Research Journal of Biotechnology

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Vol. 11(1) January 2016

Enhancement of fish growth employing feed supplemented with recombinant fish growth hormone expressed in Bacillus subtilis

Ng Alan K. L., Lam Gigi C. C., Kwong Keith W. Y., Dik Duncan W. N., Lai Nelson C. Y., Au Yong Jonathan L. W., Qian P. Y. and Wong W.K.R.

The application of fish growth hormone (FGH) to improving growth rates of fish has been a popular research topic in fish industry. Among various FGH, the tilapia (Oreochromis hornorum) growth hormone (tiGH) has captured much attention. Recombinant forms of tiGH have been expressed and employed to promote growth of not only teleost species of tilapia but also other fish such as sockeye salmon, larvae of tilapia, goldfish, trout and juveniles of carp and angelfish. In this communication, we report the use of an engineered Bacillussubtilis (well reputed as GRAS) system to express recombinant tiGH (rtiGH) which contained a 6-histidine tag at its C-terminus (rtiGH) in the cytoplasm of its B. subtilis host. The identity of rtiGH was confirmed by Western blot analysis and mass spectrometry. Using the whole cell lysate of B. subtilis comprising rtiGH, a facile method of preparing fish feed with rtiGH was developed. The efficacy of the supplemented feed was compared with that of normal feed in promoting fish growth in a randomized controlled study, employing Japanese koi as the fish model. Over the 4 months of study, the fish fed with supplemented feed (Treated group) and those with normal feed (Control group) did not display a differential preference between the 2 kinds of feed. Both groups of fish appeared to respond negatively to various effects of acclimatization in the first 2 months of the study. However, as judged by differences in overall average increases in body weight and length between the treated group (40.38g and 2.17cm respectively) and the control group (31.58g and 1.61cm respectively), it was concluded that the fish in treated group grew significantly faster than those in the control group. The fish fed with supplemented feed were shown to be healthy and physically well-developed, active and eager for food at all times.

An application of nanotechnology for the stability and sustained biological activity of tea polyphenol

Karikalan Kulandaivelu and Mandal Abul Kalam Azad

Spherical gelatin A nanoparticles were prepared with size range of 100-300 nm and loaded with tea polyphenols. FTIR peak at 1633 cm-1 confirmed the presence of major components of polyphenol. Loading as well as release of tea polyphenols was dependent on pH and duration of incubation. Our data showed the possible mechanism of interaction between constituents of tea polyphenols and gelatin during encapsulation. This encapsulation of tea polyphenols into gelatine nanoparticles will be useful in enhancement of bioefficacy of tea polyphenols.

Characterization of Streptomyces sp. from soils of Maharashtra on the basis of their morphology, functional efficiency and molecular divergence

Sonawane R. B., Deokar C. D. and Chimote V. P.

A total of seventy two Streptomyces isolates among the 131 actinomycetes recovered from soils samples from different physiographic regions of Maharashtra were evaluated for their colour, spore chain morphology, aerial and substrate mycelium and biocontrol effiency. On evaluating the spore chain morphology which is the main feature of genus Streptomyces, it was observed that majority of them (sixty six isolates) had rectiflexibles (RF) spore chains while rest six isolates showed retinaculiaperti (RA) spore chains. In the cultural studies all the isolates showed excellent growth on oat meal agar medium followed by good growth on starch casein agar media and poor to moderate growth on nutrient agar media. No variation was observed in aerial mycelium whereas slight variation observed in substrate mycelium. The H2S production was observed positive reaction in 60 isolates while 68 isolates showed gelain liquefaction ability. All the isolates tested positive in oxidase and catalase tests. Maximum in vitro inhibition was recorded in RHR-S-98 and RHR-S-23 i.e. 30.00% and 28.80% against Alternaria alternata and Fusarium oxysporum pv. ciceri respectively. Majority of isolates recorded good (56.94%) to moderate cellulose degradation activity (40.28%). Very high genetic diversity (similarity coefficient range 0.13-0.65) was observed among the eighteen efficient isolates with multiple beneficial properties on RAPD analysis using 23 random primers. These isolates formed two broad clusters with one cluster consisting of five strains of Streptomyces having good cellulose degradation activity, poor biocontrol ability and substrate mycelium was brown to dark brown varying from rest of the other isolates. The second cluster’s sub-cluster with RHR S, RHR S-40, RHR S-41 and RHR S-48 (Ghat region) and RHR S-147 (Vidarbha region) had pale yellowish substrate mycelium, good cellulose activity and poor biocontrol ability.

Genetic variation of ORF3 of porcine epidemic diarrhea virus isolated from different region in Sichuan China from 2013-2014

Li Guili, Wang Yin, Yao Xueping, Hu Ling, Li Lirui, Wang Bo, Ren Ranyang and Yang Zexiao

Positive samples had been detected by RT-PCR which were stored at the laboratory in Sichuan province from 2013 to 2014. A pair of premiers were designed to amplify and clone the ORF3 gene and the products had been sequenced. The results were compared with others PEDV ORF3 sequences which had been uploaded in the Genbank and 47 sequences included above all. Sequence analysis results showed that the homology compared the samples with the others was 89.5%-99.7% especially high in 2013-2014 but it had more difference in CV777 which is the vaccine virus.

Factors affecting Calving Ease in Romanian Spotted Cows reared under Temperate European Conditions

Neamt Radu I., Ilie Daniela E., Acatincai S. and Cziszter Ludovic T.

The aim of the current research was to evaluate the effects of several dam and calf related factors on calving ease of Romanian Spotted dairy cows reared under European temperate conditions. Mean incidence of dystocia calvings in herd was 28.3%. Calves body weight at birth influenced calving ease. Significance differences (p≤0.01) were recorded regarding body weight of eutocyal compared with dystocial calves (33.89±0.37 kg vs 35.67±0.42 kg). Small size of cows pelvic area favored occurrence of dystocia calvings. Significance differences (p≤0.05) were recorded for cows body weight (665.66±5.16 kg vs 651.83±4.56 related to eutocya vs dystocia calvings), rump lenght (55.26±0.33 vs 53.52±0.38, p≤0.001), width at ilium (57.43±0.38 cm vs 55.94±0.43 cm, p≤0.01) and width at ischia (38.03±0.34 cm vs 34.56±0.39 cm, p≤0.001).

Effect of Temperature on Stability and Nutritive Value of Soybean and Rice Bran Oil blends in varying proportions

Parikh A.D. and Parikh J.A.

Soybean oil is cultivated on large scale in recent years in India particularly in M.P., U.P. and part of Maharashtra because of its certain advantages. The direct use of soybean oil for deep frying purpose has certain limitations due to high content of linolenic acid and fishy odour during heating. Significant improvement in shelf life and thermal stability and flavour reduction can be achieved by lowering the linolenate content of soybean oil by blending in different proportions with other edible oils. Rice bran oil is popular in several countries such as Japan, India, Korea, China and Indonesia as cooking oil. It has high content of oleic acid, is rich in vitamin E, an antioxidant and high thermal stability. These oil blends could contribute as sources of important antioxidant related to the prevention of chronic diseases associated to oxidative stress such as in cancer and coronary artery disease10. In the present study , soy bean oil is blended with rice bran oil in different ratio and tested for physico-chemical properties and stability at elevated temperatures by using standard (AOCS) methods15.

Fabrication and characterization of rice like shape ZnO–ZnS nano composite by wet chemical method as photocatalyst

Farhadyar N. and Zahra Mirzaee

Semiconductor assisted photocatalysis has gained an important place among various advanced oxidation methods for wastewater treatment. In this paper, ZnO–ZnS photocatalysts have synthesized nano rice like zno/zns by simple precipitation method technology using zinc acetate as single precursor. The structure of as-synthesized photocatalysts has been analyzed and corresponding photocatalytic activities for degradation of organic pollutants have been studied under UV. As prepared material, nanosize zinc oxide loaded on the sulfide zinc was investigated by using Fourier transform infrared spectroscopy (FT- IR). X-ray diffraction (XRD), Scanning electron microscopy (SEM), Transmition electron microscopy (TEM), Photocatalytic activity of the samples, Zinc oxide loaded on the sulfide zinc were finally evaluated by degrading of the methylene blue under irradiation of UV light. The results showed that the nanosized zinc sulfide enhances the photocatalytic activity of zinc oxide when loaded on it.

Relation between Sugar Consumption and Bioethanol Production Potential in Lignocellulosic biomass

Pandey Anshika, Tiwari Shubhra, Tiwari K.L. and Jadhav S.K.

Bioethanol more appropriately termed as alternative transportation fuel play a key role in reducing the world dependence on fossil fuels. Bio-ethanol is an oxygenated fuel that contains 35% oxygen which reduces particulate and nitrogen oxides (NOx) emissions from combustion. Henceforth using bio-ethanol blended fuel for automobiles can significantly reduce petroleum use green house gas emission. Developing ethanol as fuel, beyond its current role as fuel oxygenates will require developing lignocellulosic biomass as a feedstock because of its availability and low cost. In present study Azolla, lignocellulosic biomass is used as a substrate as it is cheap and has cellulose content 15.19 ± 1.35 % of its dry weight which can be converted into the fermentable sugar through biological pretreatment. This fermentable sugar is used by the fermenting microorganism i.e. Saccharomyces cerevisiae MTCC 4780 and through anaerobic oxidation converted into bioethanol. It has been found that there is co-relation among the fermentable sugar concentration and bioethanol production. As the concentration of reducing sugar is determined by the dinitrosalicylate method (DNS Assay), a relation among the value of DNS and the bioethanol production is established. In the present study maximum sugar consumption by Saccharomyces cerevisiae MTCC 4780 is 0.42 mg/ml which results in maximum bioethanol production 4.06% (w/v). As various parameters get optimized, the bioethanol production potential from the consumed sugar level is also enhanced and the maximum production which was obtained in presence of 1 ml 1% KCl solution is 5.20%.

In Vitro role of hormones at multiplication stage of date palm (Phoenix dactylifera L.) cvs.Khalas and Sukary

Abdullatif Ali Al-Khateeb and Suliman Ali Al-Khateeb

The purpose of the present work was to investigate the physical state as affecting reducing hormone concentration on bud and shoot multiplication in date palm (Phoenix dactylifera L.) cultivars; Khalas and Sukary, BAP and 2iP in three different combinations (0.3+0.3, 0.5+0.5, 1.0+1.0 mg/l) were tested respectively. The results indicated that low (0.3+0.3 mg/l) concentrations of hormones promoted the formation of bud with significant differences while high 1.0+1.0 BAP+2iP mg/l) promoted plant length significantly. Fresh weight of the culture did not differ significantly in all the concentration used. However, when 1.0 mg/l BAP and 1.0 mg/l 2iP was added to the medium, it gave more callus fresh weigh compared to lower concentrations. Significant correlation coefficients were observed between fresh weight and bud number and fresh weight and the longest plant in both the cultivars. Khalas cultivar gave positive linear correlation but with fair to poor coefficients of determination r2 =0.48 and r2= 0.3, between fresh weight and bud formation and plant elongation respectively. On the other hand cv. Sukkary gave inverse linear correlation but with poor coefficients of determination value of r2 =0.24 which reflect the role of fresh weight on bud formation in khalas cultivar.

Cation-π interactions involved in coagulation cascade pathway-A protein stability analysis

Ali Shabana Kouser and Doss George Priya C.

The cation–π interactions are necessary for molecular recognition in biological receptors. Here, in this study we have analyzed the energy contribution of cation–π interaction in the coagulation cascade pathways proteins with ‘A’ chains of their PDB structure. The contribution of cation–π interacting residues in stabilizing residues and centres, secondary structure, solvent accessibility and conservation score has been well studied. With total data set of 190 proteins, 160 showed significant cation–π interactions. Cation–π residues pair’s involved Arg-Tyr pair and showed maximum number of cation–π interactions and Lys-Try residue pair showed minimum interactions. The cation–π interaction energy depicted that Arg-Tyr showed high energy and Lys-Tyr showed less energy. In the secondary structure, Arg and Lys preferred to be in strands because of Phe in coil and Try in helix. Residues Arg and Lys preferred to be in exposed region and π residue Phe preferred buried regions of the proteins. The significance of cation-π interaction in the stability of therapeutic proteins and pharmacology studies is important.

Enhanced and intrinsic bioremediation of TCE-contaminated groundwater

Lien P.J., Tu Y.T., Chang Y.M. and Kao C.M.

Groundwater at many industrial sites is polluted by chlorinated organic compounds. One cost-effective remedial method is the application of bioremediation technology to bioremediate the contaminated aquifers with the supplement of primary substrates. The objective of this laboratory microcosm study was to evaluate the feasibility of applying developed long-lasting substrate (LLS) to enhance the bioremediation of groundwater contaminated with trichloroethylene (TCE). The produced LLS contained vegetable oil and surfactants [Simple GreenTM (SG)]. An anaerobic microcosm study was performed to assess the capability of LLS to serve as a long-term carbon-releasing substrate for TCE dechlorination. Brown sugar was also selected as a substrate for the enhancement of TCE dechlorination as a comparison. In this study, the real-time polymerase chain reaction (PCR) was applied to evaluate the variations in TCE-dechlorinating bacteria (Dehalococcoides spp.) during the biodegradation process. Results from the microcosm study show that the total organic carbon (TOC) in brown sugar and LLS addition microcosms increased from 22 mg/L (in control microcosm) to 1,425 and 1,026 mg/L after their addition respectively. Compared to control microcosms, decreased dissolved oxygen (DO) and oxidation-reduction potential (ORP) were observed in brown sugar and LLS addition microcosms indicating that the substrates addition resulted in anaerobic conditions which favoured the reductive dechlorination process. In the LLS microcosms, results also show that the TCE concentrations dropped from 5.4 mg/L to below 1.4 mg/L after 5 days of LSS injection and then declined to 3µg/L after 60 days of incubation. In the brown sugar, TCE slowly dropped to 5µg/L after 60 days of incubation indicating that brown sugar could be used as the substrate for enhancing the reductive dechlorination process. The population of Dehalococcoides spp. increased from 4.6×101 to 7.4×106 and 4.3 ×105 cell/L after 20 days of operation in brown sugar and LLS addition microcosms respectively. This indicates that the addition of brown sugar and LLS enhanced the growth of Dehalococcoides spp. which could significantly activate the TCE dechlorination. The addition of LLS created anaerobic conditions and led to a more complete TCE removal via biodegradation and sorption mechanisms. Results show that the enhanced anaerobic bioremediation is an effective and applicable technology to remediate TCE-contaminated groundwater.

Increase of the Anti-Skin Wrinkle and Skin-Whitening Effects of Agastache rugosa Kuntze through Low Temperature Ultrasonication Extraction

Kim Nam Young, Kim Dae Kyu and Lee Hyeon Yong

The work proved that both anti-skin wrinkle and skin-whitening activities of Agastache rugosa Kuntze could be enhanced by extracting at low temperature of 600C with 70% ethanol solvent associated with ultrasonication process (UE). These results were compared with those obtained by 70% ethanol (EE) at 800C as a conventional extraction process. For antioxidant activity, the UE showed a DPPH scavenging rate of 72.55% which was higher than that of EE as 62.21%. In terms of skin whitening activities, the inhibition of tyrosinase was measured as 56.24% in treating 1.0 mgmL-1 of the UE while EE showed an inhibition rate of 31.34%. Likewise, the melanin inhibition rate using the UE was 71.09% was higher than that of EE at 53.27%. For skin anti-wrinkle effects, the production of collagen was greatly improved as 288.6 ngml-1in adding the UE and only 165.3ngml-1 from the EE. Additionally, MMP-1 production also decreased down to 969.9 pgml-1in treating 1.0 mg mL-1 of the UE and 1640.6pgml-1 from the EE. In this work, it was first shown that this improvement was caused by high elution of acacetin, one of major components inA. rugosa by ultrasonication extraction as well as less break down of the biologically active compounds at low temperature process. It is believed that these results could also be applied to improve biological activities of relatively heat labile bioresources.

Purification of fungal laccase from Mycena purpureofusca using ultrafiltration

Shan Shu-Kai, Li Zhaofeng, Hu Kaihui and Sun Shu-Jing

Laccases are copper-containing oxidase enzymes that have been found as useful biocatalysts for diverse biotechnological applications which attract attention of hundreds of scientist around the world. The separation of laccases is a key step in research on the characteristics and application of laccases. Therefore in this study, the isolation of laccase produced by M. purpureofusca has been conducted using ultrafiltration with 30 and 100 kDa molecular weight cut-off polyethersulfone membranes. Under suitable conditions, the laccase purity obtained in the retentate was about 94.9% and the recovery of laccase was close to 80% after ultrafiltration. The resulting laccase product was then analyzed by isoelectric focusing, sodium dodecyl sulfate- polyacrylamide gel eletrophoresis and circular dichroism to confirm its isoelectric point, molecular weight and molecular secondary structure. The results showed that the molecular weight of laccase was 61.7 kDa and it showed a pI value of 4.6 and a reasonable secondary structure.

Cloning and expression analysis of the Cullin gene in Citrus reticulata

Wu Xiulan and Tang Wenwu

In this study, the length cDNA and DNA of Cullin gene were cloned from Wuzishatangju (Self-incompatibilty, SI) and Shatangju (Self-compatibilty, SC). The results revealed that Cullin genes from Wuzishatangju and Shatangju were 1989 bp in ORF sequence and encoded 662 amino acid while between Wuzishatangju and Shatangju, three bases and two three amino acids were different. The expression of the Cullin gene in anthers of Wuzishatangju was 7.4-fold higher than that in anthers of Shatangju. The expression rised gradually after self-pollination. While it was first increasing, then it was decreasing after cross- pollination. The result from subcellular localization showed that the Cullin gene was localization in cell nucleus. Thus Cullin gene may participate in SI reaction in Wuzishatangju which provided a candidate gene potentially useful for self-incompatibility although its function still needs further confirmation.

Development of probioticated banana pith juice

Nithya Priya S., Ramalingam P. and Kokilavani S.

Banana pith has been studied as a low cost underutilized agricultural waste with abundant therapeutic properties. The objective of this study was to optimize the concentration of prebiotic (inulin), stevia (Stevia rebudiana) and inoculum Lactobacillus acidophilus by varying one variable at a time (OVAT) based on single factorial analysis to develop a probioticated banana pith juice. The banana pith juice with 3% prebiotic had a pH 3.87, acidity 0.45%, total sugars 143.3 μg/mL, reducing sugars 320 μg/mL and microbial viability of 3.88 X 108 CFU/mL respectively. The juice with 2 % stevia had a pH 3.9, acidity 0.42%, total sugars 250 μg/mL, reducing sugars 247.5 μg/mL and microbial viability of 7.2 X 108 CFU/mL respectively. The juice with 3% inoculum size had a pH 3.98, acidity 0.69%, total sugars 225 μg/mL, reducing sugars 352.5 μg/mL and microbial viability of 2 X 108 CFU/mL respectively. Maximum growth of the probiotic was obtained at the end of 48 h of fermentation for 1% prebiotic, 2% stevia and 3% inoculum size, thereby proving its ability and suitability to ferment banana pith juice. There was not much loss in the nutritional value and changes in organoleptic properties of the fresh (control) and probioticated juice.

Impacts of soil organic matter, iron-aluminium oxides and pH on adsorption-desorption behaviors of oxytetracycline

Feng Yao, Li Zhaojun and Hao Xiaoqing

Oxytetracycline (OTC), one of the tetracycline, was widely used in animal industry. It can enter the soil through animal feces. In order to understand its fate in soil environment, the adsorption-desorption behaviors and its influence factors in two contrast soils including black soil and red soil were investigated using the batch balancing experiments. With the increase of the OTC concentration, the adsorption and desorption quantity increased. The adsorption and desorption data in all original soils, OM-removed soils, oxides-removed soils could be well described by Freundlich and Langmuir models. The Freundlich distribution coefficient (Kf) values for the adsorption process were larger than the corresponding data for the desorption values. At the same OTC concentration, the adsorption capacity of the black soils to OTC was greater than that of the red soils. OTC adsorption amount of OM-removed black soil showed no significant difference in the black soil. However, OTC adsorption capacity of the red soil increased significantly when the organic matter was removed. OTC adsorption and desorption amount of the black soil increased after the oxide was removed while desorption capacity of the red soil decreased and adsorption capacity showed no significant difference. At the different initial pH of mixing solution, adsorption amount of OTC on the black soil was larger than those on the red soil which reached the maximum when soil pH was 3. Adsorption amount of OTC onto black soil decreased with the increase of the soil pH. There was also a desorption hysteresis for the two types of soil.