Open Access Original Research Article

Bacterial Synthesis of Silver Nanoparticles by Culture Free Supernatant of Lactic Acid Bacteria Isolated from Fermented Food Samples

Bukola C. Adebayo-Tayo, Aminat O. Popoola, Obinna M. Ajunwa

Biotechnology Journal International, Page 1-13
DOI: 10.9734/BJI/2017/34588

Aim: Biosynthesis of silver nanoparticles (SNPs) using culture free supernatant (CFS) of Lactic acid bacteria (LAB).

Study Design: To biosynthesize, characterize and to determine the antibacterial potential of silver nanoparticle using culture free supernatant of lactic acid bacteria and to determine the effect of some parameters on SNPs biosynthesis.

Methodology: Biosynthesis and characterization of SNPs using CFS of LAB and the antibacterial activity.

Place and Duration of Study: Department of Microbiology, University of Ibadan, Ibadan, Oyo state, Nigeria between Jan to October 2016.

Results: The antibacterial potential of the CFS from the LABs was evaluated on some pathogenic bacteria and antibacterial activity ranged from 0 – 20 mm. The two LAB strains LPW2 and LPF6 had 93% and 86% relatedness to Lactobacillus casei strain WK2G-3A and Lactobacillus fermentum strain E10-15. The CFS of the LAB strains and AgNO3 was able to produce SNPs. The biosynthesized SNPs had a strong Surface Plasmon Resonance (SPR) peak at 500 nm, varying shape (partially aggregated particles) and the sizes ranged from 0.7 - 10.0 nm and 1.4 – 10.0 nm for CFS SNPs from Lactobacillus casei and Lactobacillus fermentum. Carboxylic acid, protein, aldehydes, ester and hydroxyl groups are the functional groups responsible for SNPs formation. The antibacterial activity of the SNPs ranged from 11 – 29 mm. Bacillus sp. and Streptococcus pyogenes were found to be more susceptible to the biosynthesized SNPs. 28°C, pH 4 and 10 Mm AgNO3 supported the highest SNPs production.

Conclusion: In conclusion the CFS from the LABs biosynthesized SNPs which exhibited antibacterial activity against some pathogenic microorganisms.

Open Access Original Research Article

Properties of Alpha-amylase of Lactobacillus plantarum Isolated from Cassava Waste Samples

Anthony Abiodun Onilude, Gbenga Solomon Ayinla, Chika Eluehike

Biotechnology Journal International, Page 1-14
DOI: 10.9734/BJI/2017/33985

The search for various industrial enzymes including starch degrading enzymes has received a great deal of attention for their perceived technological and economic benefits. Alpha-amylase has been produced from large varieties of organisms including pathogenic ones which are not Generally Regarded as Safe (GRAS). In this study, a total of Thirty nine (39) lactic acid bacteria were isolated from the different cassava waste samples (peels, cassava waste water, fibre and soil). The isolates were screened for their ability to produce α-amylase on starch agar. The amylolytic activity of the selected isolates was determined using starch-iodine complex while the enzymes were characterized based on parameters such as pH, temperature, substrate concentration, and metal ions. Upon starch hydrolysis, 29.9% of the LAB isolates demonstrated amylolytic properties with L. plantarum having the highest occurrence. The selected amylase producer was identified both phenotypically and molecularly as L. plantarum S7. Enzyme production was found to be highest (10.573U/ml) at 30 hours of incubation. Optimum temperature and pH for the enzyme activity was found to be 40°C and 7 respectively. The α-amylase retained more than 50% of it residual activity at pH 7 and 8 after preincubation for 90mins. Calcium chloride (CaCl₂) and Ammonium disulphate (NH₄ (SO₄)₂) enhanced amylase activity however, the  activity was inhibited at 5mM molar concentration by KCl, BaCl₂, CuSO₄, NaCl, EDTA (10%) and Urea (10%). The genomic level confirmation was done with 16s rDNA and the sequence showed 90% sequence similarity with L. plantarum.

Open Access Original Research Article

Computational Molecular Analysis of the Sequences of PAPPA2 Gene of Selected Ruminants and Non Ruminants

M. Omolara Akinyemi, H. Osamede Osaiyuwu, Ismail A. Adegoke

Biotechnology Journal International, Page 1-8
DOI: 10.9734/BJI/2017/33125

Pregnancy associated plasma protein A2 (PAPPA2) is an Insulin-like growth factor binding protein (IGFBP) protease of the pappalysin family. This gene has been reported to be associated with prenatal growth, postnatal growth, skeletal growth, calving interval, milk yield, fertility and parity in cattle. The present study was undertaken to computationally investigate the attendant effects of the genetic variants of the PAPPA2 gene on its function and to gain insight into the evolutionary proximity and divergence in ruminants and non-ruminants at the studied locus. A total of fourteen (14) PAPPA2 nucleotide sequences comprising cattle (3), sheep (4), goat (1), pig (3), chicken (1) and horse (2) were retrieved from the GenBank. Functional analysis of non-synonymous single nucleotide polymorphism showed that eight amino acid substitutions (C31I, R60V, Y71G, S119L, S181Y, R190I, Q361M, P178_E180del) in goats, seven in sheep (V9E, L44D, T185N, A125W, Y78S, R194V, R240L), seven in cattle (L11F, C50_W51insTDAPK, E100L, A250T, G257L, M850V), nine in chickens (A60G, S104_R170del, A190F, G128A, I10C, S309G, F48L, Q1630L) and eight in pigs (A61L, P72D, L11Q, K184T, D110C, S193_R194insTQD, Q481_E484del and T170_V172delinsVA) were returned neutral suggesting their beneficial effect. The phylogenetic trees from nucleotide sequences revealed the close relatedness of members of the Bovidae family (sheep, cattle and goat). The present information may be exploited in research into the association between PAPPA2 genotypes and some important economical traits in farm animals.

Open Access Original Research Article

Attempt to Establish Direct Gene Transformation System to Seeds of Sweet Potato (Ipomoea batatas) Using Electroporation Method

Lanzhuang Chen, Shigeru Masuoka, Yoshiko Nishimura, Tetsufumi Sakai, Yasuhiro Takahata, Chengti Xu

Biotechnology Journal International, Page 1-8
DOI: 10.9734/BJI/2017/35275

Aims: The purpose of this study is to get transformants by using the seeds of sweet potato and the instrument of NEPA21, an electroporation method.

Study Design: As the first step, we used grafting method to get seeds from the combinations of Ipomoea crassicaulis cv. [Kidachiasagao] as stock and I. batatas (L.) Lam varieties as graft. Then, we used an electroporation method with instrument of NEPA21 to get the GUS transformants by using direct gene introduction into the seeds.

Place and Duration of Study: Faculty of Environmental and Horticultural Science, Minami Kyushu University, between September 2014 and December 2016.

Methodology: The materials for grafting between Ipomoea crassicaulis var. [Kidachiasagao] as stock and I. batatas (L.) varieties as graft to get seeds, were provided by Kyushu-Okinawa Agricultural Research Center (KOARC), and they were cultivated in growth chambers at Minami Kyusyu U. For the judgment of crossing incompatibility (CI) among the varieties of sweet potato, the standard of KOARC was adopted. For crossing experiment of sweet potato, the flowers of any two varieties were used for pollination, and the performance was stopped before 10 am. The seeds were harvested when they matured.

For the transformation of GUS into sweet potato, the seeds obtained from [Koganesengan] were used in this study. As the seeds were wrapped with hard skin, they were damaged with knife to absorb water easily. Then, the seeds were placed onto wetted filter papers laid in the dishes for 3~4 days. When the seeds germinated, they were used for transformation according to the procedures of NEPA21. After the process was finished, the container was placed in dark condition for 2 days at 25℃, and then, dyed with GUS dyeing solution.

Results: 1) According to the standard of selection for varieties having crossing ability based on the result of CI conducted by KOARC, we selected 3 varieties of [Koganesengan], [Narutokintoki] and [Beniazuma] out from 6 ones held; 2) According to the standard of selection suitable to be used as either for seed or pollen we have  obtained mature  seeds  successfully  from  the  3  varieties; 3) Using the sweet potato seeds collected from [Koganesengan] and electroporation have made GUS transformant of [Koganesengan] with GUS gene expression, for the first time, detected by GUS dyeing with blue color which appeared on the surfaces of the seeds and young buds of the transformants.

Conclusion: The results obtained in this study, provide a practical sweet potato transformation system by two steps: 1) Using the grafting between [Kidachiasagao] and sweet potato can consistently obtain mature seeds of sweet potato according to the judgmental standard for CI provided by KOARC, and 2) Using the combination of seeds of sweet potato and electroporation instrument of NEPA21, can get GUS transformants of sweet potato by using the direct gene transformation system. The establishment of GUS direct gene transformation system in seeds of sweet potato can provide a powerful tool for the ASG-1 gene transformation into sweet potato, which is considered as a crop, difficult to be cultured for plant regeneration. Furthermore, the system can be expected to open the way for all of the seed-set crops with a simplified and practical method to produce transformants.  

Open Access Original Research Article

In vitro Mass Propagation of an Epiphytic Orchid, Cymbidium aloifolium (L.) Sw., through Protocorm Culture

Tripti Regmi, Shreeti Pradhan, Bijaya Pant

Biotechnology Journal International, Page 1-6
DOI: 10.9734/BJI/2017/34891

Aim: To develop a protocol for in vitro propagation of Cymbidium aloifolium, a threatened orchid highly used for medicinal purpose through protocorm culture.

Place and Duration of Study: Tissue culture Laboratory, Plant Biotechnology Unit, Department of Botany, Tribhuvan University, Kirtipur, Nepal, between November 2013 to December 2014.

Methodology: Small, green and globular protocorms with 0.1-0.3 cm diameter were subjected to grow individually on solidified Murashige and Skoog (MS) basal medium and MS medium supplemented with various concentration of plant growth regulators, 6-Benzylaminopurine (BAP, 0.5; 1; 1.5; 2 mg/l) or α-Naphthalene Acetic Acid (NAA, 0.5; 1 mg/l) or their combination. Six replicates were used for each concentration. The data for development of shoot and root from each protocorm culture were recorded in every two weeks for upto six month.

Results: Almost all conditions favoured multiplication but MS medium fortified with BAP (1 mg/l) and NAA (1 mg/l) resulted in maximum induction of rootless healthy shoots with an average value of 8-9 shoots per culture. On this medium, shoot multiplication was initiated after 9 weeks of culture whereas MS medium fortified with BAP (2 mg/l) and NAA (0.5 mg/l) was found to be most effective condition for the shoot multiplication along with well developed roots.

Conclusion: MS medium supplemented with high concentration of BAP and low concentration of NAA was found to be efficient for maximum multiplication of shoot and root. The in vitro developed healthy rooted plantlets of C. aloifolium were successfully acclimatized in green house on potting mixture containing cocopeat and moss in the ratio of 2:1. On this condition, nearly 70% of the plantlets were successfully survived. Hence, this protocol might be useful for mass propagation and ex situ conservation of this orchid through protocorm culture.