of co-expressed genes. These results can be beneficial to characterize regulatory patterns by transcription factor binding sites which govern the process of transcriptional regulation.

Characterization and applications of protease inhibitor compound from marine yeast Candida orthopsilosis KSSB3

Banerjee Somak, Pandey Koushik, Mazumdar Soumadipta, Sarkar Anwesha and Bhaskara Rao K.V.

Page No: 43-52

Abstract:The current study was planned to screen the marine yeasts for the protease inhibitor production and to investigate its antibacterial and antioxidant activity. Among the 30 marine yeast isolates recovered from the backwater of Port Blair, Andaman-Nicobar Islands, only 4 isolates showed positive protease inhibition activity. The crude extract of the isolate KSSB3 showed 62.51% inhibition activity while the ethyl acetate extract of this isolate KSSB3 showed 59.8% inhibition activity. Medium optimization studies were carried out for better protease inhibitor production by altering various physical and chemical parameters like temperature, pH, salinity, different carbon and nitrogen sources. The optimum condition was observed at 37°C, pH 2, 15% NaCl concentration, glucose as a carbon source and casein as a nitrogen source. Structural elucidation of the potential compound was performed using HPLC, FT-IR and GC-MS analysis.

Antioxidant activity was performed to check the free radical scavenging activity of the compound. It showed good antioxidant activity in DPPH, metal chelating and hydrogen peroxide scavenging assays. Antibacterial activity was also checked for the isolate KSSB3 where it showed good antibacterial activity (22 mm) against Staphylococcus aureus. The potential yeast isolate KSSB3 was identified as Candida orthopsilosis KSSB3 strain through 18s rRNA sequencing.

Phenotypic and Genotypic Characterization of Rice (Oryza sativa L.) Genotypes for Root Morphology and Yield under Drought Stress

Dixit Shikha, Jangid Vinod Kumar, Shashidhar H.E. and Vijayakumar Swamy H.V.

Page No: 53-63

Abstract:Rice (Oryza sativa L.) is a major food crop for the world’s population which is affected adversely due to drought stress. Our investigation aimed at phenotypic and genotypic characterization of 20 rice genotypes for drought tolerance, shoot-root growth rate, root morphology and yield attributes under drought condition. Genotypes selected for study performed well under drought condition, genotypes like AM-FS-58 and AM-FS-163A exhibited lower yield loss, robust root morphology and higher shoot-root growth rate under drought stress. Genotypic characterization with SSR markers and diversity analysis of these genotypes revealed a high level of polymorphism and diverse genetic composition.

Genotypes exhibited polymorphism for SSR markers RM302 and RM212 earlier established to be associated with drought tolerance13. Thus, many of the genotypes in our study can be used as donor parents for identification and introgression of QTL for drought tolerance and robust root morphology.

Functional and structural characteristics of extruded snack product incorporated with fish powder from a lean fish

Ganesan P., Rathnakumar K., Brita Nicy A., Velayutham P., Jeyashakila R. and Anand S.

Page No: 64-68

Abstract: Extruded snack products were prepared using corn flour, rice flour, dhal, pepper, salt and water. 8% of lizardfish powder was incorporated into the extruded snack product. FTIR spectra of ingredients and extruded products were analyzed. The peaks obtained in the spectra were assigned to different functional groups and the shifts in peaks were studied. A random conformation of the protein was evident but not an aggregation of polypeptides in the extruded products.

There was no indication of retrogradation in any of the snack products, meaning good retention of the nutrients in the product and lower degradation. Microstructure analysis of control extruded snack and extruded snack with lizard fish powder was performed by Scanning Electron Microscopy (SEM). All the extruded snack products had porous, open-celled structures. All the extruded snack products had damage and breaking in continuous symmetrical structure. Large numbers of flattened and sheared granules were also seen in all the extruded snack products.

Beneficial effects of mild heat stress on the primary culture of mouse prefrontal cerebrocortical neurons

Mane Narayan R., Gajare Kavita A. and Deshmukh Ashish A.

Page No: 69-76

Abstract: In our recent investigations, we found that mouse prefrontal cerebrocortical neurons when exposed to mild heat stress of 38oC for 30min on every alternate day up to 6th day of in vitro life resulted in significantly lower accumulation of neurofibrillary tangles, senile plaques, lipofuscin granules and with well-preserved Nissl substance. In the present investigations, we have studied the effect of mild heat stress on neurodegenerative cytological alterations such as nuclear fragmentation, nuclear condensation, cytoplasmic vacuolation, apoptotic blebs, loss of dendrites, loss of axon hillock, intra-axonal aggregates, axonal swelling, loss of neuronal network in the primary culture of mouse prefrontal cerebrocortical neurons from E17 embryos.

The neurons from the experimental group were subjected to mild heat stress of 38oC for 30 min on every alternate day up to 6th day of culture and the neurons from the control group were maintained at 37oC till the end of the experiment. The neurons from the mild heat stressed group were healthier in neuronal cytoarchitecture than the neurons from the control group with significantly (p<0.001) lower incidences of the neurodegenerative alterations, reiterating that the mild heat stress exerts beneficial effects in maintaining the neuronal cytoarchitecture to youthful condition.

Seasonal dynamics of biochemical and mineral components of Himalayan cyprinid Schizothorax richardsonii (Gray, 1832)

Sharma Sharali and Singh Deepak

Page No: 77-82

Abstract: Fishes are the essential source of animal protein. Besides protein, other constituents include fats, carbohydrates and minerals. S. richardsonii (Gray, 1832), locally named 'Asela' is a common freshwater Himalayan cyprinid fish constituting about 70% of total catch in most of the Himalayan rivers, thus representing the most demanding food fish by local inhabitants of the region. Therefore, the present study is aimed at to access the biochemical composition and mineral status of Schizothorax richardsonii (snow trout). The fresh fish samples were collected from the Alaknanda, a major Himalayan river and its tributary.

Total protein, total carbohydrate, total lipid, total moisture and mineral contents were analyzed. Maximum percentage of total protein content (21.86%) was observed during summer while total carbohydrates (2.04%) and total moisture content (78.77%) were highest during monsoon season. Lipid content was highest during summer (2.21%) and monsoon season (2.21%). Minimum amount of protein (12.29%), carbohydrates (1.32%) and lipids (1.63%) were found in winter season while minimum (76.09%) moisture content was found during summer season. S. richardsonii was also found to contain important macro and micro mineral elements. Calcium was found to be highest among macroelements while iron was found among microelements. Food availability and seasonal environmental changes were attributed to influence the biochemical components in the muscle tissue.

Effect of gibberellic acid on in vitro flowering from stem node explant of Anubias barteri var. Nana

Barpete Surendra, Muhammad Aasim, Özcan Sancar Fatih and Özcan Sebahattin

Page No: 83-88

Abstract:Anubias barteri is a commercially important aquatic ornamental plant that has low multiplication rate and in vitro regeneration difficulties. Therefore, lack of scientific information is available. To increase the knowledge and function of plant growth regulators on Anubias regeneration, stem nodes explants were cultured in half and full-strength Murashige and Skoog media containing Gibberellic acid (GA3), Benzylaminopurine (BAP) and Naphthalene acetic acid (NAA). The used plant growth regulators significantly (p≤ 0.05) affected number of axillary shoots induction, shoot length, flower induction and number of flowers per explant.

The growth pattern of in vitro regenerated axillary shoots was indicated from the shoot base of the stem node. Maximum number of axillary shoots (4.25±0.38), shoots length (2.63±0.21) and flowers (4.0) per explants were achieved on full strength MS medium supplemented with 1.00 mg L-1 GA3, 0.10 mg L-1 BAP, 0.10 mg L-1 NAA and 2 g L-1 activated charcoal. The MS medium without growth regulators did not induce flower that showed the necessity of growth regulators for in vitro flowering. The root induction was statistically similar on half and full-strength MS medium containing different combination of applied growth regulators. The maximum root induction (91.66%) was noted on MS medium containing 0.25 mg L-1 GA3, 0.10 mg L-1 BAP and 0.10 mg L-1 NAA. All regenerated plantlets were successfully acclimatized in tap water filled aquarium. The present results showed importance of plant growth regulators that control growth, morphogenetic processes and micropropagation of Anubias plant.

Effect of High Hydrostatic Pressure Treatment on the Microbial Contamination and on some Chemical and Physical Properties of Minced Chicken

Hamad Siddig H., Aleid Salah M. and Aljasass Fahad M.

Page No: 89-95

Abstract:High hydrostatic pressure processing (HHPP) was tested as means for controlling microbial contamination of chicken meat under the harsh Saudi environmental conditions. Samples of minced chicken were vacuum-packaged and pressure treated at 300, 400, 450 and 500 MPa using a Stansted apparatus. Treated and untreated samples were then stored at 3°C and microbial content as well as some chemical and physical properties were monitored. The microbial load of the untreated samples reached the spoilage level of 107 cfu/g in about one week, resulting in bad smell and dark brown color. The pressure treatments reduced total bacterial counts by 1.8 to 3.2 log10 cycles and reduced counts of Enterobacteriaceae and Salmonella to non-detectable levels. The color of meat was slightly affected but pH, moisture content and the oxidation products of lipids were not substantially changed.

The treatment killed mainly gram-negative bacteria but also caused sub-lethal injury to part of the population resulting in prolonged lag phase. The population not killed by the 350 to 450 MPa treatments grew relatively slowly during storage and its load reached spoilage level in 4 to 6 weeks, while the load of the population treated at 500 MPa did not reach this level till the end of a storage period of 9 weeks. Interestingly, no signs of spoilage appeared on the pressure treated samples even when the microbial load reached the expected spoilage level. It can be concluded that HHPP can be successfully used for the control of microbial contamination of foods under Saudi and similar environmental conditions.

Cytotoxic evaluation of the GST gene expression with nano-CuO using molecular-beacon based approach for cell imaging

Rangarajulu Senthil Kumaran

Page No: 96-101

Abstract:The nanoparticle of the copper oxide (CuO) metal oxide has recently garnered much attention regarding its application as a novel contrast agent for the molecular imaging of cellular-process detection. Using molecular-beacon technology, the cytotoxicity of the CuO nanoparticles that are involved in the expression of the reactive oxygen species (ROS)-related glutathione S-transferase (GST) gene in living cells, was assessed. Monodisperse CuO nanoparticles of 40-nm size were tested at different concentrations (30, 60 and 150 µg/ml) and incubation times (12, 24 and 48 hr) with a human-cancer cell line. A molecular beacon was hybridized with the live-cell-based GST genes and these were examined using a confocal microscopic and fluorometric analysis.

The cellular-level cytotoxic-evaluation method produced consistent results that show that the GST gene expression was maximized for the 24-hr incubation at the CuO-nanoparticle concentration of 150 µg/ml. The cytotoxicity effect of the CuO nanoparticle, however, is not significant compared with the control experiments, thereby demonstrating its high potential in terms of its possible diagnostic and therapeutic use in nanomedical applications.

Effect of Plant Growth Regulator and Explant Types on in vitro Callus Induction of Gynura procumbens (Lour.) Merr

Amin Nurokhman, Hanik Faizah, Sugiharto, Edy Setiti Wida Utami and Yosephine Sri Wulan Manuhara

Page No: 102-107

Abstract:Gynura procumbens (Lour.) Merr is a medicinal plant of Asteraceae. The plant is a potent compound as pharmaceutical raw material which can be increased through plant tissue culture. This study aimed to determine the effect of various combinations of plant growth regulator and explant types on induction callus. The explants used were leaf, stem node, stem internode and petiole while the plant growth regulator used were 0.1 mg/L 2,4-D and 0.1 mg/L BAP, 0.5 mg/L 2,4-D and 1.0 mg/L Kinetin, 0.5 mg/L NAA and 0.5 mg/L BAP, 5.0 mg/L 2,4 D and 0.5 mg/L BAP, 0.1 mg/L 2,4-D and 0.1 mg/L IAA. The explants were cultured in MS medium supplemented with 30% sucrose and 8% agar for 28 days.

The results of this study indicated that the treatment of 0.5 mg/L NAA and 0.5 mg/L BAP on the petiole explants was the best combination of plant growth regulators to produce the highest callus fresh and dry weights (1478.1 mg and 40.0 mg respectively). Callus derived from leaf, petiole and internode explants was friable and compact in texture while node explant-derived callus was compact in texture.

Effect of time-course salt stress on Chlorophyll, Proline and Catalase activity in Solanum lycopersicum L.

Sivakumar J., John Elia Prashanth P., Rajesh N. and Osman Basha P.

Page No: 108-116

Abstract:Salt stress restricted the plant development through osmotic and ionic imbalance. Effect of salt stress on tomato plants happens in two phases: osmotic stress which inhibits growth of juvenile leaves in a rapid phase and ionic stress accelerating senescence of mature leaves in a slower phase. The tolerance threshold of plants differs and considerably depends on species and even cultivar in a species. To understand the response of tomato plants to salt stress, the osmoprotectants (proline), catalase and total chlorophyll content variables were evaluated using control (0 mM), 20 mM, 50 mM, 75 mM, 100 mM, 125 mM and 150 mM of NaCl treatments for 12 h, 24 h and 36 h at seedling stage and vegetative stage. The total chlorophyll content and catalase activity evaluation concluded that no significant (P<0.01) change was noted during short term salt stress. However significant change in proline was noted in the germplasm TG1, TG9 and TG10 but not in the TG7 and TG8 germplasms.

In addition, seed germination percentage was also studied and the results demonstrated that most of the germplasm showed 100% seed germination up to 50 mM salt stress whereas at 150 mM salt stress, the seeds of the TG3, TG7 and TG8 germplasm showed better germination, but the germplasms TG1, TG9 and TG10 were not germinated.

A simplified in-planta genetic transformation in soybean

Phetole Mangena

Page No: 117-125

Abstract:This review paper provides a succinct discussion on the potential use of in-planta transformation technique as an alternative for soybean genetic improvement. It discusses beneficial points, simple and straight forward procedures using Agrobacterium tumefaciens without the use of plant tissue culture conditions. Although, in-planta transformation does not regenerate multiple transgenic micro shoots, as often observed under in-vitro culture conditions, the approach has proved to significantly improve the number of glufosinate resistant soybean plants. This approach has been far neglected, with very few reports compared to in-vitro based transformation which is typically achieved through plant tissue culture.

The in-vitro based procedure follows several steps including explant tissue preparation, explant infection, co-cultivation, regeneration, selection and acclimatisation of transformed plantlets. It requires longer culture periods, aseptic conditions and the procedure is often challenged by problems such as bacterial overgrowth, poor regeneration frequencies, genotype specificity and contaminations. Some of these steps remain entirely optional and often unnecessary for plant transformation in-planta.

A review on in vitro propagation of miraculous physician Aloe vera (L.)

Sivakumar P., Ashok kumar K., Rajalakshmi K., Sangeetha R. and Muthumalai M.

Page No: 126-130

Abstract:Aloe vera (L.) is certainly a wonder herb with all its medicinal, pharmaceutical and cosmetic properties. Conventionally it is propagated by vegetative but obviously; it does not have as much immense potential as micropropagation. Therefore, investigation of most efficient in vitro propagation protocol for Aloe vera is essential. Several researchers successfully standardized the in vitro regeneration protocol in Aloe vera using different explants and different media compositions. This review article carefully reviewed the previous scientific information and gave the suitable pathway for successful development of high regeneration protocol in Aloe vera.

We suggested that the shoot tip is ideal explant for Aloe vera micropropagation rather than the other explants tried for regeneration. BAP and NAA are the two commonly used plant growth regulators for the successful micropropagation of Aloe vera with high multiplication rate. The most successful concentration of media is found to be MS medium containing 0.5 mg/l BAP and0.5 mg/l NAA. The most common constraint faced is browning of explant which is found to be overcome by using polyvinylpyrrolidone (PVP) and proline.

L-DOPA, a promising Pro-Drug against Parkinson's Disease: Present and Future Perspective

Naha Ananya, Jha Santosh Kumar and Singh Hare Ram

Page No: 131-139

Abstract:L-DOPA is the drug of choice for Parkinson’s Disease which is characterized by the decreased levels of dopamine in the brain. This review summarizes the sources of L-DOPA, the bioprocess involved in the production of L-DOPA that includes various design parameters, the advantage of enzymatic synthesis over the chemical synthesis. Certain foods were found to contain L-DOPA which can cure the disease much faster than conventional medication. The mode of action has also been discussed along with its enzyme inhibitors.

Various extraction methods have been compared and reviewed along with its efficiency, yield and purity of L-DOPA. The application of L-DOPA holds true in every field including therapeutics and herbicidal effects in case of weeds. Recent researches are being focused on the L-DOPA therapy for the treatment of Alzheimer’s disease.