Open Access Original Research Article

Nutrients and Microbial Evaluations of Ginger Pre-treated Smoke-dried African Lungfish (Protopterus annectens Owen, 1883)

L. A. Agbabiaka, B. C. Umeh, O. A. Kuforiji, A. O. Atanda

Biotechnology Journal International, Page 1-6
DOI: 10.9734/BBJ/2016/24119

The nutrients assay and microbial evaluation of ginger pre-treated smoked African lungfish (Protopterus annectens) stored for a week at room temperature were studied. Forty five fish samples weighing 1.0 kg each were purchased, killed, eviscerated and washed thoroughly under tap water and grouped into three treatments of fifteen fish each coded as treatments A, B, and C respectively. Fish in treatment  A were immersed in 5% brine without spice (ginger) pre-treatment served as the control, treatment B was soaked in mixture of 5% brine and 2.5% ginger extract while treatment C was immersed in a solution containing mixture of 5% brine and 5% ginger extract. Each batch was smoke-dried for a period of six hours. The samples from each batch were subjected to proximate composition within 48 hours and microbial analysis on 7th day of storage at ambient temperature. The results of the proximate analysis showed a significant difference (P<0.05) among the pre-treated samples. Sample A had the highest moisture content (11.85%). The highest concentration of crude protein was obtained in sample C (63.94%) while samples B and A recorded the values of 63.8 and 59.51% respectively (p<0.05). The highest crude fat concentration was obtained in sample C (8.46%), followed by sample B (8.44%) and the least was recorded in sample A (5.78%). The microbiological analysis showed that sample A (control) favoured the proliferation of microorganisms such as E. coli, Staphylococcus aureus, Bacillus spp and Micrococcus spp more than samples treated with ginger extract. The overall microbial population ranged between 3.0x104 and 2.0x105 which are within the safety limit (≤ 105 cfu/g) for total bacterial plate count for microbiological food. This result therefore has indicated that the use of brine with ginger extract could be suitable for prolonging the shelf-life of smoked lungfish without negative effects on the nutrients composition and storage quality under ambient conditions.

Open Access Original Research Article

Molecular Characterization of Selected M5 Lines of Rice after TILLING for Salinity Tolerance Using 20 SSR Primers

Adeeba Raihan, Abu Shamim Mohammad Nahiyan, Ataur Rahman, Lutfur Rahman

Biotechnology Journal International, Page 1-11
DOI: 10.9734/BBJ/2016/23868

About 20% of the net cultivable land is affected by various levels of salinity, which leads to season loss and crop loss. A rice variety BINA 7 was used to develop saline tolerant, high yielding varieties through TILLING. 10 rice lines including 5 from TILLING (Targeting Induced Local Lesions in Genomes) population were studied in Patuakhali, the coastal zone of Bangladesh between July to December, where maximum salinity reached during harvest was close to 8 ds/m2. Traits like plant height, effective tillers, thousand seed weight on randomly selected plant basis and yield (kg/plot) were recorded. Appropriate statistical analysis was done. This was followed by Genetic Fingerprinting using saline trait specific, 20 SSR primers of the 5 TILLING population along with parent. Data Analysis of band position and creation of dendogram along with genetic distance between the materials have been reported. Indications of positive relationship between the molecular charactization and morphological studies gave direction towards possible saline tolerant lines. It was clearly seen from the 20 SSR markers used that the lines developed through the TILLING technique has more possibility of being saline tolerant than the parent,e.g. primer RM 585 bound between 175-650 bp to all except the parent. Such results indicated the TILLING lines to be diverse than the parent.

Open Access Original Research Article

Apoptotic Gene Expression in Sheep Hepatocytes during Fasciola hepatica Infection (Fascioliasis)

Wafa A. I. Al-Megrin, Wedad S. Al-Qahtani

Biotechnology Journal International, Page 1-11
DOI: 10.9734/BBJ/2016/24186

Aims: The main objective of this work was to investigate the apoptotic genes of sheep liver hepatocytes to elucidate the apoptosis pathway mechanisms during Fasciola hepatica infection using molecular and serological techniques.

Study Design: This is a laboratory based study whereby F. hepatica infected liver specimens were used.

Methodology: Total RNA was extracted from fresh-frozen liver tissue. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to investigate the genes encoding the mRNA for following proteins: 18S; Bax; Bcl-2; Caspase-3. Additionally, the concentration of total protein in each sample was estimated spectrophotometrically. Polyclonal antibodies were produced by immunizing rabbits and diluted with 5% skimmed milk in PBS containing 0.1% Tween 20 (1:1000). The detection of the reacted antigen/antibody products was performed using immunoblotting technique were used to assess the quantification of protein kinetic to apoptotic genes. Data obtained from this investigation were analyzed using Portable IBM SPSS 2006 Statistics software.

Results: We investigated the apoptotic gene expression in sheep liver hypatocytes during                F. hepatica infection by mRNA expression of three genes involved in apoptosis; Bax, Bcl-2 and Caspase-3 and histopathological parameter after infection with F. hepatica in the liver cells. Quantitative real-time PCR, histological examination and immunoblotting were used to quantify apoptotic genes, histopathology and protein kinetic, respectively. F. hepatica infection induces apoptosis in the liver cells via Bax, Bcl-2 and Caspase-3 genes.

Conclusions: F. hepatica infection induces apoptosis in the liver cells via Bax, Bcl-2 and Caspase-3 genes and it precedes necrosis. Thus, this study suggests that the induced apoptotic gene expression was due to the outcome of F. hepatica.

Open Access Original Research Article

Isolation and Purification of Lipase from the Midgut of Fifth Instar Larvae of Antheraea mylitta drury

Lakshmi Marepally, G. Benarjee

Biotechnology Journal International, Page 1-9
DOI: 10.9734/BBJ/2016/24442

Lipases obtained from healthy and pebrinised larvae were purified by 45-85% (NH4)2SO4 fractionation followed by Sephadex S-100 gel filtration and CM-Sepharose. In both the samples final enzyme purification reported was 19.02 and 17.7 folds(magnitude) and the recovery of final purified enzyme was 19.32% and 17.14% with a specific activity of 7.87 and 7.52 µmol/min/mg. Results also show that activity of purified lipase was highest at pH 8 in both the samples. Highest lipase activity was recorded between 37°C to 40°C temperature and lipase activity was maximum at 37°C temperature in healthy sample and 38°C temperature in pebrinised sample. The enzyme activity reduced with addition of NaCl, Urea and MgCl2 whereas EDTA and CaCl2 increased the activity. The molecular weight of the purified enzyme was 30 kDa as determined by SDS-polyacrylamide gel electrophoresis.

Aim: The present study was conducted to isolate, purify and characterize lipases from the midgut of both healthy and pebrinised fifth instar larvae of Antheraea mylitta drury.

Study Design: Study involves dissection of midgut from the fifth instar larvae of fourth day of both healthy and pebrinised larvae, Lipases obtained from both the samples were purified by 45-85% (NH4)2SO4 fractionation followed by Sephadex S-100 gel filtration and CM-Sepharose and specific activity was measured at various temperatures and different pH. Molecular weight of lipase was measured by SDS PAGE.

Place and Duration of Study: Healthy and pebrinised fifth instar larvae of fourth day were collected from the forest patches of Jakaram (18.1E and 79.8 N), Warangal in August 2015.

Methodology: Lipase was isolated from the midgut of both healthy and pebrinised larvae and purification of enzyme was done by (NH4)2SO4 fractionation, Sephadex S-100 gel filtration and CM-Sepharose. Temperature, pH suitable for highest lipase activity was measured and specific activity against various chemicals was also measured. Kinetic parameters like Km and Vmax were estimated by Sigmaplot software version 11. SDS Polyacrylamide gel was used to determine the molecular weight of lipase.

Results: In both healthy and pebrinised larvae final enzyme purification reported was 19.02, 17.7 folds and the recovery % of final purified enzyme was 19.32, 17.14 with a specific activity of 7.87, 7.52 µmol/min/mg. Results also show that activity of purified lipase was highest at pH 8 in both the samples. Highest lipase activity was recorded at temperatures between 37 and 40°C with a remarkable activity at 37°C and 38°C in healthy and pebrinised samples. The enzyme activity reduced with addition of NaCl, Urea and MgCl2 whereas EDTA and CaCl2 increased the activity. The molecular weight of the purified enzyme was 30 kDa as determined by SDS-polyacrylamide gel electrophoresis.

Conclusion: Pebrine disease has reduced the recovery percentage and also the specific activity of the enzyme. Maximum activity was recorded at high temperature in both the samples. During pebrine infection the midgut of fifth instar larvae has got influenced highly with a significant variation in many biochemical components including enzymes. Pebrine spores are the indicators of disease incidence.

Open Access Original Research Article

DSPC Liposomes Improve Transport of L-cysteine and Reduce Metabolic Activity

Ramiro M. Perrotta, M. Jimena Prieto, Silvia del V. Alonso, Nadia S. Chiaramoni

Biotechnology Journal International, Page 1-11
DOI: 10.9734/BBJ/2016/24723

Aims: In this work, we developed and characterized liposomal formulations that encapsulate L-cysteine to study their further application in drug delivery and amino acid supplementation. The lipids used were 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

Methodology: Encapsulation efficiency and amino acid release were determined. For biophysical characterization of the three formulations, the size, surface charge and surface packing were also studied. Cell viability was analyzed with MTT reagent after treatments with formulations ir order to study efficiency of these systems in induce changes in metabolism.

Results: Results showed that L-cysteine interacts at the polar head level and that this interaction stabilizes the surface charge and prevents aggregation. We also determined the influence on cell metabolism in all formulations. The presence of L-cysteine in the DSPC formulation induced deeper changes in metabolism, evidencing that this formulation provides better transport of this amino acid.

Conclusion: Liposomes developed herein are well suited for the application in the delivery of             L-cysteine. Particularly, they can encapsulate nearly all the L-cysteine and can retain it for 6 hours. Also, L-cysteine stabilized liposomes, preventing their aggregation. L-cysteine encapsulated in the DSPC formulation induced deeper changes in cell metabolism, causing a decrease in metabolic activity; this was probably due to a higher entry, thus a better liposome-mediated transport. Considering that the smaller the particle, the better the circulation, we believe that the stabilization of the vesicle by L-cysteine may allow these transporters to have higher circulation times. Based on the above, we conclude that the DSPC formulation is the best suited for further application in          L-cysteine delivery.