Research Journal of Biotechnology

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Research Journal of Biotechnology

Antioxidant activity of ethanol extract of Poncirus fructus immaturus

Hui-Fang Guo, Kandasamy Saravanakumar, Myeong-Hyeon Wang and Hye-Young Kim

Page No: 1-4

Abstract: The present study investigated the antioxidant activity of ethanol extracts derived from Ponciri fructus immaturus using various in vitro antioxidant assays. Poncirus fructus immaturus samples were pulverized and extracted using 70% of ethanol and it was concentrated using a rotary evaporator. The concentrated samples were dissolved in respective solvent for antioxidant tests including DPPH radical scavenging, superoxide radical scavenging, metal-chelating reducing power and total antioxidant assays. Analytical results revealed that ethanol extract of Poncirus fructus immaturus contains the phenolic content of 60.81 mg TAE.g-1 and flavonoid content of 17.50 mg QE.g-1.

Further, same extracts exhibited the concentration dependent antioxidant activity. Among the different level of concentrations tested, 800 μ of Poncirus fructus immaturus extract bared the higher 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide radical scavenging, metal-chelating, reducing power and total antioxidant activities. Present findings revealed the importance of Poncirus fructus immaturus as a potent source for antioxidant ingredient.

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Isolation of benzene tolerant bacteria and Production of organic solvent stable enzymes from Bacillus species

Periasamy Anbu and Jae-Seong So

Page No: 5-11

Abstract: In the present study, the organic solvent stable protease and lipase producing bacterial strains were isolated from soil by benzene enrichment media. The positive strains were identified as Bacillus sp. PAP08 (Protease) and Bacillus subtilis LAP04 (lipase) by 16S rRNA sequencing method. The high level of protease and lipase was produced at 48 h of incubation. In addition, the enzyme stability was checked in different concentrations of benzene (1, 5, 25 and 50%).

The result showed that the protease was completely stable and the activity was stimulated in all the tested concentration of benzene. The lipase activity was completely stable and enhanced up to 25% benzene. However, the enzyme activity was inhibited by 18% at 50% benzene. These organic solvent stable enzymes from Bacillus species may be useful for industrial applications in the presence of non-aqueous media.

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Evaluation of pathogenicity level of three different strains of Fusarium oxysporum in Musa acuminata cv. Berangan

Umaiyal Munusamy, Yusmin Mohd-Yusuf, Kamilatulhusna Zaidi, Nadiya Akmal Baharum and Rofina Yasmin Othman

Page No: 12-20

Abstract: Fusarium oxysporum is one of the most adaptive and pathogenic fungus that causes wilting in banana. It is regarded as one of the economically destructive fungus and control of this disease is always ineffective once the disease becomes established in banana plantations. Various researches have been carried out, but sustainable solution is yet to achieve. In this study, we identified the infection phenotypically through LSI and RDI which can be only done after 5th-week of post inoculation. With qPCR application, we identified the infection level much faster within 2 to 96 hrs. Disease severity index obtained from LSI and RDI value showed that Berangan is highly susceptible towards C1 HIR, 9888 and 2296.

Meanwhile, qPCR analysis besides showing infection pattern as early as 2 hrs in Berangan was infected with C1 HIR, 9888 and 2296. We also found that additional information such as specific genes were expressed in the Berangan infected with C1 HIR, 9888 or 2296. This information can be used to identify the fusarium strain that is actually infecting Berangan. This could help planters to identify early infection of fusarium as well as strain recognition.

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Molecular characterization and identification of virulence genes in Escherichia coli isolated from patients with urinary tract infection

Alsanie Walaa, Felemban Ebaa, Hassan Mohamed and Gaber Ahmed

Page No: 21-30

Abstract: The aim of this study was to analyze the molecular characteristics and survey the frequency of antibiotic and virulence gene genotypes in Escherichia coli isolated from hospitalized patients with urinary tract infection and to determine their antibiotic resistance patterns. A total of 250 E. coli strains were isolated from urine specimens obtained from suspected cases with urinary tract infections at different hospitals located in Taif city, Saudi Arabia. Antibiotic susceptibility testing for these isolates was executed using the disk diffusion method. The frequency of different virulence genes expressed in E. coli was detected by polymerase chain reaction (PCR) assay. Repetitive sequence repeat PCR (Rep-PCR) was used for genotyping all isolates. In addition, phylogenetic grouping was also performed on E. coli isolates.

Out of 250 isolates, 42 isolates (16.8%) belonged to the emerging pathogens such as necrotoxigenic Escherichia coli (NTEC). The prevalence of the genes Afa, pyelonephritis associated pili (pap genes) and S-family adhesions (sfa gene) among the isolated strains was 100% in each of the 42 tested isolates. TheStx gene was present in 11 (26.2%) isolates, Stx1 in 4 (9.5%) and Stx2 in 7 (16.7%) isolates. Interestingly, the cytotoxic narcotizing factor type 1 (cnf) gene was not found in any isolate. All isolates (100%) were sensitive to fosfomycin, imipenem and tigecycline and the majority of them were highly resistant to piperacillin (76%). Due to the increase of E. coli with multiple virulence genes, continuous investigation about the efficacy of antibiotics is necessary.

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Combined post-harvest process through juice blending and directed vat set fermentation for developing high quality ready-to-serve vegetable beverages

Zhongke Sun, Dayi Ji, Shuangshuang Lou, Qingzhi Li, Lili Li and Chengwei Li

Page No: 31-39

Abstract: Large amounts of vegetables require in-time processing after harvest to avoid spoilage due to their perishable nature. Juicing and fermentation are common techniques for vegetable processing. However, many vegetables are not ready-to-serve after simple juicing or fermentation as they may have either an unpleasant smell or a bad taste. In this study, we try to solve this problem through a combination of scientific blending and fermentation.

Using some commonly available or quickly spoiled vegetables, we blended several juices and one blended juice with a composition of 6:1:6:3 (celery: potato: carrot: cucumber, in volume), has the highest sensory score, though still tastes terrible for direct drinking. Then, we prepared direct vat set lactic acid bacteria (DVSL) for further fermentation of the blended juice. Inoculation of DVSL containing Lactobacillus plantarum WCSF1, Lactobacillus delbrueckii subsp. bulgaricus S-1 and Streptococcus thermophilus G1208 resulting in significant improvement of the total sensory score (P < 0.01, especially in aroma and taste). Optimization of parameters indicates that the optimum fermentation conditions are 0.20% inoculum (m/v), 37°C, static, lasting for 5 days. Comparison of the chemical and nutritional attributes before and after biotransformation suggests DVSL fermentation can greatly decrease sugar content, pH, acidity and vitamin C, but increase the content of protein as well as vitamin B2. In contrast to blended juice that spoils within one week, fermented juice can be stored at room temperature for more than one month without obvious putrefaction. This study supplies a technique for potential process of vegetables, especially for those under-processed resources.

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The Weaning Stress Effect on Calf Behaviour and Performances

Neamț R.I., Ilie D., Enculescu M., Săplăcan S. and Cziszter T.L.

Page No: 40-43

Abstract: Good practices in animal rearing have been proved to be essential conditions in order to guarantee the animal welfare, health and attenuate the stress. The aim of the study was to analyse the effects of weaning method on calf welfare based on body weight post weaning and behaviour patterns. The study sought to set the most appropriate weaning method in order to ensure the calves welfare. Study was carried out on 63 Romanian Brown calves weaned at 90 days of age either abruptly or with a 10-day pre-weaning period.

Comparisons between the weaning methods for body weight and feeding behaviour were performed using one-way ANOVA assay with categorical factor “weaning method”. A positive effect of the gradual weaning was observed on calves’ body weight post calving. The abrupt weaning induced a 6.2 % loss in calves body weight within the first 10 days after weaning compared to 3.6 % recorded in gradual weaning (P<0.001). Gradually weaned calves allocated 151.47±4.28 min. / day for feeding divided into 8.1±0.31 meals compared to 93.87±6.11 min. / day feeding and 6.3±0.17 meals (P<0.001) recorded in abruptly weaned calves. Performing gradual weaning decreases loss of calves’ body weight also increasing the feeding behaviour patterns.

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Organogenesis and anatomical study of gamma rays induced mutant of chrysanthemum (Chrysanthemum morifolium Ramat.) from ray florets

Verma A.K. and Prasad K.V.

Page No: 44-53

Abstract:We developed an efficient in vitro regeneration protocol for the isolation, rapid multiplication and field establishment of Pink Incurving novel mutant from ray florets of chrysanthemum. Earliest (8.33 ± 1.17 d) and highest (86.67 ± 2.03%) callogenesis was observed on Murashige and Skoog (MS) medium supplemented with 10 mg l–1 kinetin (KIN) and 2 mg l–1 α-Naphthalene acetic acid (NAA). MS medium supplemented with 10 mg l–1 KIN + 0.1 mg l–1 NAA was found most effective to induce maximum regeneration of micro shoots (96.33 ± 1.95%) while maximum shoot proliferation (98.33 ± 0.88%) was obtained when the medium was supplemented with 5 mg l–1 KIN + 0.01 mg l–1 NAA + 0.2 mg l–1 GA3. Good shoot elongation was noticed after 30 days of transfer onto a medium comprising of 0.50 mg l–1 GA3. The elongated microshoots were best rooted on half-strength MS medium supplemented with 0.5 mg l–1 NAA. The rooted plantlets were successful acclimatized in 3 weeks in glass jar with polypropylene cap filled with peat + Soilrite® (1:1) and transferred to greenhouse for flowering.

The plants obtained through this technique produced the true-to-type flowers in υM2 generation. The isolated mutant was found stable for the selected characters. Histological investigations revealed that unorganized callus got organized and turned into meristematic zone to produce shoot primordia and further developed into multiple shoots as the concentration of growth regulators changed.

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Photo catalytic Applications of FeTiO2 Nanoparticles in Dissipation Kinetics of Herbicide Residues in Soils

Rao Tentu Nageswara

Page No: 54-63

Abstract: The dissipation kinetics activity of FeTiO2 nanoparticles on benzoic Acid class herbicide (dicamba), Imidazolinone class herbicide (imazethapyr), sulfonylurea class herbicide (metsulfuron-methyl) and Aryloxyphenoxy-propionate class herbicide(clodinafop-propargyl) in soil was investigated. The decontamination effect of catalyst on the residues of dicamba, imazethapyr, metsulfuron-methyl and clodinafop-propargyl was studied at four different soils (Sandy loam, Loamy sand, Sandy clay and Clay soil). The decontamination of herbicides on soil surface in sunlight under natural climatic conditions was studied. Kinetic parameters such as rate constant (k), DT50 and DT90 were calculated using the dissipation data of all herbicides. The dissipation of herbicide follows first order reaction rate. The degradation was high in clay soil for dicamba, metsulfuron-methyl and clodinafop-propargyl when compared to other soil, the degradation order was Sandy loam < Loamy sand < Sandy clay < Clay soil.

The degradation was high in sandyloam soil for imazethapyr when compared to other soil, the degradation order was Sandy clay < Loamy sand < Clay < Sandy loam soil. The optimum concentration of catalyst required for the rapid decontamination of herbicide residues on soil was established by varying the quantity of catalyst within 0.02 to 0.1 g/L. The optimum concentration of catalyst was 0.6% w/w. When compared with photolysis, photo catalytic degradation of herbicides in soils was faster than photolysis and reduced the toxicity of herbicides in soils under photocatalytic degradation.

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Characterization of Melanin producing Bacteria isolated from 96m Depth Arabian Sea Sediments

Kurian Noble K., Nair Harisree P. and Bhat Sarita G.

Page No: 64-71

Abstract: Marine bacteria are barely explored for useful metabolites. Melanins are indispensable pigments with diverse bioactivities present in almost all living forms. This study investigates melanin producing bacteria in benthic sediments of Arabian Sea, the isolated melanin producers were characterized by biochemical and molecular techniques. Their antibiotic resistance pattern and ability to produce extracellular enzymes were also explored. Among the 40 isolates, 11 produced considerable quantity of melanin ranging from 27 μg/mL to 504 μg/mL. The organisms identified included Marinobacter alkaliphilus, Leclercia sp, Halomonas meridiana which were reported as melanin producers. Very high antibiotic resistance was exhibited by the isolates with 72.77% having MAR index value > 0.2. The melanin producers also secreted multiple extracellular enzymes, adding to its ability to produce diverse bioproducts.

This study reveals melanin production could contribute to antibiotic resistance to the bacteria and the mechanism underlying need to be explored further. As melanin being a pigment with great pharmacological and cosmetological importance, the melanin produced by the bacteria in the study can be utilized in developing cosmetics and future drugs.

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Bioprospecting potential of mangrove fungus from Vellar estuary, Southeast coast of India for biocontrol of damping off on mustard

Pandian Sureshkumar and Subbiah Kavitha

Page No: 72-78

Abstract: Pre- and post-emergence damping off caused by Rhizoctonia solani is a serious disease on vegetable crops. The strain of fungus Cladosporium oxysporum RM1, isolated from mangrove plant Rhizophora mucronata was assessed as a potential biocontrol agent. This study demonstrated that this fungal species would be considered as an efficient and environment friendly bio-agent to prevent damping off and root rot diseases caused by R. solani in mustards. The pot experiment confirmed its effectiveness and final results indicated significant reduction of disease pathogenicity at the end of 4th week.

The plant supplied with our isolate has comparatively shown superior growth than the control. Hence, the fungus is also screened for in vitro plant growth promoting activity. The present study forms the ground work suggesting that the mangrove-associated fungus with bioprospecting and biocontrol potential deserves further studies to explore and reveal their hidden capability.

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Cladistic phylogeny of Calanoid copepods (Crustacea: Arthropoda) using 18S rRNA gene

Pachiappan Perumal, Chandrasekaran Rajthilak, Perumal Santhanam, Manickam Raja, Thodhal Yoganandham Suman and Govindasamy Balasubramani

Page No: 79-87

Abstract:This study establishes a molecular phylogeny for calanoid copepods based upon 18S rRNA gene including 4 families from 2 super families. For this investigation, copepods were collected from different habitats of the Tamil Nadu Coast (Southeast India). The tree topology confirmed the monophyletic state of the families Pontellidae and Pseudodiaptomidae with high bootstrap values of 82 and 98% respectively.

The family Acartiidae derived from the clade Pontellidae with a genetic distance ranged from 11.2 to 14.5%. However, the Paracalanidae family showed the highest genetic distance of 17.5% to the superfamily Centropagoidea and formed a seperate clade. We have also reconstructed phylogenetic trees using neighbor-joining (NJ)), maximum-likelihood (ML) and minimum evolution (ME) methods by retrieving 10 reference sequences from the NCBI that were compared with our own 15 calanoid copepod sequences. All these trees resolved clear separation of calanoid copepods based on family and superfamily level and hence 18S rRNA gene is a suitable molecular marker to study the relatively recent cladogenetic events among calanoid copepods.

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Transposon Mutagenesis and its insertion mapping for enhanced nitrogen fixing ability and nodulation by Bradyrhizobium japonicum infecting Vigna radiata

Ghatage Abhay, Pandey Rachna and Khetmalas Madhukar

Page No: 88-92

Abstract:This study deals with a transposon mutagenesis procedure for Bradyrhizobium japonicum infecting Mung bean (Vigna radiata). Transncongugate of 800 was produced by biparental mating between Bradyrhizobium japonicum and E.coli with frequency of 3.7x 10-6. These 800 mutants were screened for nodulation which showed 100 mutants with pink coloured nodulation to mung bean plant. Out of 100 mutants, 2 were promising for nitrogen fixation. These two mutants were further studied for Tn3 specific sequence amplification and DNA sequencing of amplified PCR product.

Blast alignment of generated sequences of strain compared with Bradyrhizobium japonicum indicates that Tn3 occurred in the genes Aldehyde Oxidase of mutant strain AVR040 and RND Transporter of AVR 063.

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Biodegradation Promising solution to the Rubber waste and Rubber Industry by the Streptomyces diastaticus from Soil

Vidya T.V. and Lali Growther

Page No: 93-99

Abstract:The rubber biodegradation activity of Streptomyces diastaticus was recently determined and was tested with vulcanized natural rubber products and non-vulcanized rubber products such as fresh latex as well as latex gloves. The degradation activity was confirmed by scanning electron microscopy by the colonization and disintegration as well as the oxidative cleavage of the rubber by the organisms on the surface of the gloves on the 3rd month of the inoculated sample was confirmed by SEM. The degradative products were identified by GCMS. FTIR analysis on the inoculated samples reveals insight to the biodegradation mechanism. Decrease in the Cis -1,4 double bonds in the polyisoprene chain, the appearance of the keto and aldehyde group formation indicate the oxidative attack of the double bond of the rubber hydrocarbon.

The strains were able to degrade both the natural and the vulcanized rubber with low complexity. Polyisoprene product is a recalictrant material which can’t melt, reshape or recycle on melting to cause serious health hazards to the human beings. Biodegradation is the only solution for this process. Even though during the processing of rubber and the expulsion of its waste dumped to the open air, it also affected the humans residing near. By the strong activity of the biodegrader, it is a promising solution for the environment to ecologically balance the environment. Actinomycetes are aerobic and potent biodegraders comparing with the other organisms, Streptomyces sp are one among those. In this study Streptomyces diastaticus is the more potent isolate which could degrade rubber.

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High-frequency Somatic Embryogenesis and Plantlet Regeneration in Papaya

Shukla Manish, Tiwari Rajesh K. and Al-Busaidi Khair Tuwair

Page No: 100-108

Abstract:Papaya is an important fruit crop, grown commercially in tropical and subtropical regions of the world. Efficient and genotype independent somatic embryogenesis, plantlet regeneration, robust rooting and acclimatization protocol in papaya are essential for mass multiplication and crop improvement program. Explants type, explanting season, optimum 2,4-D (2,4-Dichlorophenoxyacetic acid) concentration, callus plating density, application of osmoticum and ideal rooting media have a direct effect on somatic embryogenesis and plantlet regeneration in papaya. Immature zygotic embryos as explants at 10 mg/l 2,4-D concentration with a placement of single callus in the medium induced the highest callusing (68.47%) and produced maximum somatic embryos (18.75) per explants. 68.86 % somatic embryos were matured and 35.25 % regeneration efficiency was observed in 45 mg/l poly ethylene glycol along with Murashige and Skoog basal medium. 2 mg/l Indole butyric acid and 8 gm/l powdered cocopeat in MS basal medium produced high frequency rooting and regeneration of papaya plantlets.

Rooted papaya plantlets were survived successfully in sand: soil: cocopeat (1:1:1 v/v) after transplantation. Acclimatized plants were transferred to open field conditions after 2 weeks in green house. Efficient somatic embryogenesis, plantlet regeneration, rooting and acclimatization for papaya have been developed.

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