Research Highlights in 4Bs: Biosensors, Biodiagnostics, Biochips and Biotechnology

Research Highlights in 4Bs
Biosensors, Biodiagnostics, Biochips and Biotechnology
Proceedings of the 3rd Regional Conference on Biosensors,

Biodiagnostics, Biochips and Biotechnology 2016 (3rdRC4Bs-2016) held
on the campus of AIMST University, Kedah, Malaysia, in April 2016
Editor
Subhash Bhore
2016

Subhash Bhore (Editor)
Published by AIMST University
2016
ISBN: 978-983-43522-8-8 (Print version)
eISBN: 978-983-43522-7-1 (e-Book version)
Financial support for the 3rd Regional Conference on

2016 (3rdRC4Bs-2016) and for this Book was provided
by:
Conference was jointly organized by:
Conference was supported by:
Published by
AIMST University
Printed by
AIMST University
Copyright
Copyright 2016: This book is an Open Access type of publication for the free and
permanent unrestricted online access to scholarly research articles and or abstracts.
Authors retain copyright to their work, and a license is applied which allows users to
download, copy, reuse and distribute data provided the original article is fully cited.
This open access aims to maximize the visibility of research articles and abstracts,
much of which is from publicly funded projects.
Disclaimer: The information provided in this book is designed to highlight the
research findings, views and or perspectives of respective researchers. While the
best efforts have been used in preparing this book, Editor and or Publisher make no
representations or warranties of any kind and assume no liabilities of any kind with
respect to the accuracy or completeness of the contents and specifically disclaim any
implied warranties. Neither the Editor nor Publisher of this book shall be held liable or
responsible to any person or entity with respect to any loss or incidental or
consequential damages caused, or alleged to have been caused, directly or
indirectly, by the information highlighted herein. Readers should be aware that the
information provided in this book may change.
All full articles and abstracts published in this book are deemed to reflect the
individual views of the authors and not the official points of view, either of the Editor
or of the Publisher.
Edited by
Dr. Subhash J. Bhore
Senior Associate Professor
Chairman, the 3rd Regional Conference on Biosensors, Biodiagnostics, Biochips and
Biotechnology 2016 (3rdRC4Bs-2016)
Department of Biotechnology, Faculty of Applied Sciences, AIMST University,
Bedong-Semeling Road, 08100 Bedong, Kedah Darul Aman, Malaysia; Telephone
No.: +604 429 8176; e-mail: subhash@aimst.edu.my / subhashbhore@gmail.com
Front Cover Design

Mr. Mahes D.S.
AIMST University, Malaysia
Edition
First; July 26, 2016
Dedication
This book is dedicated to all speakers,
participants and organizing committee
members of the 3rd Regional Conference
on Biosensors, Biodiagnostics, Biochips
and Biotechnology 2016 (3rdRC4Bs-2016)
in recognition of their contributions for
making
event.
this
conference
successful
Conference Organizing Committee
Chairman
Dr. Subhash J. Bhore
Co-chairman
Dr. Matiullah Khan
Secretary
Ms. Kalaiselvee Rethinam
Scientific Committee
Snr. Prof. Dr. M. Ravichandran
Prof. Dato' Dr. Mohd Zaki Salleh
Prof. Dr. Uda Hashim
Prof. Dr. Teh Lay Kek
Dr. Subhash J Bhore
Dr. K. Marimuthu
Dr. Lee Su Yin
Dr. V. Ravichandran
Dr. Werasak S.
Dr. P. Balakumar
Dr. Benchaporn L.
Dr. S. Kathiresan
Dr. Kazi Selim Anwar
Dr. Sivakumar Pendyala
Dr. Ramesh Kumaresan
Secretariat
Dr. Annie Jeyachristy
Dr. Heera Rajandas
Treasurer
Mr. Arvinth Murugiah
Mr. Vijayan Krishnan
Logistics Committee
Ms. Musalinah Buzri
Mr. V. Krishnan
Ms. Yoganandhini
Mr. Christapher V.
Mr. Mahes D.S.
Mr. G. Prabhakaran
Dr. D. Jawahar
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Publicity & Exhibitions Committee

Dr. Sivachandran P.
Mr. Siventhiran B.
Mr. M. K. Faiz
Mr. Dhanaraj R.
Mr. P. K. Karuna
Reception Committee
Ms. Sridevi Visvanathan
Ms. Elil Suthamathi
Dr. Tahmina Monowar
Ms. Azdlina
First Aid and Safety Committee
Dr. Sawri Rajan
Dr. Leela A. J.
Mr. Maheswaran S.
iv
Foreword
It is my great pleasure to write this foreword for this

book; because, I have attended this conference and
witnessed the success of the whole event. First of all, I
would like to thank all speakers, delegates, young
researchers and participants of the 3rd Regional
Conference on Biosensors, Biodiagnostics, Biochips
and Biotechnology 2016 (3rdRC4Bs-2016) for sharing
their research findings, views and perspectives in the
domains of Biosensors, Biodiagnostics, Biochips and
Biotechnology.
The 3rdRC4Bs-2016 brought together the leading
scientists, academicians, researchers, students, entrepreneurs and industry players in
the field of Biosensors, Biodiagnostics, Biochips and Biotechnology to discuss the
latest developments in the respective fields. This conference provided a wonderful
opportunity for all the participants not only to present their research contribution and
interact with eminent colleagues but also to widen their professional network.
I want to convey my special thanks to YBhg. Dato' Prof. Dr. Asma Binti Ismail,
Honourable Director General, Department of Higher Education, Ministry of Higher
Education Malaysia and Prof. Eiichi Tamiya, Osaka University, Japan for delivering a
key note address and visiting the AIMST University. I wish to thank all the scientists
and researchers who have travelled from 13 countries to participate in 3rdRC4Bs2016. I also wish to thank all co-organizers and supporters for supporting the
3rdRC4Bs-2016.
A proper documentation of scientific information and or events is highly important in
this modern world and I am extremely happy to know that full-length articles and
abstracts received from the participants of the 3rdRC4Bs-2016 are being published in
this book. I want to record my special thanks to Senior Associate Professor Dr.
Subhash Bhore, a highly committed organizing Chairman and Editor of this book for
his efforts in bringing out this book to document the event. I also thank the purpose
driven scientific committee, organising committee members and volunteers for their
contribution.
I am very sure that this book will serve as a reference to students, researchers,
scientists and all other stakeholders of the Biosensors, Biodiagnostics, Biochips and
Biotechnology sectors.
Thank you,
Senior Professor Dr. M. Ravichandran

Chief Executive & Vice-Chancellor, AIMST University, Malaysia
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Preface
The 21st century is the age of biological sciences and a

special emphasis on biotechnology is clearly noticeable
woldwide. Biotechnology do have a tremendous potential
not only to address the challenges in agriculture and health
care sectors but also to generate the new jobs and wealth
(bio-economy). In fact, biotechnology has become an
integral part of the knowledge-based economy of
developing and developed countries.
In biotechnology industry and health care sector, biosensors, biodiagnostics and
biochips based technologies are playing an important role in providing the most
innovative products and services. In April 2016, the 3rd Regional Conference on
Biosensors, Biodiagnostics, Biochips and Biotechnology 2016 (3rdRC4Bs-2016) was
held at the AIMST University which provided a platform for scientists, researchers,
academicians, students and stakeholders to share their knowledge, challenges, recent
advances and future perspectives in the multidisciplinary areas of biosensors,
biodiagnostics, biochips and biotechnology (4Bs). The scientific programme of the
conference was rich and wide-ranging with 2 keynote talks, 14 invited plenary talks,
38 technical papers and about 50 posters presentation.
Prof. Asma (Ministry of Higher Education, Malaysia) delivered a keynote address and
highlighted various aspects of Moving the Regional Biotechnology and Bioeconomy
Forward. By giving examples of policies and long term vision plan implemented by
Chinese Government for moving their national bio-economy forward, she highlighted
that most of the Asian countries including Malaysia have a great potential and
resources to boost regional bio-economy; however, the lack of alignment of the
numerous policies, strategies and initiatives makes it a challenge to coordinate
implementation.
In a keynote address, Prof. Eiichi Tamiya (Osaka University, Japan) highlighted the
achievements, challenges and latest trends in nanotechnology oriented biosensors and
biomedical applications. In a plenary talk, Prof. Prakash Kumar (NUS, Singapore)
highlighted that we need to use modern biotechnological approaches to enhance the
agricultural productivity for the global food security and sustainability. All 14 plenary
talks were very comprehensive and highlighted the latest trends, achievements and
challenges in various domain of biosensors, biodiagnostics, biochips and
biotechnology. This book contains full-length articles, talk abstracts of all invited
speakers, and abstracts of all technical papers presented in oral and poster sessions.
I wish to thank and acknowledge the support of all co-organizers, supporters, and
AIMST University. Nevertheless, the success of the conference was possible because
of the hard efforts of dedicated and committed members of the organizing committee
team as well as volunteers and they deserve the appreciation for it.
Subhash Bhore
July 26, 2016
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vi
Contents
Foreword ....................................................................................................................... v
Preface .......................................................................................................................... vi
Contents ......................................................................................................................vii
Full Length Articles ..................................................................................................... 1
Relevance of Biotechnological Applications for Global Food Security and
Sustainability.............................................................................................................. 1
Nano- and Bio-technological Advancement to assist in the Determination of Halal
Products.................................................................................................................... 10
Bacteriophages for Biocontrol of Foodborne Pathogens: An Overview ................. 20
Cloning and Expression of the Urease Operon from Helicobacter pylori J99 ........ 33
Production of Butter Flavour Concentrate from Butter fat with Lactic Acid Bacteria
by Solid Substrate Fermentation .............................................................................. 42
Reconfigurable Filter Bank for Accurate Spectral Decomposition of EEG Signals57
Coenzyme Q10 Dietary Supplementation during Antitubercular Therapy Prevents
Renal Damage in Rats .............................................................................................. 67
Safe Water as the Key to Food Safety and Global Health ....................................... 77
The Kratom Plant [Mitragyna speciosa (Korth.)] Paradox: Beneficial or
Detrimental? ............................................................................................................. 84
Abstracts (Keynote and Plenary Talks) ................................................................... 90
Moving the Regional Biotechnology and Bioeconomy Forward ............................ 90
Nanotechnology Oriented Biosensors and Biomedical Application ....................... 91
Current Progress in Cholera Diagnostics ................................................................. 92
Supercomputing in Biotechnology: Making Sense of Big Data .............................. 93
Molecular Approaches to Fundamental Studies on Biomarkers and Development of
Sustainable Rapid Nano-biodiagnostics to Enteric Diseases for Low Resources
Settings ..................................................................................................................... 94
Bio-Applications of Innovative Nano-materials ...................................................... 95
Aptasensors: Bench to Bedside and Beyond ........................................................... 96
Recent Progress in the Production of Biodegradable Plastics from Palm Oil in
Malaysia ................................................................................................................... 97
Recent Advances in Biosensors Based on Enzyme Inhibition ................................ 98
Genomics of the Endangered Orang Asli: Disease Susceptibility and Sustainability
.................................................................................................................................. 99
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vii
Highly Sensitive Detection of DNA Hybridization and Immunoassay Based on

Nanomaterials ........................................................................................................ 100
Fungal Secondary Metabolites - A Pharmaceutical Chemist Perspective ............. 101
Foot-and-Mouth Disease: Current Scenario in Asia and Bangladesh ................... 102
Abstracts (Oral Presentations) ............................................................................... 103
Paper-based visual detection of Salmonella bacteria using Isothermal DNA
amplification and magnetic beads aggregation ...................................................... 103
Development of a Reverse Hybridization Assay (RHA) for Simultaneous
Identification of Salmonella Serotypes Causing Enteric Fever ............................. 104
Decrypting the Evolutionary Path of Antimicrobial Resistance of Acinetobacter
baumannii via Next-Gen Sequencing .................................................................... 105
Isoluminol-functionalized gold nanoparticles and graphene oxide nanoribbons
composite for development of enzyme-based electrochemiluminescence biosensors
................................................................................................................................ 106
Disposable Screen-Printed Electrodes Modified With Nanoparticles for Sucrose
Sensor..................................................................................................................... 107
Analysis of Chalcone-Flavanone Isomerase (CHI) Gene cDNA Isolated from
American oil-palm (Elaeis oleifera) Mesocarp Tissue cDNA Library ................. 108
Non-Protein coding RNA genes as novel diagnostic markers to detect pathogenic
bacteria ................................................................................................................... 109
Herbal Based Stabilizers of Native and Misfolded State of Nuclear Co-repressor
(N-CoR) ................................................................................................................. 110
Hepatoprotective effect of methanol extract of Polygonum minus leaves in carbon
tetrachloride-induced liver damage in rats ............................................................. 111
Molluscicidal Effect of Poly herbal Extracts on Golden Apple Snail, Pomacea
maculata ................................................................................................................. 112
Design and Characterization in Time of an On-off DNA Biosensor ..................... 113
Optimization of PCR for Rapid Detection of CTX-M Gene in ESBL Producing
Klebsiella pneumoniae Clinical Isolates ................................................................ 114
Umami Tasting Detection Based Electrochemical Sensor .................................... 115
Detection of Salmonella enterica serovar Typhi Form Water Samples and Its
Association with Geographical Clustering of Enteric Fever ................................. 116
The Fabrication of Membrane-Based Pneumatic Microvalves in Microfluidic
System .................................................................................................................... 117
Role of Outermember Proteins (OMP) and Lipopolysaccharides (LPS) in Antibody
Response Against Pasteurella multocida type B-2 in Bovines ............................. 118
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viii
Exploration of Novel Endophytic Bacterial Isolates for Their Antioxidant and Prooxidant Properties .................................................................................................. 119
Sensitivity Analysis of Graphene Based Surface Plasmon Resonance Biosensor 120
Ameliorative Effect of Curcumin on Olanzapine Induced Obesity in Sprague
Dawley Rats ........................................................................................................... 121
Study of Nanoparticle-Modified Screen-Printed Electrodes for detection of Sudan I
contamination in chili ............................................................................................ 122
Bioengineering of Tacca integrifolia (Bat flower): Effects of Hormones on in vitro
Rooting and Production of Taccalonolides ............................................................ 123
Isolation, characterization and potential application of bacteriophages for phage
therapy.................................................................................................................... 124
Eco-friendly Biosynthesis of Atrocarpus altilis Mediated Silver Nanoparticles Characterization and Evaluation of its Antimicrobial and Antioxidant Potential . 125
Mutiplex Isothermal Amplification for Detection of Melioidosis ......................... 126
Effective Pulmonary Therapeutic Delivery via Surface Acoustic Waves
Nebulization and Phononic Crystal Structures ...................................................... 127
Development of a Novel Duplex PCR Assay for Specific Detection of Salmonella
enterica subspecies enterica serovar Typhi Based on Single-Gene Target ........... 128
Assessment of Biodiesel Properties From the FAME Composition of a Malaysian
Rhodophyte (Kappaphycus sp.) ............................................................................. 129
Generation of RNA Aptamers Against Mycobacterium tuberculosis Secretory
Protein ESAT-6 - a Preliminary Study .................................................................. 130
An Expression Analysis of Salmonella Pathogenicity Island (SPI)-Derived NonProtein Coding RNAs in S. Typhi Biofilm formation ........................................... 131
Quantitative, Single-Step Measurement of Hemoglobin A1c in Whole Blood for
Personalized Medicine ........................................................................................... 132
Development of Rapid Diagnostic Detection for Salmonella enterica Subspecies
enterica Serovar Paratyphi A using Cross Priming Amplification ........................ 133
Conversion of Rice Husks to Polyhydroxyalkanoate (PHA) ................................. 134
Salmonella typhimurium Detection Based on Electrochemical Immunoassay using
Methylene blue/MWNTs/Magnetic Particle .......................................................... 135
Electrochemical Characterisation and Determination of Mycobacterium
tuberculosis by Voltammetry at Polymer Nanocomposite modified Platform ...... 136
Abstracts (Poster Presentations) ............................................................................ 137
Cloning, Over-expression, and Purification of Hfq Protein from Klebsiella
pneumoniae ............................................................................................................ 137
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ix
In vitro Anti-oxidant Assay, HPLC Profiling of Polyphenolic Compounds, AAS

and FTIR Spectrum of Malaysian Solanum torvum Swartz fruit .......................... 138
Phytochemical Analysis and Antioxidant Activity of Malaysian Medicinal Plant
Abroma augustum Leaf Extract ............................................................................. 139
Reduced Reproductive Function up to Three Generations of Rats Due to Paternal
Heroin Addiction ................................................................................................... 140
Optimization of Cryopreservation Using Different Cryoprotective Agents and
Differential Temperatures on Freeze Dried Probiotics .......................................... 141
Development of a Reusable Electrochemical Immunosensor for Direct Detection of
Small Organic Molecules ....................................................................................... 142
Over-expression and Purification of an RNA Chaperone, Hfq Protein of Proteus
mirabilis ................................................................................................................. 143
Peritoneal Mast Cell Stabilization and Toxicological Properties of the Ethanolic
Extract of Solanum trilobatum Linn. ................................................................... 144
Understanding the Host-Pathogen Interaction in Klebsiella pneumoniae Infected
Rat Model via Metabolomics Approaches ............................................................. 145
New PDE4 Inhibitors: Design, ADMET and Docking Studies on Chalcones and
Flavones for Anti-Inflammatory Activities ........................................................... 146
Effect of Telfaira occidentalis in Mice Fed Aflatoxin Contaminated Feed .......... 147
Accuracy of Rapid Point-of-Care Diagnostic Tests for Acquired Immune
Deficiency Syndrome - A Systematic Review and Meta-analysis ........................ 148
Biocontrol of Macergen Infestation on Plants using Bacteriophage Cocktail ....... 149
Oral Bacterial Diversity Study in Malay Ethnic Group in Malaysia ..................... 150
Isolation and Characterization of Seven Lytic Bacteriophages As Candidates for
Phage Therapy ....................................................................................................... 151
Transcriptome Analysis for the Identification of Novel ncRNAs in Acinetobacter
baumannii .............................................................................................................. 152
Computational Modelling of the Newly Synthesized Chalcone Derivatives in
Inhibiting 5-lipoxygenase ...................................................................................... 153
Computational Design of Flavone and Chalcone Derivatives as Cyclooxygenase-2
(COX-2) Inhibitor .................................................................................................. 154
Identification of Novel npcRNA Candidates in Klebsiella pneumoniae ............... 155
Arduino Microcontroller Based Heart Rate Monitor using Fingertip Sensors ...... 156
Transcriptome Analysis of Proteus mirabilis during Oxidative Stress Adaptation
................................................................................................................................ 157
Safety and antiobesity effect of Garcinia atroviridis ............................................ 158
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Development of Ochratoxin A Detection Based on Electrochemical Sensor by

Using Au-ball Labels ............................................................................................. 159
Effect of Different Diets on the Growth and Survival of Silver Arowana
(Osteoglossum bichirrosum) .................................................................................. 160
Effect of Aqueous and Methanol Extracts of Polygonum minus Leaves on DrugInduced Hepatotoxicity in Rats .............................................................................. 161
Identification of Novel Non-protein Coding RNAs (ncRNAs) in Staphylococcus
haemolyticus Biofilm ............................................................................................. 162
Application of a Novel Lytic Bacteriophage Strain as Biocontrol Agent for Water
Sanitization ............................................................................................................ 163
Phytochemical Analysis and Pharmacological Screening (Dopamine level) of the
Ethanolic Extract of Solanum trilobatum Linn. ..................................................... 164
Computational Analysis of Common Bean (Phaseolus vulgaris L.) S-adenosyl-Lmethionine dependent methyltransferase gene cDNA Isolated from Bean-pod-tissue
cDNA Library ........................................................................................................ 165
Biochemical Changes in African catfish, Clarias gariepinus Exposed to
Buprofezin.............................................................................................................. 166
Haematological Changes in African catfish, Clarias gariepinus exposed to
Buprofezin.............................................................................................................. 167
Identification of Novel Non-coding RNA (ncRNA) from Tannerella forsythia ... 168
Microbial Load, Antimicrobial Sensitivity and Plasmid Profiles of Vibrio cholerae
in Fruit Juice .......................................................................................................... 169
Isolation of Arsenite Resistant Bacteria from Ground Water and Soil of Dhaka,
Bangladesh ............................................................................................................. 170
Compared to Serum Media, Serum-Free Medium Enhanced the Generation of
Mesenchymal Stem Cells Derived from Full-Term Human Amniotic Fluid ........ 171
Hybrid Assembly of Salmonella enterica subsp. enterica ser. Typhi Isolates
PM016/13 and B/SF/13/03/195 from a Typhoid Outbreak in Pasir Mas, Kelantan in
2013........................................................................................................................ 172
Identification and Characterization of Novel Non-protein Coding RNAs in
Salmonella serovar Typhi Biofilm ........................................................................ 173
Identification of Novel Non-coding RNA (ncRNA) from Bacillus thuringiensis . 174
Morphological and Differential Protein Expression Analysis of Placentas from
Term and Spontaneous Preterm Labor with Intact Membrane .............................. 175
Expression of Salmonella Typhi Ty21 TolC Protein in Different Host Cells ....... 176
Stemness of Spontaneously Transformed Murine Bone Marrow Mesenchymal Stem
Cells is Maintained Upon Prolonged In Vitro Expansion ...................................... 177
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xi
16S Ribosomal DNA Sequence Based Identification of Bacterial Endophytes

Isolated from Seeds of Starfruit (Averrhoa carambola L.).................................... 178
An Alternative Method of Screening the M2/ANXA5 Haplotype for Repeated
Pregnancy Loss ...................................................................................................... 179
Computational Analysis of Class IV Chitinase Gene cDNA Isolated from American
Oil Palm (Elaeis oleifera) Fruit Mesocarp Tissue cDNA Library......................... 180
In Silico Analysis of Omega-6 Fatty Acid Desaturase Gene cDNA Isolated from
Common Bean (Phaseolus vulgaris L.) Pod Tissue cDNA Library ...................... 181
Computational Analysis of Common Bean (Phaseolus vulgaris L.)
Palmitoyltransferase Gene cDNA Isolated from Bean Pod Tissue cDNA Library
................................................................................................................................ 182
Annotation of Elaeis oleifera CAP cDNA and its Deduced Amino Acid Sequence
using Computational Tools .................................................................................... 183
Comparison of Methods for Pure Quality RNA Isolation from Polysaccharide Rich
Averrhoa carambola L. Fruits (Starfruits) ............................................................. 184
Appendices ................................................................................................................ 185
Appendix I: Brief biography of keynote and plenary speakers who delivered their
talk in 3rdRC4Bs-2016. .......................................................................................... 185
Appendix II: A copy of scientific programme of the 3rdRC4Bs-2016................... 198
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xii
Men love to wonder, and that is the seed of science.

--- Ralph Waldo Emerson
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xiii

Res. Highl. 4Bs (2016), P1-9
Relevance of Biotechnological Applications for Global Food Security and

Sustainability
Prakash P. Kumar*
Department of Biological Sciences, National University of Singapore, 10 Science Drive 4,
Singapore 117543;*corresponding author, e-mail: dbskumar@nus.edu.sg
ABSTRACT
In this review, I will briefly discuss the use of biotechnology in the recent past for crop plant
improvement with some specific examples of successes. These will include brief discussions on
herbicide/insect resistance, drought tolerance, and golden rice. Some of the main objections
raised against GM crops will be examined briefly to see if these objections have reliable
scientific basis. Breeding in a number of animal and plant species has been revolutionized by the
emergence of DNA markers such as single nucleotide polymorphism (SNP) arrays. The method
is based on the prediction of genetic merit by incorporating relationships among individuals
based on SNP array data. This process is known as genomic selection. Some of the current
developments in the field, including genome-wide association studies (GWAS) tools applied for
crop plant improvement will be discussed with an example of a recent study on oil palm.
Another area that is making rapid progress in biotechnology is genome editing. Genome editing
tools that are currently being developed include transcription activatorlike effector nucleases
(TALENs), zinc-finger nucleases (ZFN) and Clustered regularly-interspaced short palindromic
repeats (CRISPR) paired with CRISPR-associated (Cas) nuclease system. Of these, the
CRISPR/Cas9 tool, which is based on bacterial immune system, holds great promise for crop
biotechnology as well as biomedical fields. The clustered repeats in the bacterial DNA
correspond to captured DNA fragments of pathogens that invaded the cells. The Cas9
endonuclease detects these and using a guide RNA molecule with complementarity to the DNA,
it serves to cut the new invaders with similar DNA material. This is now adapted to create
specific breaks and repairs in the genomic DNA of eukaryotic cells, thereby achieving DNA
editing. The tools are being refined such that when genome editing is finished, the resulting
plants will be treated just as natural mutants rather than transgenic plants. Highlights of these
biotechnological tools will be examined with a discussion on how these can contribute to future
global food security.
Keywords: Agriculture; Biotechnology; DNA; Food; GM technology; Sustainability.
PREAMBLE
The use of genetically modified (GM) crops
has been made into a controversial topic that
elicits quite polarized views. This article is
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based on a plenary talk delivered at a

conference. Based on scientific evidence and
first-hand experience with GM plant
production, the intended take-home message
of this talk was that we should not hesitate to
Res. Highl. 4Bs (2016)
Global Food Security and Sustainability

use modern technologies for crop
improvement. It is acknowledged that
having adequate tests to verify the general
safety of the GM crops is important, but
once proven safe (or not significantly
different from the conventional crop
variety), the GM crop should be used as a
valuable alternative. With judicious use of
technology and crop management practices,
we can achieve global food security.
GM FOOD
What is Genetic Modification (GM) and is
it vastly different from conventional plant
breeding in its principle?
Crop modification has been ongoing for
thousands of years. The use of everchanging technologies has been a constant
theme in this as with all other modern
human endeavors. We recognize that use of
any novel technology involves risks and
benefits, it is up to us to evaluate them and
decide to adopt them if the benefits vastly
outweigh risks. Also, human ingenuity is
such that technology can be constantly
refined to minimize the initial risks to
negligible levels as was done with the
minimization of radiation emission in the
present day cell phone or mobile phone
technology. The yield enhancement as a
result of the Green Revolution has reached
its limit now. Although it is known that crop
plants have not reached their biological
upper limits for yield increase, any further
increases in crop yield will have to rely on
novel tools. One of the relatively recent
novel technologies is genetic modification or
genetic engineering (also known as
biotechnology,
recombinant
DNA
technology, etc.).
Two main approaches in GM plant
production are: a) Modifying existing genes
in plants - e.g., fruit ripening by modifying
ethylene biosynthesis or ethylene action, and
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Kumar
b) Expression of introduced (foreign) gene
to confer a new/modified trait to the crop
plant, e.g., carotenoid production in the
endosperm tissue of the Golden Rice grains.
The main examples of GM plants
The main examples of GM plants that have
been commercially cultivated for the last 20
or so years include herbicide resistant and
insect resistant plants. The major crops with
such GM varieties include corn, soybean,
canola and cotton. In 2013, GM crops were
grown in about 175 million hectares of land
by 18 million farmers in 27 countries
(James, 2013). The global market value of
GM crops for 2013 was estimated to be
about US$15.6 billion.
Some of the other traits being introduced to
crop plants include drought tolerance,
disease tolerance (both fungal and viral),
resistance towards root nematodes, modified
nutritional parameters such as altered lipid
profiles in oil crops and fortified grains.
Although the Golden Rice has been fully
developed and safety tested it has gone
through the so called deregulation process
mandatory for GM crops it has not yet
received
the
necessary Government
approvals. The Golden Rice has been touted
as a staple grain that will lead to vast
improvements in the nutritional status (with
respect to vitamin A and iron) of millions of
poor people in Asia and Africa.
Nevertheless, it has not been released to the
growers. Scientists at the International Rice
Research Institute, Philippines (IRRI) have
developed Golden Rice varieties suitable for
several rice growing regions with the help of
generous funding from philanthropic
organizations such as the Bill and Melinda
Gates
Foundation.
Therefore,
when
approved, the Golden Rice seeds can be
distributed to growers without profiteering.
The IRRI has established a highly positive
and remarkable reputation for successfully
2

releasing the flood tolerant SUB1 rice that
was developed by non-GM methods (marker
assisted breeding). However, it must be
highlighted that this could not have been
achieved without data from GM tools. Prior
studies using molecular biological tools
helped to identify the flood tolerant variant
of the Sub1a gene in rice. Experimental GM
rice plants demonstrated that when this
resistant form of the gene was introduced to
flood sensitive rice varieties they could be
turned into flood tolerant forms. Therefore,
without GM tools scientists could not have
so easily developed the marker assisted
SUB1 rice varieties in such a short time
span. These varieties dubbed Scuba Rice
are being grown for the past several years by
thousands of happy farmers in millions of
hectares of land, for example, in the flood
prone areas of India and Bangladesh.
OTHER GM FOOD
Besides the examples cited above, there are
several other minor successes in GM plants
including the virus resistant GM papaya in
Hawaii, the delayed ripening tomatoes, as
well as some ornamentals such as long-vaselife carnation flowers and the Blue Rose
marketed by Suntory Biotechnology
company.
The use of GM products such as
recombinant proteins, mainly enzymes in
food industry is noteworthy development.
GM or Recombinant proteins from bacteria
and fungi are used in cheese making. Cheese
production requires the use of a protein
called chymosin (also known as rennin or
rennet). Chymosin was previously obtained
only from calf stomachs, which had several
implications including health concerns,
conflict with nutritional preferences due to
religious restrictions or personal choices of
various groups of people. The use of
chymosin from GM microbes has become
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Kumar
common in cheese making now, with
estimated 70% to 90% of cheeses in the
USA manufactured using such microbial
GM rennet now. Also, the US Food and
Drug Administration (FDA) has approved
over 30 other recombinant enzymes for use
in food production (including -amylase,
used to make glucose or fructose syrups).
Another noteworthy development in the
recent years is the FDA approval of the first
transgenic animal for food use, namely, GM
salmon. After an exhaustive and rigorous
review that lasted nearly twenty years, the
FDA has approved the use of fast growing
GM salmon (AquAdvantage) in November
2015.
FDA
determined
that
the
AquAdvantage salmon is as safe to be
consumed by humans as the commercial
non-GM Atlantic salmon. This was a longawaited positive decision welcomed by
biotech scientists around the world.
AGRICULTURAL ECONOMY
How important are GM crops to the
agricultural economy?
An economic assessment of the impact of
commercial agricultural biotechnology on
global agriculture was done (Brookes and
Barfoot, 2009). The study highlighted the
significant impacts on the production of
soybeans, corn, cotton and canola crops.
According to this study, GM technology led
to net economic benefits at the farm level of
about US$10.1 billion in 2007. The other
benefits associated with the use of GM had a
positive impact to the tune of 25% of the
total direct farm income benefit in the USA.
Biotech crops contributed to increasing
global production levels of the four main
crops examined. For example, the use of
GM soybeans corresponded to adding 68
million tons and GM corn added 62 million
tons to global production levels in 2007.
Their use was equivalent to 172 million kg
3

reduction in pesticide use (which
corresponds to 14% reduction in the
environmental footprint associated with
pesticide use). In addition, other factors
from growing these biotech crops
significantly reduced greenhouse gas
emissions from agriculture equivalent to
removing 5 million cars from the roads
(Brookes and Barfoot, 2009).
THE NEED TO INCREASE FOOD
PRODUCTION
Why not just stick to old ways of producing
food? Why resort to new tools? According
to a UN study, the world population is
expected to grow to ~9.7 billion by 2050.
According to that study, Africa and Asia
would be adding 1.14 billion more people to
their population. By 2050, Africa (2478
million) and Asia (5267 million) collectively
will have a population size larger than the
current world population.
Kumar
Further complications are already evident
from the global climate change. The
predicted impacts of climate change include,
reduced wheat production in South Asia by
2030, and rice production in Southeast Asia,
particularly in the Greater Mekong
Subregion (ADB, 2009). These will almost
certainly lead to increased food prices. The
ADB estimates that rice, wheat, and soybean
prices could increase by 10%50%, while
corn price may double by 2050. This should
be recalled in connection with the global
food and energy price surge in 20072008,
which exposed the vulnerabilities of
households and of the international food and
nutrition insecurity. Increase in extreme
weather events (floods, droughts, typhoons)
will have serious consequences for
agriculture, food, and forestry production in
the coming decades. Therefore, it is
imperative that crop yield has to be raised to
ensure food security.
FOOD PRODUCTION AND WATER
Average Asian rice consumption is about

100 kg per person per year. The number of
undernourished people in the world is close
to 1 billion and nearly two-thirds of the
worlds hungry people reside in Asia and the
Pacific (FAO, 2009).
POPULATION
AND
CLIMATE
CHANGE V/S FOOD SECURITY
It is well recognized now that crop yield has
to be raised to ensure food security in the
coming decades. Global food production
will need to increase by 40% by 2030 to
keep pace with global demand (FAO, 2009),
because the world population is increasing.
The maxim Less food, more mouths to
feed is made relevant when we realize the
fact that the land area available for crop
cultivation is continually decreasing as the
population keeps increasing.
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The growing worldwide shortage of water is

extremely worrisome. The global demand
for water is estimated to double by 2050.
However, about 35% of the world
population is already facing water shortages.
Asia is facing acute water shortage, with
available freshwater being unevenly
distributed. Under these circumstances, it is
obvious that to grow more food with less
water, crop productivity needs to be
improved. However, the traditional breeding
tools are often inadequate to achieve such
spectacular increases in yield.
Water and rice - how are the two related? To
grow the current world production of
approximately 500 million tons of rice it is
estimated that about 1,750 km3 fresh water
is needed per year [1 km3 = 1000 billion
liters (=1012 liters)]. If we take overall
agriculture, it accounts for about 80% of
4

world
water
use
(www.adb.org/publications/asian-waterdevelopment-outlook-2013).
Currently,
~40% of this water is lost due to inefficient
agricultural practices (crops and farm
animals). The water required for production
of some of the key agricultural produce
illustrate the vastness of water needed in
agriculture. Liters of water necessary per kg
of produce (indicated in parentheses): for
rice (2,500), other grains in a favorable
climate (2,000), grains in arid climate
(5,000) and beef (15,000). Therefore, the
total world water use is 7,450 to 8,160
km3/year, and the water used for food
production is estimated to be 6,390 to 7,200
km3/year, with domestic needs accounting
for 180 to 344 km3/year (IRRI, and various
other sources).
Some of the major challenges for rice and
grain production include abiotic and biotic
stresses. The major abiotic stresses are (1)
cold (a climatic factor that affects ~66% of
land worldwide), (2) drought, (3) flood
(~10% of yield loss, ~25% of the global rice
crop lands sees floods each year with ~20
million hectares of Asian rice land prone to
flooding), (4) salinity (~10% of worlds land
affected and ~12 million hectares in Asia
affected by increased soil salinity).
SUSTAINABLE FOOD PRODUCTION
STRATEGIES
There are several ways to increase crop
yield. Four of the prominent options are
mentioned here. (1) Expanding the
cultivated area: land expansion can account
for ~ 20% increase in yield. (2) Increasing
the cropping intensity can contribute about
10% increase. (3) Reducing pre- and postharvest loss and wastage: 20 to 30% increase
if all wastage is prevented, (4) Improving
crop yields using latest knowledge and
technologies. Therefore, about 40% increase
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Kumar
in crop yield has to be through plant
breeding and biotechnology.
In view of the above, the answer to the
question of whether we should use novel
technologies (e.g., GM technology) for crop
improvement
should
be
obviously
affirmative! I cite the example of breast
feeding v/s infant formula in support of this
view. We all know breast feeding is the best
nutritional option for infants. But, global
market for baby food is huge (over US$10
billion/year) with 50% to 70% of it as the
value of infant formula! North America and
Europe constitute 33% of this market, while
Asia forms 53% of the infant food market.
Over 120 companies manufacture and sell
infant formula! What has happened here is
the need for convenience (in most cases,
necessity in some) has led to the adoption of
an alternative to breast feeding as an
acceptable alternative for our infants. In a
similar rational manner, we need to develop
modern technologies for crop improvement
and ensure food security. We should not
hesitate to use new technologies to develop
alternative options that should coexist with
the established techniques.
What are the other alternatives?
Certainly, GM crops are not the only option
to increase crop yield they are an efficient
and complementary tool to the established
tools at our disposal. It is recognized that
efficient crop management will improve
yield to some extent. These include the use
of right amount of chemical fertilizers and
pesticides, because excess use of such
agricultural chemicals leads to yield loss as
well as air and water pollution. Ecological
engineering is another concept being tested
with positive effect. For example, the use of
beneficial insects for pest control by
growing flowering plants around the fields is
one such ecological modification tool. This
method helped to reduce pesticide use by
5

~21% in Vietnam according to a study
coordinated by IRRI. Alternate Wetting and
Drying (AWD) method is another crop
management tool. AWD can lead to ~35%
less water requirement for rice growing
compared to the traditional farming method,
with ~10% yield increase and attendant
reduction in greenhouse gas (methane)
emission from the fields. The reduction in
methane emission is due to the reduction in
methanogenic bacteria growing in the root
zones of rice plants when cultivated under
permanent submergence (leading to
anaerobic soil favoring the methanogenic
bacteria). The AWD management practice
leads to reduced growth of such bacteria
along with reduced water needs.
Marker-assisted breeding, genome-wideassociation study (GWAS) leading to
Genomic Selection (GS) are established as
non-GM alternatives for crop improvement
in the past decade or so (Meuwissen et al.,
2001, Desta and Ortiz, 2014). Genomic
Selection is based on the powerful tools of
genome-wide
prediction
in
plant
improvement. GWAS helps to generate
statistical models to predict how a plant will
perform before conducting a genetic cross
and field-testing of actual crops. This
involves the combined use of genomic
information, statistics, bioinformatics tools,
and prediction-based breeding for crop
improvement. Of course, this requires that
the scientists establish robust association
between DNA seqence (SNPs) and specific
traits for a large number of breeding lines
(1000s of individual plants). This will then
be used to estimate Genomic Breeding
Values (GEBV). New plants to be used for
breeding will then be sequenced; for each
new breeding line a DNA-profile will be
determined and using such profiles from
each line scientists will estimate GEBV for
every trait of interest. Based on the genetic
potential for the different traits the breeder
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Kumar
can design a new variety on the computer
which combines plant lines with highest
scores (using the predictive models built).
One of the more recent GWAS studies with
impressive results is on oil palm (Teh et al.,
2016). Also, several improved crops
generated using these techniques are being
released and more will be coming in the near
future. The gene that facilitates deep rooted
phenotype beneficial to develop drought
tolerant crops, e.g., DEEP ROOTED1 gene
or DRO1 of rice was identified by GWAS
approaches while as mentioned above, the
SUB1 rice was developed by marker
assisted breeding.
Genetically modified (GM) crops: to use or
not?
Based on ample evidence from the use of
GM crops during the past couple of decades
it is safe to conclude that GM food can save
millions of kids from dying due to
malnutrition. The use of GM crops can also
help to reduce pesticide usage and
environmental pollution. In this connection
it is worth remembering that technological
advances
are
continually occurring.
Accordingly, new genome editing methods
may be seen as good alternative tools to
generate the new generation, advanced crop
plants. Besides GM, several non-GM
strategies are being developed and some of
the novel strategies for crop breeding by
genome editing include: (a) TALENs (b)
Zinc Finger Nucleases (ZFN) (Petolino and
Kumar, 2016) and (c) CRISPR-Cas9 system
(Chen and Bailey, 2016; Kleinstiver et al.,
2016).
TALEN stands for Transcription Activatorlike
Effector
Nucleases;
while
CRISPR/Cas9
stands
for
Clustered
Regularly Interspaced Short Palindromic
Repeats/CRISPR-associated(Cas)
system.
CRISPR/Cas9 was first named as Short
Regularly Spaced Repeats (SRSR) in 2000.
6

It was renamed as CRISPR in 2002.
CRISPR/Cas9 induces double-strand break
(DSB), generates deletion and/or insertion of
short sequences at the break leading to
altered gene of interest. In fact, the common
feature of all these genome editing tools is
that they induce DSBs in the genomic DNA
at specific locations (gene of interest) with
or without short insertions. The DSB will
then be repaired by one of two repair
processes, namely, Non-Homologous End
Joining (NHEJ), or Homology Dependent
Repair (HDR). This results in gene editing.
FUTURE PERSPECTIVES
Gene-edited CRISPR mushroom has been
generated recently and it was exempted from
having to go through US Regulatory
process; because, such organisms do not
harbor foreign genes and are similar to
induced mutants. The white button
mushrooms generated have their polyphenol
oxidase gene edited, which helps to prevent
browning
(Waltz,
2016).
Scientific
American (14 April 2016) also indicated that
this is one of almost 30 genome edited
organisms to avoid regulatory requirement
currently. These represent the first few
improved crops and more such crops will be
released by various companies in the years
to come (Wolt et al., 2016).
Kumar
direct beneficiaries until now. But, when we
carefully examine it there are already plenty
of indirect benefits for all. GM papayas
(ring-spot virus resistant) saved the papaya
industry in Hawaii, GM crops will play an
important role in protecting soil and water
resources, minimizing crop diseases and
attendant loss of produce, and responding to
the pressures of climate change. Cultivation
of Bt-cotton has reduced the amount of
neurotoxic insecticide use by millions of
kilograms every year in many countries
where it is grown. Hence, one way of
looking at it is that the concept of organic
crops and GM crops coexist! GM crops do
not pose any conflict with traditional or
organic farming. A book published by
Oxford University Press in April 2008 by
authors Pamela Ronald and Raoul
Adamchak (UC, Davis) advocates a food
system that is organic and genetically
engineered! They argue that a judicious
blend of GM and organic farming
representing two important strands of
agriculture is key to helping feed the world's
growing population in an ecologically
balanced manner.
If they are safe why is there hesitation to

use GM crops?
One of the possible contributing factors is
that the general public and most of the
opponents of GM crops do not fully
understand the underlying process. Perhaps
equally importantly, consumers are not
direct beneficiaries of the GM technology so
far. GM crops of the past have been
designed to enhance the productivity of
industrial farming so only the farmers who
adopted the GM crops and the seed
companies that sell them have been the
Even newer GM crops that will directly

benefit the consumers are coming, for
example, the purple tomatoes produced by
Prof Cathie Martins lab, John Innes Centre,
UK. These GM tomatoes have high amounts
of plant nutrients (e.g., anthocyanins found
in some berries), which are super foods that
will confer tremendous health benefits to the
consumers. The purple tomatoes were grown
in controlled greenhouses in Canada and
juice from these GM tomatoes is undergoing
tests and evaluations. Clearly, this represents
a super food that could not be otherwise
made available to us in plentiful quantities.
They have already shown that inclusion of
these high-anthocyanin GM tomatoes in the
diet of cancer-prone mice can extend their
healthy life-span by 30% - a most welcome
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prospect indeed! It is a firm belief of the
author that people will line up for this type
of GM superfoods and demand that more be
supplied!
From the foregoing discussion it is clear that
the future global food supply can benefit
greatly by the use of new initiatives in
research. We should be open to employ a
multidisciplinary
approach
involving
molecular genetics, cell and developmental
biology, systems biology, as well as
ecology. GM crops have been grown and
consumed for over 20 years now and the
underlying technology is continually being
improved. Hence, the emerging novel tools
such as genome editing will help us alleviate
food scarcity in the future decades. We
should be mindful to develop safe
technology and develop suitable alternative
strategies such as GM, molecular marker
assisted breeding and genome editing.
Collectively, these will serve us well in
ensuring future food security.
REFERENCES
ADB
(2009). Operational plan for

sustainable food security in Asia and
the Pacific. Document downloadable
from
ADB
web
site.
http://www.adb.org/publications
Brookes G, Barfoot P (2009). Global

impact of biotech crops: Income and
production
effects,
1996-2007.
AgBioForum 12(2), 184-208.
Chen H, Bailey S (2016). Cas9, poised for
DNA
cleavage.
Structural
rearrangements explain how Cas9 cuts
DNA. Science 351, 811-812.
Desta ZA, Ortiz R (2014). Genomic
selection: genome-wide prediction in
plant improvement. Trends in Plant
Science 19, 592-601.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Kumar
FAO paper (2009). How to Feed the World
in 2050? Document available at the
FAO
web
site.
http://www.fao.org/fileadmin/template
s/wsfs/docs/expert_paper/How_to_Fee
d_the_World_in_2050.pdf
James, C. (2013). Global status of
commercialised biotech/GM crops:
2013, ISAAA Brief No. 46.
International
Service
for
the
Acquisition
of
Agri-Biotech
Applications, Ithaca, NY. ISBN 9781-892456-55-9.
www.
isaaa.
org/resources/publications/briefs/46,
2014.
Kleinstiver BP, Pattanayak V, Prew MS,
Tsai SQ, Nguyen NT, Zheng Z,
Joung JK (2016). High-fidelity
CRISPRCas9 nucleases with no
detectable genome-wide off-target
effects. Nature 529, 490-495.
Meuwissen THE, Hayes BJ, Goddard ME
(2001). Prediction of total genetic
value using genome-wide dense
marker maps. Genetics 157, 1819
1829.
Pamela C. R. and Raoul, W. A. (2008).
Tomorrow's Table: Organic Farming,
Genetics, and the Future of Food,
Oxford University press (OUP).
Petolino JF, Kumar S (2016). Transgenic
trait deployment using designed
nucleases.
Plant
Biotechnology
Journal 14, 503509.
Teh CK, Ong AL, Kwong QB, Apparow
S, Chew FT, Mayes S, MohamedM,
Appleton D, Kulaveerasingam H
(2016). Genome-wide association
study identifies three key loci for high
mesocarp oil content in perennial crop

oil palm. Scientific Reports 6, 19075 |
DOI: 10.1038/srep19075
Waltz E (2016). Gene-edited CRISPR
mushroom escapes US regulation. A
fungus engineered using CRISPR
Cas9 can be cultivated and sold
without oversight. Nature 532, 293.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Kumar
Wolt JD, Wang K, Yang B (2016). The
regulatory status of genome-edited
crops. Plant Biotechnology Journal
14, 510518.

Res. Highl. 4Bs (2016), P10-19
Nano- and Bio-technological Advancement to assist in the Determination

of Halal Products
Quamrul Hasan1, 2, *
1
College of Business, Universiti Utara Malaysia (UUM), 06010 Sintok, Kedah, Malaysia; 2Japan
Halal Research Institute for Products and Services (JAHARI), Kobe, Japan; *corresponding
author, e-mail: quamrul@uum.edu.my / quamrulhasan@gmail.com
ABSTRACT
Aims: Halal industry science can be defined as the experimental investigation of the consumable
product by using scientific (analytical) method to reveal its contents, thus assisting to determine
the product is halal or not. This scope can be further extended to address any relevant issues on
the halal products involving scientific and technological advancements. This definition is
contrived for the first time in this article by the author. The present study was conducted in
collaboration with university, industry, professional laboratories and governmental organization,
which was aimed in finding out how effective the currently available analytical methods
especially when the results were targeted to be used in the determination of the Halal products.
Furthermore, in this study, the successful development of a protein-based (immunochromatography) test kit for the purpose was explained. Methodology and results: DNA
extraction was performed using commercially available DNA extraction kit from QIAGEN or
NEOGEN. The DNA extraction was performed in duplicate for each sample. PCR-conventional
was performed using Thermal cycler (GeneAmp PCR System 9700, Applied Biosystems).
Porcine and Bovine specific primers for mitochondrial DNA sourced from Food and Agricultural
Materials Center, Japan (FAMIC) were used. In addition, for comparison NEOGEN primer
specific for porcine genomic DNA was used for one sample. A total of 4 commercial products (3
food/snack, 1 functional cosmetic) were tested in this study. Among these, except 1
marshmallow product which might be fish DNA positive, 3 were found as porcine DNA positive.
One of the porcine DNA positive product failed to show the same result when it was tested using
the commercial kit-NEOGEN- containing porcine genomic DNA. None of these products were
found as bovine DNA positive. Conclusion, significance and impact of study: The determination
of the Halal is a very sensitive issue. Therefore, in this study, we have concluded that in
determination of the Halal in a processed and commercialized product by employing a single
approach or method especially when targeting DNA is not enough to confirm the authenticity of
the test result due to possible limitation of the method used. We have proven that the primers for
mitochondrial DNAs sourced from FAMIC, Japan could be more reliable for the purpose. The
effective collaboration between industry, academia and related professional organizations for
developing innovative Halal test kit successfully is critical.
Keywords: Biotechnology; Determination of halal products; Halal industry science;
Nanotechnology.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
10
Determination of Halal Products

INTRODUCTION
As processed and prepared ready-to-eat food
products with animal origins have become
increasingly available to consumers, due to
technological advances, the possibility of
fraudulent adulteration and substitution of
the expected species (source) with other
sources has also increased. The same goes
to confectionery and functional cosmetic
products especially containing gelatin or
collagen peptides. Such practices pose a
substantial concern to consumers in terms of
economic
loss,
allergies,
religious
observance, loss of traceability, and food
safety. In many cases, porcine derivatives
are used due to cheaper price and readily
available. For Muslim consumers, the major
authenticity concerns are in meat and meat
products include porcine gelatin, collagen,
fat, and so on. The analytical methods used
for Halal authentication of meat and meat
products include: Polymerase Chain
Reaction
(PCR);
Enzyme
Linked
Immunosorbent Assays (ELISA); Mass
spectrometry; Chromatography, Electronic
nose and Spectroscopy. An overview of the
currently available analytical methods or
techniques is given in Table 1. The
analytical methods currently in use to detect
the presence of porcine materials are mainly
protein and DNA-based. These are described
below.
DNA-based detection
PCR is capable of amplifying very few
copies of DNA and its detection limit is
much lower than what is observed with
protein based assays. PCR amplication is
based on hybridization of specic oligonucleotides to a target DNA and synthesis of
million copies anked by these primers. The
simplest PCR strategy applied to evaluate
presence of any species in a meat product is
the amplication of DNA fragments,
followed by agarose gel electrophoresis for
fragment size verication. To successfully
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Hasan
detect a species with PCR, adequate genetic
markers are chosen to develop the assay.
Either nuclear or mitochondrial genes can be
targeted (Fajardo et al., 2008). However, the
use of mitochondrial DNA (Mt DNA) offers
a series of advantages over cell nucleus
DNA. Mitochondrial DNA facilitates PCR
amplication, even in cases where the
availability of DNA template after its extraction is insufcient for detection
(Murugaiah et al., 2009). This is attributed to
the fact that Mt DNA is several fold more
abundant than that of nuclear genome; each
mitochondrion is estimated to contain 2 to
10 Mt DNA (Murugaiah et al., 2009).
Furthermore, Mt DNA evolves much faster
than nuclear DNA and henceforth contains
more sequence diversity facilitating the
identication of phylogenetically
related
species (Fajardo et al., 2010; Girish et
al., 2005; Murugaiah et al., 2009). Among
the mitochondrial genes, cytochrome b (cyt
b) (Aida et al., 2005; Murugaiah et al.,
2009) and 12S rRNA (Chen et al., 2010;
Girish
et al., 2005) are the most
commonly used markers
in
the
development of DNA methods for meat
species authentication.
Protein-based detection
Porcine protein, due to it is being cheap and
readily available, might fraudulently be used
to substitute other animal proteins. ELISA is
the most commonly used method to detect
animal proteins and a number of commercial
immunoassays are available. Chen and
Hsieh (2000) were the first ones to develop
the immunoassay (ELISA) using a
monoclonal antibody to a porcine
thermostable muscle protein for detection of
pork in cooked meat products. They
observed no cross-reactivity with common
food proteins. By employing this technology,
the first pork detection kit was developed in
Japan by a Japanese company (Okamoto,
2016). This kit is immuno-chromatographic
employing nano-sized colloidal gold
11

particles to detect presence of pork in food
samples. It can detect pork in both raw and
cooked food. It allows rapid detection of
pork in food samples at low cost without
Hasan
using any special equipment or requiring
skill. The basic principle of immunechromatographic kit is shown in Figure 1.
Table 1: Summary of analytical techniques applicable in the halal authentication of meat

products.
Authenticity issue
Pork adulteration
Species identification
Analytical Techniques
PCR-RFLP
Real time PCR
Species-specific PCR
Pork protein
Pork fat (lard)
RAPD
PCR sequencing
ELISA
Chromatography
Peptide examination
Isoelectric focusing
FTIR spectroscopy
DSC
Electronic nose
Blood plasma
Isoelectronic focusing
ELISA
Immunodiffusion
LC MS/MS
References
Murugaiah et al. (2009), Aida, Che Man, Raha,
and Son (2007), and Aida et al. (2005)
Martn et al. (2009), Kesmen, Gulluce, Sahin,
and Yetim (2009), Tanabe et al. (2007),
Fumire, Dubois, Baeten, von Holst, and
Berben (2006), and Lpez-Andreo, GarridoPertierra, and Puyet (2006)
Soares, Amaral, Mafra, and Oliveira (2010),
Alaraidh (2008), Che Man et al. (2007) and
Montiel-Sosa et al. (2000)
Martinez and Malmheden Yman (1998)
Karlsson and Holmlund (2007)
Chen and Hsieh (2000); Chen and Hsieh (2000)
Chou et al. (2007)
Aristoy and Toldra (2004)
Hofmann (1985)
Rohman, Sismindari, Erwanto, and Che Man
(2011a, 2011b), Che Man, Abidin, & Rohman,
2010, Rohman and Che Man (2011a, 2011b),
Rohman and Che Man (2009), Che Man, Gan,
NorAini, Nazimah, and Tan (2005), Che Man,
Syahariza, Mirghani, Jinap, and Bakar (2005)
and Che Man and Mirghani (2001)
Marikkar, Ghazali, Man, and Lai (2003) and
Marikkar, Lai, Ghazali, and Che Man (2001)
Nurjuliana, Che Man, and Mat Hashim
(2011a), Nurjuliana, Che Man, Mat Hashim,
and Mohamed (2011b), Che Man, Gan, et al.
(2005), and Che Man, Syahariza, et al. (2005)
Bauer and Stachelberger (1984)
Church and Hart (1995)
Price, Hart, and Church (1992)
Grundy et al. (2007) and Grundy et al. (2008)
Source: Nakyinsige et al. (2012).
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Hasan
Figure 1 The basic principle of the immuno-chromatographic kit.
MATERIALS AND METHODS

Detection of Porcine and Bovine DNAs
from
different
products
containing
gelatin/collagen from different sources
DNA extraction was performed using DNA
extraction kit from QIAGEN. About 0.25g
DNA per sample was extracted. The DNA
extraction was performed in duplicate for
each sample. The conventional-PCR was
performed
using
Thermal
cycler
(GeneAmp PCR System 9700, Applied
Biosystems). Porcine and Bovine specific
primers for mitochondrial DNA were used.
Each primer sequence is shown in Table 2.
In addition, in order to check that DNA was
extracted successfully, the PCR using the
primer for vertebrate detection was also
performed. The PCR condition is
summarized in Table 3.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Table 2 Sequence of Porcine and Bovine

specific primers.
Target Primer
Sequence
Porcine Forward
GAC CTC CCA
GCT CCA TCA
AAC ATC TCA
TCT TGA TGA
AA
Reverse
GCT GAT AGT
AGA TTT GTG
ATG ACC GTA
Bovine Forward
GAC CTC CCA
GCT CCA TCA
AAC ATC TCA
TCT TGA TGA
AA
Reverse
CTA GAA AAG
TGT AAG ACC
CGT AAT ATA
AG
13

Table 3 Condition of PCR
Temperature
Step
(C)
Initial
94
denaturation
Denaturation
94
Annealing
60
Extension
72
Final
72
Extension
Hold
4
Hasan
B. FAMIC method:
Time
Cycle
1 min
30 sec
30 sec
30 sec
30
7 min
Primer:FAMIC
Porcine
mitochondrial DNA
DNA:
20 ng
PCR steps:
Step
Comparative study on the PCR-based

detection of the Porcine and Bovine genes
using different primers
Initial
denaturation
Denaturation
Annealing
Extension
Final
Extension
Hold
primer-
Temperature
(C)
Time
Cycle
95
9 min
92
60
72
30 sec
1 min
1 min
45
72
5 min
A. Neogen kit method:

Primer:
Neogen
genomic DNA
DNA:
20 ng
PCR steps:
Porcine
primer-
Step
Temperature Time
Cycle
(C)
Initial
94
10
1
denaturation
min
Denaturation
94
15
sec
Annealing
64
15
30
sec
Extension
72
15
sec
Final
72
3
1
Extension
min
Hold
4
Primer:FAMIC
Bovine
mitochondrial DNA
DNA:
20 ng
PCR steps:
Step
Initial
denaturation
Denaturation
Annealing
Extension
Final
Extension
Hold
Temperature
(C)
primer-
Time
Cycle
95
9 min
92
55
72
30 sec
30 sec
30 sec
45
72
5 min
RESULTS
Detection of Porcine and Bovine DNAs
from different products containing
gelatin/collagen from different sources
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
14

Table 4: Summarized results of the
detection of porcine, bovine and vertebrate
DNA
Porcine
Bovine
Vertebrate
Sample 1 Not
Not
Detected*
detected
detected
Sample 2 Detected*
Not
Not
detected
detected
Sample 3 Detected*
Not
Not
detected
detected
Sample 4 Detected*
Not
Detected*
detected
*When one of the test samples from
duplicate was found positive, it was taken as
the positive (as shown in the following three
figures).
Hasan
3. Two products (one marshmallow,
one functional cosmetic) showed
positive result for vertebrate material.
Only one of the two marshmallow products
might contain fish material (gelatin) since it
was found porcine negative.
Figure 3 Result of PCR using Bovine

specific primer; Lane 1: Marker (25bp
ladder), 2: Negative control, 3: sample 1
(n=1), 4: sample 1 (n=2), 5: sample 2 (n=1),
6: sample 2 (n=2), 7: sample 3 (n=1), 8:
sample 3 (n=2), 9: sample 4 (n=1), 10:
sample 4 (n=2), and 11: Positive control
[Microchip
electrophoresis
system
(MultiNA), Shimadzu Corporation].
Figure 2 Result of PCR using Porcine
specific primer; Lane 1: Marker (25bp
ladder), 2: Negative control, 3: sample 1
6: sample 2 (n=2), 7: sample 3 (n=1), 8:
sample 3 (n=2), 9: sample 4 (n=1), 10:
[Microchip
electrophoresis
system
Assumptions on the detection of porcine,
bovine and vertebrate DNA from four
commercialized products are listed below:
1. Not a single product showed positive
result for bovine material.
2. Three products (one marshmallow,
one Jelly, one functional cosmetic)
showed positive result for porcine
material.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Figure 4 Result of PCR using the primer

that common to a vertebrate; Lane 1: Marker
(25bp ladder), 2: Negative control, 3: sample
1 (n=1), 4: sample 1 (n=2), 5: sample 2
8: sample 3 (n=2), 9: sample 4 (n=1), 10:
[Microchip
electrophoresis
system
15
Hasan
Comparative study on the PCR-based

detection of the Porcine and Bovine genes
using different primers
Result-1 (DNA concentration)
The values are shown in the following table
DNA extraction kit: Neogen (Speciation)
Samples:
No
Sample ID DNA
260/280
(ng/l)
1
M-A
12.66
1.50
2
M-B
22.44
1.38
3
T-A
10.49
1.63
4
T-B
12.19
1.52
Result-2 (PCR-Electrophoresis)
A. Neogen kit method: As shown in the
Figure 5, no porcine DNA was detected in
any of the two samples tested in duplicates.
B. FAMIC method: As shown in the Figure
6, Porcine DNA was detected for the two
samples though in single from duplicate (MB and T-A). No bovine DNA was detected
from the two samples tested.
Figure 5 Porcine DNA; PCR product size:

380-bp (housekeeping fragment), 314-bp
(porcine fragment); from left: marker, M-A,
M-B, T-A, T-B, positive control (380-bp,
314-bp).
Assumptions on the comparative study using

different primers are listed below:
1. The porcine DNA was detected in the
both samples (marshmallow M-B and
T-A) when using only the FAMIC
method. These results indicate that
both the marshmallows might have a
small amount of porcine DNA.
2. The bovine DNA was not detected in
any of the two samples tested under
the same condition.
DISCUSSION
In this study, we have reported on comparing
the specific and qualitative detection
methods for porcine and bovine materials in
the processed food products (marshmallows
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Figure 6 Porcine DNA; PCR product size:

126-bp; from left: M-A, M-B, T-A, T-B,
positive control.
16
Hasan
tested. PCR-based detection was performed
by using PK mastermix/control POD from
Neogen Europe Ltd., UK, and porcine and
bovine specific primers from Food and
Agricultural Materials Inspection Center
(FAMIC), Japan.
Figure 7 Bovine DNA; PCR product size:

126-bp; from left: M-A, M-B, T-A, T-B,
positive control
and jelly containing gelatin) and functional
cosmetic product containing collagen
peptides by employing conventional PCR
and two different primers (nuclear and
mitochondrial genes). Earlier, two other
groups also reported about the successful
application of the conventional PCR in meat
species detections including from the
processed foods (Tanabe et. al., 2007;
Matsunaga et. al., 1999). These conventional
PCR methods are simple and useful. That is
why we have employed these. The choice of
the target gene and the design of the primers
have a great impact on the sensitivity and
specificity of a detection system. It is wellknown that very sensitive PCR assays can be
established when the primer target is a
multicopy gene, such as a mitochondrial
gene (Holzhauser et. al., 2006). In this study,
we chose both mitochondrial and nuclear
(genomic) genes as the target to detect
porcine and bovine materials for comparing
the efficiency of these two primers while
using the processed products. DNA
extraction was performed by using the
commercial kit from Neogen Europe Ltd.,
UK and following the manufacturers
instructions.
The DNA extraction was
carried out in duplicate for each sample
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Among the four products tested, in this study,

we found out at least three were porcine
DNA positive (one marshmallow, one jelly,
one functional cosmetic). These result
surprised us since all these products were
sourced from a strictly Halal-regulated
Muslim dominated country though these
were imported from other countries.
Moreover, one product (functional cosmetic)
claimed that it contained fish collagen from
Japan, which we found porcine positive.
Our results from this study proved that
selection of the right primer is critical to
obtain the authentic result: the porcine DNA
was detected positive only when the FAMIC
primer (mitochondrial DNA) was used and,
in contrast, the result was negative using the
NEOGEN kit primer (genomic DNA). This
might be due to the higher sensitivity of
mitochondrial gene target primer, which is a
multicopy gene. In another study, we
attempted to develop a rapid method for
meat species identification based on the loop
mediated isothermal amplification and
electrochemical DNA sensor (Ahmed et al.,
2010).
In a separate effort, a protein-basedimmuno-chromatographic porcine detection
kit was successfully co-developed under a
collaborative effort between two universities
(in USA and Japan) and two companies in
Japan (the methods and results were not
available for this paper). This development
was owned and sponsored by a Japanese
company having a leading position in the
precious metal business including the
expertise in nano-gold technology. This
company
obtained
the
immune17

chromatographic
know-how
from
a
university in Japan. However, to develop a
porcine detection kit, the missing part was
the biotechnology: a monoclonal antibody to
a porcine thermostable muscle protein for
detection of pork in cooked meat products.
To address this issue, the author of this paper
was approached by the Japanese company
who successfully made the connection with a
university in USA already reported on the
availability of this antibody and he
coordinated the collaboration between the
Japanese company and the US University.
Finally, the effort was paid-off. About 2
years later, the Japanese company confirmed
the successful development of porcine
detection kit and announced it. Immediately,
they were approached by a large US
company (renowned analytical equipment
manufacturer) for this technology, which
resulted
in
to
the
successful
commercialization of this protein-based
(immuno-chromatographic)
porcine
detection kit in the global market.
ACKNOWLEDGEMENTS
The author gratefully acknowledges the cooperations from Professor Eiichi Tamiya at
Osaka University, Japan; Professor Emerita
Peggy Hsieh at Florida State University,
USA; Mr. Masatoshi Watai at Japan Food
Research Laboratories, Japan; Dr. Hiro
Haraguchi at FASMAC Co., Japan; Dr. Koji
Okamoto at Tanaka Precious Metal Co.,
Japan; and the Ministry of industry and
primary resources, Brunei Darussalam
without which this study would not have
been possible. The author is thankful to Mr.
Haikal Ismail at STML, Universiti Utara
Malaysia for kindly assisting him in the
preparation of this manuscript.
REFERENCES
Hasan
Meat species
the loop
amplification
DNA sensor.
605.
identification based on
mediated isothermal
and electrochemical
Food Control 21, 599-
Aida, A. A., Che Man, Y. B., Wong, C. M.

V. L., Raha, A. R. and Son, R.
(2005). Analysis of raw meats and
fats of pigs using polymerase chain
reaction for halal authentication.
Meat Science 69(1), 4752.
Chen, F. C., and Hsieh, Y. H. P. (2000).
Detection of pork in heat-processed
meat products by monoclonal
antibody-based ELISA. Journal of
AOAC International 83(1), 7985.
Chen, S. Y., Liu, Y. P. and Yao, Y. G.
(2010). Species authentication of
commercial beef jerky based on
PCRRFLP
analysis
of
the
mitochondrial 12S rRNA gene.
Journal of Genetics and Genomics
37(11), 763769.
Fajardo, V., Gonzlez, I., Martn, I.,
Rojas, M., Hernndez, P. E.,
Garca,
T.
et
al.
(2008).
Differentiation of European wild
boar (Susscrofa scrofa) and domestic
swine (Susscrofa domestica) meats
by PCR analysis targeting the
mitochondrial D-loop and the nuclear
melanocortin receptor 1 (MC1R)
genes. Meat Science 78(3), 314322.
Fajardo, V., Gonzlez, I., Rojas, M.,
Garca, T. and Martn, R. (2010).
A review of current PCR-based
methodologies for the authentication
of meats from game animal species.
Trends in Food Science &
Technology 21(8), 408421.
Ahmed, M. U., Hasan, Q., Hossain, M. M.,

Saito, M. and Tamiya, E. (2010).
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
18

Girish, P. S., Anjaneyulu, A. S. R., Viswas,
K. N., Shivakumar, B. M., Anand,
M., Patel, M. et al. (2005). Meat
species identification by polymerase
chain reaction-restriction fragment
length polymorphism (PCRRFLP)
of mitochondrial 12S rRNA gene.
Meat Science 70(1), 107112.
Holzhauser, T., Stephen O., and Vieths, S.
(2006). Polymerase chain reaction
(PCR) methods for the detection of
allergenic foods. In Detecting
Allergens in Food, S.J. Koppelman
and S.L. Hefle, edc. CRC Press,
Boca Raton, pp. 125-143.
Matsunaga, T., Chikuni, K., Tanabe, R.,
Muroya, S., Shibata, K., Yamada,
J., & Shinmura, Y. (1999). A quick
and simple method for the
identification of meat species and
meat products by PCR assay. Meat
Science 1, 143-148.
Hasan
Radu, S. (2009). Meat species
identification
and
halal
authentication
analysis
using
mitochondrial DNA. Meat Science
83(1), 5761.
Nakyinsige, K., Che Man, Y.B., & Sazili,
A.Q. (2012). Halal authenticity
issues in mean and meat products.
Meat Science 91, 207-214.
Okamoto, K. (2016). Development of
Porcine
Immunochromato.
Bioindustry, April 2016, Vol. 33(4),
26-32, CMC Books, Tokyo. (in
Japanese).
Tanabe, S., Miyauchi, E., Muneshige, A.,
Mio, K., Sato, C., and Sato, M.
(2007). PCR method of detecting
pork in foods for verifying allergen
labeling and for identifying hidden
pork ingredients in processed foods.
Bioscience, Biotechnology, and
Biochemistry 71(7), 16631667.
Murugaiah, C., Noor, Z. M., Mastakim,

M., Bilung, L. M., Selamat, J., &
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19

Res. Highl. 4Bs (2016),P20-32
Bacteriophages for Biocontrol of Foodborne Pathogens: An Overview

Bhandare Sudhakar Ganapati*
Faculty of Veterinary Medicine, University Malaysia Kelantan, Locked Bag 36, Pengkalan
Chepa, 16100 Kota Bharu, Kelantan, Malaysia; *corresponding author,
e-mail: sudhakar@umk.edu.my
ABSTRACT
This article is an overview to depict the potential of bacteriophages as biocontrol agents in
controlling the foodborne pathogens for enhancing the food safety. Pathogenic strains of
Salmonella spp., Campylobacter spp., E. coli, Listeria spp., Vibrio spp. and many other
foodborne bacteria are a significant cause of foodborne illnesses in humans worldwide and
especially in the developing world. Antibiotic resistance is increasing in many foodborne
pathogens and the development pathway for new antibiotics is time consuming. Thus very few
new antibiotics are discovered in recent years. Hence there is an urgent need for alternatives to
antibiotics and bacteriophage biocontrol is one of the viable alternatives to antibiotics especially
for the multiple drug resistant pathogens. The concept of using bacteriophages as food safety tool
is emerging rapidly and they are becoming the logical agents for targeted control of pathogenic
foodborne bacteria to overcome the food safety concerns regarding the entry of such pathogens
in food chain and thereby affecting the public health. Thus, the bacteriophage biology, their
structure, morphology, classification, mode of action, their usage as biocontrol agents to control
foodborne bacteria and their advantages are discussed in this paper.
Keywords: Antibiotic resistance; Bacteriophages; Biocontrol; Food safety; Foodborne bacterial
pathogens.
INTRODUCTION
Antimicrobial or antibiotic resistance
(AMR/ABR) is an increasing concern world
over. The World Health Organization in its
Global Surveillance Report on ABR states
that The problem is so serious that it
threatens the achievements of modern
medicine. A post-antibiotic era - in which
common infections and minor injuries can
kill is a very real possibility for the 21st
century. The US President (Whitehouse,
2014) and UK Prime Minister (ONeill,
2014) have already asked their Governments
to prepare a roadmap to tackle this issue.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
The European parliament has asked the

member states to specifically prioritize the
development
of
phage
therapy
(Parliamentary Assembly, 2014). Pathogenic
strains of Salmonella spp., Campylobacter
spp., E. coli, Listeria spp., Vibrio spp. and
many other foodborne bacteria are a
significant cause of foodborne illnesses in
humans worldwide and especially in the
developing world. Such illnesses can be
treated with antibiotic chemotherapy but
over the years, there has been considerable
misuse of antibiotics. Many strains of
foodborne pathogens are becoming resistant
to antibiotics. The progressive resistance of
20
Phage Biocontrol of Foodborne Pathogens

Salmonella to the antibiotics has been
reported (Fluit, 2005). The multiple drug
resistance in zoonotic non typhoidal
Salmonella from food animals and food
sources is of great concern all over the world
due to their entry into human food chain
(OMahony et al., 2005). Similar antibiotic
resistance is noticed in Campylobacter
species (Bester and Essack, 2008), E. coli
(Diarrassouba et al., 2007 and Saenz et al.,
2001) and Listeria species (Li et al., 2007).
Drug resistance emerges through excessive
usage of antimicrobials in humans and in
food animals, which imposes a selection
pressure for bacteria that are resistant to
antibiotics (Huges and Heritage, 2006).
Particularly, in fish farming antibiotics are
given to the whole population with
inaccurate doses and thus healthy fishes
along with sick ones are exposed to
antibiotics and thus development of
antibiotic resistance is inevitable (Sorum,
2008). In USA about 40% of total antibiotics
produced are used on livestock and about
80% of that is used as growth promoters
(Hileman, 1999). Thus, considering the
threat to public health the European
Commission has imposed an EU wide ban
on the use of antibiotics as growth
promoters in animal feed from January 1,
2006 (European Commission, 2006). The
development pathway for new antibiotics is
time consuming and very few new
antibiotics are discovered in recent years.
Hence, there is an urgent need for
alternatives to antibiotics and bacteriophage
biocontrol is one of the viable alternatives to
antibiotics especially for the multiple drug
resistant
pathogens
(Barrow,
2001;
Matsuzaki et al., 2014). Lytic phages are
responsible for limiting bacterial numbers in
an aquatic environment with up to 80 % of
mortality evidenced in the bacterial
population (Weinbauer, 2004). Phages are
strain specific and thus attempting a
biocontrol of single strain of bacteria at a
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Bhandare
time is advisable and if successful another
strain can be tackled. Eventually, a cocktail
of phages will give a broad-spectrum
activity.
Recently, the scientific community has
witnessed the sudden surge of interest in
bacteriophage
research.
Bacteriophage
therapy is a promising and natural way of
reducing Salmonella (Atterbury et al.,
2007), Campylobacter (Atterbury et al.,
2005) and E. coli (Raya et al., 2006) as a pre
harvest treatment. While, post harvest
application of phages to reduce Listeria
monocytogenes (Leverentz et al., 2003)
contamination in later stages of food
production is logical. Phages can help to
reduce specific bacterial load in food
animals through proactive ante mortem
interventions rather than reactive end
product testing or treatment of patients.
They can be applied directly to foods to
extend the shelf life and also as hygiene
indicators in assessing food quality (Hudson
et al., 2005). The isolation and
characterization of bacteriophages is
uncomplicated and is possible on large scale
(Toro et al., 2005). Hankin in 1896 first
noticed the bactericidal activity of phages
from the Ganges and Jumna river waters in
India, which when filtered had antibacterial
properties against Vibrio Cholerae. Phage
therapy was pioneered by Felix DHerelle,
in early 19th century and his major
contributions came from his work in India
and he could discover the phages in Ganges,
the holy river in India (Summers, 2001). The
former Soviet Union has been using phage
therapy since 1920s (Chanishvili et al.,
2001) but after the discovery of antibiotics
in early nineteenth century scientists stopped
working with phages until recently. The
reappraisal of phage therapy in the Western
world was done by H. Williams Smith and
his colleagues for oral E. coli infection
control in neonatal animals and for systemic
21

infection control of E. coli in mice (Smith
and Huggins, 1982; Smith and Huggins,
1983). The concept of using bacteriophages
as food safety tool is emerging rapidly
(Hagens and Offerhaus, 2008) and they are
becoming the logical agents for targeted
control of pathogenic foodborne bacteria to
overcome the food safety concerns
regarding the entry of such pathogens in
food chain and thereby affecting the public
health. There are regulatory concerns over
the human phage therapy but their
application for food safety has relatively less
regulatory concern owing to the presence of
phages already in the food environment.
Bhandare
Phage structure and morphology
They consist of a nucleic acid genome
surrounded by a protein coat called as
capsid. Many phages contain additional
structures such as collar, tails, basal plate
and spikes or fibers (Figure 1). Structure of
phages may be icosahedrons, spherical
shapes consisting of triangular faces, or
filamentous or complex structures consisting
of icosahedral heads with helical tails. Their
genomes can consist of either DNA or RNA,
single (ss) or double (ds) stranded, circular
or linear (Nicklin et al., 1999).
BACTERIOPHAGE BIOLOGY
Bacteriophages
(in
Greek
language
phagein means to eat or to devour, thus
they are bacteria eaters), normally
abbreviated to phage are a family of
naturally occurring viruses that can be
isolated from all those habitats where
bacteria can thrive. They are the most
abundant in the environment and almost ten
bacteriophages are supposed to be there for
each bacterial cell (Skrunik and Strauch,
2006). Phages can persist outside the host
cells under a great variety of conditions, and
usually persist much better than their
bacterial hosts under adverse conditions.
Most phages are far more resistant to heat,
freezing, radiation, chemical disinfection
and natural inactivation than their host
bacteria (Jofre and Muniesa, 2000). They
can infect and kill specific bacteria and do
not affect other bacteria or cells, meaning
the dysbiosis (imbalance of commensal gut
flora) often resulting from the use of broad
spectrum antibiotics can be avoided. They
are obligate intracellular parasites that are
capable of existing as phage particles
outside the bacterial cell but can only
reproduce inside the cell.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Figure 1: Schematic representation of

bacteriophage.
Phage classification
The morphology of the phages helps in their
classification. The International Committee
on Taxonomy of Viruses (ICTV) has
presently classified bacteriophages in to one
order, ten families and forty genera (ICTV,
2011) based upon their symmetry, nucleic
acid and other morphological features
(Ackermann, 2006) (Table 1 and Figure 2)
22

Table 1: Classification of bacteriophages as per ICTV (Source: Bhandare, 2015).
Order
Caudovirales Myoviridae
dsDNA linear
Genome size
(Kb)
31-317
Caudovirales Podoviridae
dsDNA linear
Caudovirales Siphoviridae
dsDNA linear
Unassigned
Unassigned
Unassigned
Unassigned
Family
Genome
Corticoviridae dsDNA circular

supercoiled
Plasmaviridae dsDNA circular
supercoiled
Tectiviridae
dsDNA linear
Inoviridae
ssDNA (+)
circular
Unassigned
Microviridae
Unassigned
Cystoviridae
Unassigned
Leviviridae
ssDNA (+)
circular
dsRNA three linear
segments
ssRNA (+)
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Bhandare
Envelope Morphology
Virion size
No
Icosahedral head with

tail
Icosahedral heads: 60-45

nm;
Elongated heads: 80-110
nm;
Tail: 16-20 80-455 nm
nm;
Tail: 10-20 nm
nm;
Tail: 5-10 100-210 nm
60 nm
16-78
No

short tail
21-134
No

long tail
10
No
Icosahedral
12
Yes
15
Inoviruses: 5.812.4
Plectroviruses: 4.58.2
4.4-6.1
No
No
Quasi-spherical,
pleomorphic
Icosahedral
Inoviruses: filamentous
Plectroviruses: rod
shaped
No
Icosahedral
66 nm
Inoviruses: 7 700-3500
nm;
Plectroviruses: 15 200400 nm
25-27 nm
6.4-7.1;
3.6-4.7;
2.6-3.2
3.5-4.3
Yes
Spherical
85 nm
No
Icosahedral
23
26 nm
50-125 nm
Bhandare
Figure 2: Schematic representation of major phage groups. (Source: Ackermann, 2006)

Phages have binary (consisting of two
parts), cubic, helical and pleomorphic
symmetries. Most phages have double
stranded (ds) DNA, but there are few phages
with single stranded (ss) DNA, ssRNA or ds
RNA. Few phages contain lipid containing
envelope surrounding the capsid (the protein
coat covering viruses). The majority (96%)
of the phages are tailed phages (binary
symmetry), which are classified into the
order Caudovirales and three very large
phylogenetically related families, while
remaining 4% are other types. Taxonomic
names of orders, families, and genera are
typically constructed from Latin or Greek
roots and end in -virales, -viridae and
virus, respectively (Ackermann, 2006).
Phage mode of action
Phages possess the ability to infect a

bacterium and redirect the cell to synthesize
phage components for replication. A typical
life cycle of a phage (Figure 3) starts by
adsorption of the phages on to a specific
receptor structures on the surface of the
bacterium. After attachment they inject their
genetic material inside, which reprogrammes the bacterium for synthesis of
phage nucleic acid and other molecules
required for reproduction of complete viral
particles and thereby stopping synthesis of
host cell components. Then new phage
particles are assembled and when the new
viruses are mature, an enzyme is produced
that digests the cell wall, allowing the phage
to burst out of the cell. If bacteriophage
greatly outnumber their hosts, with many
phages attached to each cell, the bacteriums
Figure 3: Replication cycle of Bacteriophage.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
24

cell wall can become unstable and rupture,
without replication of the viruses inside, this
phenomenon is called passive inundation
(Atterbury, 2006).
The stages in the life cycle of the phages are
recognized when virus particles infect cells
in culture and are illustrated in Figure 4,
which exhibits a one-step growth curve.
This graph displays the results of a single
round of viral multiplication in a population
of cells. Following adsorption, the virus
particles undergo uncoating and viral
nucleic acid is injected into bacterial cell,
thus there is no growth observed and the
phenomenon is called eclipse. During the
latent period, replication of viral nucleic
acid and proteins occurs. The maturation
period follows, when virus nucleic acid and
protein are assembled in to mature virus
particles. At this time, if the cells are
prematurely lysed (by the use of chloroform
for example), mature virus particles can be
detected. Finally, release occurs, either with
cell lysis (e.g. lytic phage) or without cell
lysis (e.g. temperate phage). The timing of
the one-step growth cycle varies with the
Eclipse
Bhandare
viruses as different viruses have different
latent periods and burst sizes. With many
bacterial viruses, the whole cycle may be
complete in 30-60 min (Madigan et al.,
2003). The burst size is the average yield of
phage particles per cell, which is very
important for determination of inoculum
size in phage therapy. Many times small
doses are not sufficient and even at large
doses some times there is no active
replication and therapeutic benefits are
obtained by using very large or repeated
doses of phage (Payne and Jansen, 2001).
Large dosage may cause lysis from without
so that bacterial cells are destroyed without
any phage replication if the phages are
applied at high (> 100) MOI i.e. Multiplicity
of Infection, which means number of phage
per bacterium. The latent period is the
minimum length of time from the adsorption
of phage to its host, until the release of
newly formed phage particles, which is
crucial for phage therapy because the
inoculations given in right time taking latent
period in to account would be more
effective.
Rise
Plaque forming units
Latent period
Extracellular phage
Burst size
Intracellular phage
Time
Figure 4: One-step growth curve of bacteriophage replication (Source: Bhandare, 2015).
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
25

BACTERIOPHAGES
APPLICATION
AS BIOCONTROL AGENTS TO
CONTROL FOODBORNE BACTERIA
There are several reports on successful
usage of bacteriophages in controlling the
foodborne pathogens and some of the
important reports are discussed in this
section. Phages can help to reduce
Salmonella in poultry through proactive
antemortem interventions rather than
reactive end product testing or treatment of
sick birds. Berchieri, et al. (1991)
successfully used bacteriphages to control
Salmonella colonization in the poultry
gastrointestinal tract. The mortality was
reduced from 60% to 3% although large
numbers of phages were needed. Their study
revealed that the production of large
numbers of phages is practicable on farms. It
was also noticed that phages spread readily
between bacterially infected birds. Similarly,
E. coli infection in the poultry can be
controlled by using phage therapy. Toro, et
al. (2005) stated that there is a beneficial
effect of the phage treatment on weight gain
performance in chickens along with
reduction of Salmonella colonization. Huff,
et al. (2006) used aerosol spray of phages to
bring down the mortality of birds due to E.
coli infection. The mortality was brought
down to 7% in phage treated birds while it
was 48% in the untreated ones.
Campylobacter spp., the celebrity bug in
poultry as referred by Butzler (2004) is also
being targeted by the phage therapy.
Wagenaar et al. (2005); Loc et al. (2005)
and Atterbury et al. (2005) have carried out
successful phage therapy trials against
Campylobacter spp. in poultry.
Amongst the phage therapy reports in large
food animals Smith and Huggins (1983)
showed an effectiveness of phages in
treating experimental E. coli diarrhea in
calves, piglets and lambs. Infections were
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Bhandare
caused by three different E. coli strains in
calves, piglets and lambs and a mixture of
two phages were given orally to protect
them. E. coli infection was reduced to cure
the diarrhea in all three species. The phage
resistant mutants were emerged in the calves
and piglets but they were less virulent than
their parent strains. A cocktail of phages
significantly reduced the numbers of E. coli
O157:H7 in the intestinal tract of sheep
(Callway and colleagues, 2008). Even in the
fisheries the successful phage therapy is
carried out by Karunasagar et al. (2007).
They could achieve biocontrol of pathogens
in
shrimp
hatcheries
by
using
bacteriophages (Douglas, 1974). Also, the
Japanese researchers (Park et al., 2000; Park
and Nakai, 2003) studied the potential use of
phages to control the fish infections caused
by Lactococcus and Pseudomonas by giving
phages orally as phage impregnated feed.
Largely it is a win-win situation for the
mankind if phage therapy is thoroughly
scrutinized for applications in the food
animals and then used because it will help to
reduce the antibiotic usage to avoid multiple
drug resistance along with the reduction in
foodborne pathogen load.
Rather than applying the phages in live
animals before their slaughter the
application of phages after slaughter to the
carcasses and to the food products during
their processing prior to the consumption is
one more option to get rid of foodborne
pathogens (Thorns, 2000). The significant
reductions in the pathogenic bacteria can be
achieved by their bacteriophage biocontrol
in food processing. In number of studies the
poultry products are used for their surface
decontamination by phages. Atterbury et al.
(2006) achieved a reduction in S. enteritidis
and S. typhimurium below the detectable
levels by applying phages to the chicken
skin. Similarly, Goode and colleagues
(2003) could reduce the Campylobacter
26

jejuni by 1-1.3 log10 CFU within 24 hours
by application of phages to the surface of
chicken skin. The phage biocontrol of
Salmonella upon whole carcasses of broiler
chickens and turkeys was achieved by
Higgins et al. (2005). Phages have also been
used to reduce the numbers of Salmonella
from chicken sausages (Whichard et al.,
2003).
In case of beef the bacteriophage biocontrol
has been attempted to extend shelf life by
reducing spoilage organisms (Greer and
Dilts, 2002). The numbers of E. coli
O157:H7 from the beef surfaces were
brought down to below the detectable levels
by OFlynn et al. (2004). A 3 log10 CFU
reduction in the Listeria counts on salmon
was achieved by Hagens and Loessner
(2007) after applying high titres of phage. L.
monocytogenes, is more likely to occur
during food processing, and thus at this
point of time phage biocontrol of this
pathogen can be done successfully. AntiListeria phages as food additives have been
approved by the US FDA by according it a
Generally Recognized As Safe (GRAS)
status and such products are available in the
market for use in food processing.
For control of biofilms in the food
processing
environment
the
phage
application is being envisaged. Roy et al.
(1993) and Hibma and colleagues (1997)
have showed that the formation of Listeria
biofilms
in
the
food
processing
environments can be reduced by application
of phages.
A great deal of research is being carried out
for non-thermal ways of controlling
foodborne pathogens during processing for
the want of maintaining nutritive values of
food
and
phage-based
non-thermal
intervention is an ideal way in such
situations.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Bhandare
ADVANTAGES OF USING PHAGES
FOR FOOD SAFETY
Phages are ubiquitous in the environment.
Every known bacterium in nature is
supposed to have its complementary phage
and thus it is possible to use phage
biocontrol against any type of bacteria.
Bacteriophages are strain specific to an
individual bacteria and they dont harm the
other beneficial bacterial flora of the gut as
is the case with antibiotics which harm the
commensal gut flora. They can also be given
in cocktails for broad spectrum effect.
Bacteriophages are self replicating and
thus the given dosage can self amplify in
due course of the treatment so that they
effectively tackle the target bacteria. Also,
they will only replicate till the target
bacterium is present and thus they are
naturally self limiting.
As there is resistance development in
bacteria for antibiotics, the phage resistance
may also develop but it has been reported
(Loc Carrillo et al., 2005; Atterbury et al.,
2007 and Smith and Huggins, 1983) that
such bacteria would have low virulence or
less survival rate owing to the fitness
penalty. Also, the resistance to phage does
not affect their sensitivity to antibiotics and
the relevant phages naturally evolve
alongside as bacteria evolve resistance.
There are no reports of allergic reactions
to phage as in antibiotics and no serious side
effects have been described so far either in
animals or humans. This may be due to the
abundance of phages in our environment and
regular exposure of animals and humans to
them.
27

It is relatively cheaper and easier to
produce phage preparations especially as
topical
applications
for
surface
decontamination of foods.
As the human phage therapy is fraught
with the limitations of lack of public
confidence and more regulatory concerns,
the application of phages as biocontrol
agents in food animals and in food
processing is an ideal way of harnessing the
potential of this natures weapon for food
safety.
Though not totally eliminated, the foodborne
pathogens can be reduced to the acceptable
levels by application of bacteriophages as
pre-harvest and post-harvest interventions.
Phages can be effectively used along with
other food safety tools to protect public
health.
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32

Res. Highl. 4Bs (2016), P33-41
Cloning and Expression of the Urease Operon from Helicobacter pylori J99
Mohamad CWSR.1, 2, *, Abdul-Manaf U.2 and Mat-Arip Y.2
1
Biomedical Electronic Engineering, School of Mechatronic Engineering, Pauh Putra Main

Campus, Universiti Malaysia Perlis, Arau, 02600 Perlis, Malaysia;
2
School of Biology Science, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia;
*corresponding author, e-mail: robiah@unimap.edu.my
ABSTRACT
Aims: Helicobacter pylori urease is one of the antigens found in H. pylori with strong
immunogenic property. This present study was to clone the whole of urease operon
(pET32UOA6) with biologically active recombinant urease enzyme complex (UreA/UreB).
Methodology and results: A recombinant molecule of the full-length urease operon was
constructed in vitro from the H. pylori J99 and expressed in Escherichia coli cells, BL21 (DE3).
The potential colonies were screened for inserts by performing colony PCR using specific
primer, restriction enzyme digestion and nested PCR. A positive urease operon transformed
using pET32Ek/LIC vector carrying the recombinant gene of the full-length urease operon, 5.9
Kb. This positive urease operon was growth in LB-medium and at exponential-phase culture of
recombinant urease operon was induced with 0.4mM isopropyl-beta-D-thiogalactoside (IPTG).
Positive urease clone pET32UOA6 expressed both ureases, UreA and UreB. This meant that the
cloned urease operon was functioning in E. coli cell. Therefore, cloning of the whole of urease
operon (pET32UOA6) produced biologically active recombinant urease enzyme complex
(UreA/UreB) and verified by immune functioning assay using commercial antibody H. pylori
urease- and commercial antibody H. pylori urease-. Other than that, the confirmation of
recombinant protein of urease operon was demonstrated by protein sequencing. The results of
amino acid alignment based on BLASTp between recombinant urease against H. pylori J99
urease show high percent identities, 99-100%. Conclusion, significance and impact of study: The
production of recombinant UreA/UreB complex indicates that a fully functional urease operon or
urease operon was successfully constructed. In addition, the constructed H. pylori urease
replicon opened an opportunity for developing a genetically modified animal model to study H.
pylori pathogenesis.
Keywords: Escherichia coli; Helicobacter pylori, pET32 Ek/LIC; Recombinant protein; Urease.
INTRODUCTION
Helicobacter pylori is one of the common
bacterial infections in human and recognized
as the etiologic agent for majority of upper
gastro duodenal diseases. H. pylori has been
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
established as the causative agent for acute

or chronic gastritis (Mitchell et al., 1999)
and could be further developed into peptic
ulcer disease, gastric carcinoma and others
upper gastro duodenal diseases (Kiesslich et
al., 2005; Ardekani et al., 2013). According
33
Cloning and Expression of the Urease

to the World Health Organization (WHO)
statistic, H. pylori infection is on the rise and
proportional to the progress of a country.
Almost 50% of the world's population is
infected by H. pylori (Sasidharan et al.,
2008). In Malaysia, H. pylori infection is on
the raise as well. From the year 2000 until
2007, patients infected by H. pylori were
30.4% of the gastro duodenal cases reported
(Sasidharan et al., 2008).
Urease is one of the pathogenic factors that
help H. pylori colonizes the epithelium in
the acidic environment of the stomach
(Ardekani et al., 2013). H. pylori urease
displays enzyme-independent effects in
mammalian models, mostly through
lipoxygenases-mediated pathway (Uberti et
al., 2013). The urease would induce edema,
neutrophil chemotaxis and shows apoptosis
inhibition reverted in the presence of the
lipoxygenase inhibitors esculetin (Uberti et
al., 2013).
In addition to its involvement in the
pathogenesis process of H. pylori infection,
urease is also a target for vaccination
development (Voland et al., 2006) besides
being a suitable marker to use as a target
protein for detecting presence of H. pylori
infection among suspected gastrointestinal
patient.
In this study, urease operon were isolate and
examined by colony PCR, restriction
enzyme digestion and nested PCR with
specific primer to confirm orientation of
urease operon was done before continue to
protein expression. The sodium dodecyl
sulphate polyacrylamide (SDS-PAGE) gel
was performed to examine the immune
functioning assay of recombinant urease
operon of Helicobacter pylori J99 and
protein sequencing to make sure this protein
was functioning.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
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Escherichia coli growth and maintenance
The E. coli strains (Merck Inc.) used in this
study and their genotypes are shown in
Table 1. Escherichia coli strains were
grown on LB broth at 37C for 16 hours in a
shaker incubator (Shel Lab, UK). For
storage purposes, E. coli strains were stored
in LB broth containing 50% glycerol (50%),
then, kept at -80oC for long term storage.
Table 1: Genotypes of E. coli strains.
Strain
Genotype
Nova Blue
endA1 hsdR17(rK12 mK12+)
supE44 thi-1 recA1gyrA96
relA1 lac [F proA+B+
lacIqZ M15 ::Tn10]
BL21 (DE3) F ompT hsdSB (rB mB)
gal dcm (DE3)
Helicobacter pylori J99 growth and
maintenance
Helicobacter pylori J99 (ATCC 700824)
was grown on Eugon agar with 10% human
expired blood at 37C, 10% CO2 and 100%
humidity in an incubator (Shel Lab, UK).
Subcultured was performed every four to
seven days to maintain fresh bacterium. For
storage purposes, H. pylori J99 strain was
stored in TSB containing 20% glycerol
(20%), then, kept at -20oC for short term
storage and -80oC for long term storage.
Plasmid cloning vector
The plasmid cloning vector used in this
study
was
pET-32-Ek/LIC
(Merck,
Germany). This vector contains 109aa
TrxTagTM which encodes for the 109 amino
acid of thioredoxin protein. The pET-32Ek/LIC vector was specially designed for
cloning and high-level expression of target
protein. Thioredoxin protein and histidine
tag would be fused to the target protein to
enhance purification of the expressed
proteins.
34

Genomic extraction of H. pylori J99
Genomic of H. pylori was extracted by
employing GENEAll ExGene Cell SV kit
(Intron, Korea). The extracted genomic
DNA was then stored at -20oC for further
analysis. This genomic extraction of H.
pylori J99 was used as a template for
amplification of urease gene. Primer pair
(pET32UOHP-F & pET32UOHP-R) would
amplify the urease operon. The specific PCR
primers for urease operon with pET-32
Ek/LIC complimentary overhangs are shown
in Table 2.
Table 2: Specific PCR primers sequence
with vector-compatible overhang.
Primer
Sequences
pET32UOHP-F
5 GAC GAC GAC
AAG ATG AAA
CTC ACC CCA AAA
GAG 3
pET32UOHP-R
5 GA GGA GAA
GCC CGG TTC
AAA CCT TTT GCG
TGG TGG 3
Amplification of urease gene
The total volume for PCR reaction was 25
l [1x PCR buffer, 0.1 mM dNTPs (NHK
Inc), 0.4 pmol of each pET32UOHP-F,
pET32UOHP-R primers, 1 unit of i-Taq
DNA polymerase (NHK Inc) and 50 to 100
ng of H. pylori J99 DNA template].
Gradient temperatures for amplification of
urease operon were from 64oC to 74oC by
using gradient PCR thermal cycler
(Biometra, USA).
PCR cycler was programmed for 30 cycles
with 95oC for 1 minute, 57oC for 2 minutes
for full length urease operon and 72oC for 1
minute. The final elongation was set at
72oC for 10 minutes. Negative control was
included consisting of all mixture except the
DNA template. After PCR ended, 1 l of
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Mohamad et al.
the PCR product was analyzed on 1%
agarose gel then stained with ethidium
bromide (EtBr) and 1Kb DNA ladder
(Promega, USA) was used as a marker.
Preparations of pET32 Ek/LIC insert
The purification PCR product of H. pylori
urease operon by employing PCR DNA
Extraction System (Intron, Korea). After
that, the annealing procedure between
purified PCR products (Ek/LIC insert) with
pET32 Ek/LIC vector was follow according
to manufacturing protocol (Merck Inc.).
Transform recombinant into cloning host
Three microliter (3 l) of amplification
product was transferred into a new 1.5 ml
Eppendorf tube which had been cooled on
ice and the remaining annealing product was
stored at -20C. An uncut plasmid (0.1 ng)
was used as a control for transformation
efficiency of the competent cells.
Escherichia coli NovaBlue competent cells
were thaw on ice before transformation
procedure. Transformation involved mixing
50 l of the competent cell with 2 l of
annealing product and mixed gently. After
that, the mixture was incubated on ice for 5
minutes, then, heat-shocked for 30 seconds
at 42C. Immediately after heat-shock, the
tube was placed on ice for 2 minutes. Next,
80 l of LB was added and the mixture was
incubated at 37C, shaker at 250 rpm for 60
minutes. The mixture was spread onto LB
agar
plate
containing
ampicillin
(100g/mL). The plates were incubated
overnight (16 - 20 hours) at 37C.
Screening of recombinant urease operon
Colonies that formed had the possibilities of
carrying the insert. Thus, the colonies were
screened for inserts by performing colony
PCR using specific primers as in Table 3.
The potential clones were also subjected to
restriction enzyme digestion to confirm the
35

presence of the inserts.
The used of
restriction enzymes either as single (Sal1) or
double digestions (EcoR1/ Pst1 and
EcoRV/Bgl II) followed the manufacturer
protocol (Promega, Inc.).
The orientation of the urease gene was
determined so that it was in the correct
reading frame by nested PCR. The specific
primers cws1, cws2 and cws3 were used to
confirm the target insert as shown in Table
3. Finally, the clones were verified using
DNA sequencing which was carried out by a
service provider, NHK Bioscience Solution
Sdn Bhd. The selected recombinant plasmid
was named as pET32UOA6 for urease
operon recombinant for expression urease
UreA/UreB.
Table 3: Specific nested PCR primers used
for insert confirmation.
Gene
Sequences
cws-1-F 5-CGT TAT GTC CTT AAG
GAA AAA AC-3
cws-1-R 5-CCC ATG AGC GAT CGC
TGG GTT AAT GG-3
cws-2-F 5- CCA TTA ACC CAG CGA
TCG CTC ATG GG -3
cws-2-R 5- CTA TGG GGC ATG CTT
ACG GTT AAG -3
cws-3-F 5-CAA AGC TGA ATT CCA
ACG ATC GCT TAA CCG
TAA GCA TGC C-3
cws-3-R 5-GGT TAA AAA GAC TCG
AGG GTT TTT TAA TC-3
Transformation of pET32UOA6 into
expression host
The recombinant plasmids were sub-cloned
into an expression host, E. coli BL21 (DE3)
following the manufacturer protocol
(Promega Inc.)
Expression of urease operon
A single colony was inoculated into a
universal bottle containing 5 ml of LB broth
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Mohamad et al.
with ampicillin (100g/mL) and incubated
at 250 rpm, 37oC for 3 to 4 hours until
OD600 reached 0.4 to 1.0. Next, 2 ml of this
culture was aliquot into Mc Courtney bottles
and kept at 4oC overnight. The next day, 2
ml pre-culture from 4oC was centrifuged at
13 000xg for 15 seconds, the supernatant
was discarded and the cell pellet was
suspended with 2 ml LB broth with
ampicillin (100g/mL). The suspended cell
with fresh media then inoculated into a 250
ml flask containing 50 ml LB broth with
ampicillin (100g/mL). The cells were
grown at 37oC, 250 rpm until OD600 reached
approximately 0.6 (~3 to 4 hours). One ml
of this culture was removed as un-induced
sample for SDS-PAGE analysis. Cells were
induced with 0.4mM IPTG at 37oC, 250 rpm
for 3 hours.
Subsequently, the culture was incubated on
ice for 10 minutes and harvested by
centrifugation at 5000 x g for 20 minutes at
4oC. The cell pellet was washed with 0.25
culture volume of cold 20 mM Tris-HCl pH
8.0, then centrifuged at 5000 x g for 20
minutes at 4oC. The supernatant were
discarded and the cells pellet was kept at 80oC for further analysis.
The expression of urease protein was
detected
by
Sodium-dodecyl-sulphate
polyacrylamide gel electrophoresis (SDSPAGE). The preparation and the assembly
of SDS-PAGE electrophoresis was carried
out according to the manufacturer protocol
(Bio-Rad, USA). Briefly, the resolving gel
was at 12.5% with standard 4% stacking gel.
Cell pellets were suspended with PBS before
5X SDS-PAGE sample buffers at the ratio
3:1 was added. Next, the sample was heated
at 95oC for 5 minutes. After heated, this
sample became viscous and 29cc gouge
needle was used to reduce the viscosities of
the sample. Twenty microliter of sample
36

was loaded per lane on the SDS-PAGE gels.
The SDS-PAGE running buffer was filled
into the bottom and upper chambers and all
the bubble were removed from the wells of
the gels using a syringe. The gel was run at
80V constant current for 35 minutes and
100V for 2 hours until the stacking dye had
reached the end of the gel. After that, the gel
was carefully removed from the glass plates
and stained with Coomassie Blue stain for 1
hour and destained with destining solution
overnight (~16 hours). The expression of
constructed recombinant urease operon was
important to measure the urease expression
level and to know the biological active of
the recombinant urease.
Immune functioning assay
The immune functioning assay was used to
verify the recombinant protein of
pET32UOA6. The electrophoresis transfer
of proteins to 0.45 m Polyvinylidene
fluoride (PVDF) membrane (Millipore Inc.)
was accomplished by a modification of the
method described by Towbin et al. (1979).
Protein was electro blotted from the gel onto
PVDF membrane by electrophoresis transfer
using a Mini Trans-bolt Electrophoretic
Transfer Cell (Bio-Rad, USA). Lastly, the
membrane was put in a plastic wrap and the
image was captured within 1 minute using
Gene Genius Bio Imaging System.
Protein sequencing
Recombinant urease clones were grown,
induced with 0.4 mM IPTG, followed by
SDS-PAGE as described in Section 3.6.9.
After SDS-PAGE, the band which
represented the recombinant UreA and UreB
was purified and sent to 1st BASE, Inc. for
protein sequencing. Finally, the amino acid
sequence resulted from protein sequencing
was aligned using Protein Basic Local
Alignment Search Tool (NCBI BLASTp) of
National
Center
for
Biotechnology
Information (Stephen et al., 1997) and this
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Mohamad et al.
protein sequencing was carried out to ensure
the expressed recombinant urease was
identical to H. pylori J99 urease protein.
RESULTS AND DISCUSSION
Determine of recombinant urease operon
The urease operon was determined by their
size based on urease gene sequence
information, accessed from NCBI database
(Accession No. NC_000921.1). Complete
urease operon that encode for urease
enzymes and accessory proteins. As shown
in Figure 1, the presence of the urease
operon PCR amplified was detected with the
expected size of 5974 bp.
Figure 1: Amplification of urease genes

from ATCC 00824 H. pylori J99. Lane 1:
1Kbp DNA ladder; Lane 2 to 7:
amplification of urease genes; Lane 8:
Negative control (lack of i-Taq DNA
polymerase).
Recombinant clone screening using PCR
The positive colonies were selected and
subjected to PCR reaction using specific
primers (Table 3) to confirm the presence of
the inserts. All selected colonies was had
inserts with the expected size that represent
the size of urease operon fragment.
Restriction enzyme digestion analysis
Two clones from each of the recombinants
were further selected for verification. The
plasmids from potential recombinant clone
pET32UO were digested with restriction
enzymes to further confirm the presence of
urease gene fragments. A single digestion
(Sal I) and double digestion (EcoRI & Pst1
37

or EcoRV & Bgl II) produced different
restriction enzyme patterns due to the unique
sites of these restriction enzymes on the
plasmids. Table 4 shows the expected
fragment sizes resulted from the chosen
restriction enzymes. Furthermore, agarose
gel electrophoresis verified the expected
fragment sizes from restriction enzyme
digestions, as shown in Figure 2. All of the
selected clones indicated the presence of
recombinant plasmid harbouring urease
genes.
Table 4: Restriction enzyme digestions of a
selected potential recombinant clone.
Expected
Recombinant
Restriction size
clones
enzyme
fragment
(kbp)
pET32UO
EcoRI &
1332 &
Pst I
10559
EcoRV &
Bgl II
5865 &
6026
Sal I
11891
Figure 2: Restriction enzyme analysis of

potential recombinant clones. The restriction
enzyme digestion of urease operon
fragment. Lane 1: 1 Kb DNA ladder; Lane
2 and 6: undigested plasmids; Lane 3 and 7:
EcoRI & Pst I digestion; Lane 4 and 8:
EcoRV & Bgl II digestion; Lane 5 and 9: Sal
I digestion and Lane 10: Hind III DNA
ladder.
DNA Sequencing
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Mohamad et al.
Final verification of the recombinant
plasmids was made by DNA sequencing on
clone pET32UOA6. The DNA sequencing
was performed using specific primers as
shown in Table 3. The results of the DNA
sequencing showed 99% nucleotide
similarity for urease operon to H. pylori J99
genome when analyzed using NCBI BLAST
program (Zheng et al., 2000).
Expression of urease genes
Plasmid pET32 Ek/LIC carries IPTG
inducible T7lac promoter for protein
expression (Merck, Inc.). The expressed
protein from this plasmid would be a fusion
protein of 109 amino acids thioredoxin to
the protein of interest. Thus, the
recombinant urease produced would be
slightly bigger than the native urease
Induction study of ureases production
In this study, the expression of recombinant
urease was used 0.4 mM and/or 1.0 mM
IPTG and 2 and/or 3 hours induction time.
As shown in Figure 4, bands representing
both ureases, UreA and UreB, were detected
on SDS-PAGE with approximate sizes of 45
kDa and 74 kDa, respectively. Clone
pET32UOA6 expressed both ureases, UreA
and UreB (Figure 4). This meant that the
cloned urease operon was functioning in E.
coli cell. The selected recombinant clones
carrying correct urease gene fragments, as
well as, complete urease operon were
subjected to expression study. As shown in
Figure 3, bands representing recombinant
UreA and UreB were detected indicating the
clones were carrying functional urease
genes.
Determination of immune functioning of
the expressed ureases
The sizes of H. pylori recombinant UreA
and UreB (Figure 4) were bigger, more than
29 kDa and 62 kDa respectively due to the
fused amino acids thioredoxin. Sometime,
38

the presence of additional amino acids fused
to protein of interest could change the
protein conformation. Therefore, it was
essential to determine whether the expressed
recombinant UreA and UreB still
maintained their immune properties.
Figure 3: SDS-PAGE analysis for

expression of recombinant urease protein.
The
expression
profiles
for
(A)
pET32UOA6 (B) pET32ureA3 and (C)
pET32ureB2. Lane 1: DGelTM Marker;
Lane 2: Pre-culture urease gene; Lane 3 and
6: uninduced culture; Lane 4: Induced
culture with 0.4 mM IPTG at 2 hours;
Lane7: Induced culture with 0.4 mM IPTG
at 3 hours; Lane 5: Induced culture with 1.0
mM IPTG at 2 hours and Lane 8: Induced
culture with 1.0 mM IPTG at 3 hours.
Figure 4 shows immune functioning of
UreA and UreB from cell crude of
recombinant urease clones compared with
other bacterial crude cells, as negative
controls and H. pylori J99 crude cell as the
positive control. Helicobacter pylori urease and urease- antibodies (Santa Cruz, Inc.)
were very specific and sensitive to UreA and
UreB of H. pylori J99 crude cell (lanes 8 and
18). Similar specificity and sensitivity were
observed for recombinant UreA and UreB,
expressed in full operon unit (lanes 2-3 and
12-13). As suggested by the manufacturer,
crude cells from Salmonella (lanes 4 & 14),
Pseudomonas (lanes 7 & 17) and E. coli
(lanes 9 & 19) gave negative results that
indicate H. pylori urease- and urease-
antibodies were very specific and sensitive
to the native, as well as, the recombinant
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Mohamad et al.
ureases.
These results confirmed the
recombinant UreA and UreB maintained
their immune properties.
Figure 4:
Western blot analysis for
determination of recombinant ureases
immune functioning in of UreA (A) and
UreB crude cell (B). Lane 1, 10, 11 and 20:
Kaleidoscope Prestained protein ladder;
Lane 2-3 and 12-13: pET32UOA6 cell
crude; Lane 5-6: pET32ureA6 cell crude;
Lane 15-16: pET32ureB2 cell crude; Lane 4
and 14: Salmonella cell crude; Lane 7 and
17: Pseudomonas cell crude; Lane 8 and 18:
H. pylori J99 cell crude; Lane 9 and 19: E.
coli cell crude.
Immune functioning assay verified the
recombinant UreA and recombinant UreB
still maintained their antigenicity, equivalent
to native enzyme regardless of the presence
of additional amino acids fused to them.
These were supported by immune
functioning assay through Western blotting
and previous work by Hu et al. (1992).
Purification of both recombinant ureases did
not affect the antigenicity, as evidence by
Western blotting (Figure 4). Regardless of
this condition, both recombinant ureases still
maintained their antigenicity in immune
functioning assay. Purification process
failed to separate UreA from UreB since H.
pylori urease- and urease- antibodies
(Santa Cruz, Inc, USA) still detecting both
39
Mohamad et al.
of them in the immune functioning assay.

Thus, clone pET32UO expressed and
assembled the urease apoenzyme and
perhaps together with the accessory proteins
to form the holoenzyme.
This study was supported by the Universiti

Malaysia Perlis for Academic Training
Scheme Funding (SLAI).
The confirmation of recombinant urease

protein was demonstrated by protein
sequencing. The results of amino acid
alignment based on BLASTp between
recombinant urease against H. pylori J99
urease show high percent identities, 99100%. Thus, these results further confirmed
the authenticity of the recombinant ureases
similar to H. pylori J99 urease.
Ardekani, L. S., Gargari, S. L. M.,

Rasooli,
I.,
Bazl,
M.
R.,
Mohammadi, M., Ebrahimizadeh,
W. and Zare, H. (2013). A novel
nanobody against urease activity of
Helicobacter pylori. International
Journal of Infectious Diseases 17(9),
e723-e728.
Confirmation of recombinant ureases by

protein sequencing
Figure 5 shows the results of protein identity
based on BLASTp between recombinant
urease and H. pylori J99 ureases. These
results further confirmed the authenticity of
the recombinant ureases expressed by
pET32UOA6.
Figure 5: The percentage identity of

recombinant ureases against H. pylori J99
ureases, pET32UOA6.
CONCLUSION
In this study, the availability of a functional
replicon carries urease operon has potential
benefit related to H. pylori pathogenesis. A
through and better understanding of H.
pylori pathogenesis process could contribute
to the improvement of diagnostic methods.
ACKNOWLEDGEMENT
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
REFERENCES
Hu, L. T., Foxall, P. A., Russell, R. O. B.

E. R. T. and Mobley, H. L. (1992).
Purification
of
recombinant
Helicobacter
pylori
urease
apoenzyme encoded by ureA and
ureB. Infection and Immunity 60(7),
2657-2666.
Kiesslich, R., Goetz, M., Burg, J., Stolte,
M., Siegel, E., Maeurer, M. J. and
Neurath, M. F. (2005). Diagnosing
Helicobacter pylori In Vivo by
Confocal
Laser
Endoscopy.
Gastroenterology 128(7), 21192123.
Mitchell, H.M., Hazell, S.L., Bohane,
T.D., Hu P., Chen, M. and Li, Y.Y.
(1999). The prevalence of antibody
to cagA in children is not a marker
for specific disease. Journal of
Pediatric Gastroenterology and
Nutrition 28, 71 75.
Sasidharan, S., Uyub, A.M. and Azlan,
A.A. (2008). Further evidence of
ethnic and gender differences for
Helicobacter pylori infection among
endoscoped patients. Transactions of
the Royal Society of Tropical
40

Medicine and Hygiene 102, 12261232.
Stephen, F., Altschul, Thomas, L.,
Madden, Alejandro, A., Schffer,
Jinghui Zhang, Zheng Zhang,
Webb, M. and David, J. L. (1997),
Gapped BLAST and PSI-BLAST: a
new generation of protein database
search programs. Nucleic Acids
Research 25, 3389-3402.
Towbin, H., Staehelin, T. and Gordon, J.
(1979). Electrophoretic transfer of
proteins from polyacrylamide gels to
nitrocellulose sheets: procedure and
some applications. Proceedings of
the National Academy of Sciences
76(9), 4350-4354.
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Uberti, A. F., Olivera-Severo, D.,
Wassermann, G. E., ScopelGuerra, A., Moraes, J. A.,
Barcellos-de-Souza, P. and Carlini,
C. R. (2013). Pro-inflammatory
properties and neutrophil activation
by Helicobacter pylori urease.
Toxicon 69, 240-249.
Voland, P., Zeitner, M., Hafsi, N. and
Prinz, C. (2006). Human immune
response
towards
recombinant
Helicobacter pylori urease and
cellular fractions. Vaccine 24(18),
3832-3839.
Zheng, Z., Scott, S., Lukas, W. and Webb,
M. (2000). A greedy algorithm for
aligning DNA sequences. Journal of
Computer Biology 7(1-2), 203-214.
41

Res. Highl. 4Bs (2016), P42-56
Production of Butter Flavour Concentrate from Butter fat with Lactic Acid
Bacteria by Solid Substrate Fermentation
Nadaraj Sivan1, Thambirajah, J. J.2 and Guruswamy Prabhakaran*1
1
Department of Biotechnology, AIMST University, Bedong 08100, Kedah Darul Aman,

Malaysia; 2Faculty of Business Management, AIMST University, Bedong 08100, Kedah
Darul Aman, Malaysia; *corresponding author, e-mail: prabhakaran@amist.edu.my
ABSTRACT
Aim: The aim of this study was to investigate the fermentation of butter fat with different
lactic acid bacterial strains by solid substrate fermentation (SSF) for the production of butter
flavour concentrates. Methodology and results: Lactic acid bacteria (LAB) were isolated from
dairy products and environmental samples using Mann Rogosa and Sharpe (MRS) agar.
These, together with two reference ATCC lactic acid bacterial strains were evaluated for the
production of flavor components which were determined by GC-MS. The SSF was found to
be effective in producing sweet and buttery notes within 24 hours of fermentation. Scale up
studies with 120 g of butter fat supplemented with 10% galactose enhanced butter flavour
production. Butter oil recovered from the fermented samples was subjected to sensory
evaluation by 120 volunteers. The butter flavour compounds in butter oil samples were
quantitatively analyzed in GC-MS. The untreated butter fat recorded the lowest concentration
of diacetyl (211.5 ppm) and acetoin (161.7 ppm) whereas, butter fat supplemented with
galactose and fermented showed a significant increase in concentration of acetoin (1321.2
ppm) and diacetyl (511.4 ppm). The formulation of butter powder with maltodextrin was
investigated. Conclusion, significance and impact of study: The results obtained from this
study will pave the future investigations for development of a microbial process for
production of butter flavour concentrate from butter fat.
Keywords: Acetoin; Butter fat; Diacetyl; Butter flavor; GC-MS; Lactic acid bacteria; Solid
substrate fermentation.
INTRODUCTION
Flavour is a combination of taste and
aroma. It results from the perception of
odor-active volatile compounds. Food
flavours are mixtures of natural and/or
artificial aromatic compounds. They are
designed to impart, modify, or even mask
an undesirable flavour. Flavours along
with fragrances are highly prized in the
global market. Currently there are three
known methods of acquiring flavour
compounds (Bicas et al., 2010). These
include, extraction from pre-existing
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
natural sources, synthesis by chemical

precursors and by biotechnological routes
via de novo or biotransformation.
The milk fat component of dairy source
largely contributes to the release of
flavour. Butter possesses its own distinct
flavour. The flavour compounds usually
are aldehydes, ketones and lactones of
which diacetyl and acetoin play important
roles in parting the well-known buttery
flavour. Lactones play important role in
conferring the buttery taste and sweet
aromas altogether (Hua et al., 2007).
42
Microbial Production of Butter Flavour

Currently, diacetyl and acetoin, the flavour
compounds are chemically synthesised at
commercial scale. The inhalation of
intense synthetic butter flavour at the
source of manufacturing and application
results
in
bronchiolitis
obliterans,
commonly termed as popcorn lung
disease (Morris and Hubbs, 2009).
The increasing demand for natural butter
flavour has lead to the development of
biotechnological processes. However, the
fermentation processes which involve
lactic acid bacteria in obtaining high yields
of desirable enantiomeric butter flavour
compounds from butter fat are yet to be
established. The development of solid state
fermentation
techniques
for
the
bioconversion of butter fat and the
recovery of butter flavour compounds are
important challenges for researchers in this
field. Hence, this study was undertaken to
optimize the fermentation conditions for
the production of butter flavour
concentrate from butter fat by lactic acid
bacteria in solid substrate fermentation.
Nadaraj et al.
The process flowchart in Figure 1
highlights the major stages in the
production of butter flavour concentrate.
Isolation of Lactic Acid Bacteria (LAB)
from various samples
Raw milk was purchased from a local
market. Soured milk was prepared by
allowing the raw milk to sour for a day.
Pasteurized milk (Marigold, Dutch Lady),
cheese (Emborg, Kraft), unsalted butter
(Devondale,
Tatura),
salted
butter
(Devondale, Anchor), cultured drink
(Solivite, Nutrigen), yoghurt (Dutch Lady)
were purchased from TESCO supermarket
in Alor Setar, Kedah Darul Aman,
Malaysia. Soil, grass and water samples
were collected from a nearby cattle farm.
Lactic Acid Bacteria (LAB) were isolated
from these samples as per the method
(Bettache et al., 2012). The isolates were
stored in agar slants at 4C. The individual
isolates were tested for their ability to
ferment butter to produce flavour
concentrates.
Figure 1: Flowchart for the production of butter flavour concentrate.

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43
Nadaraj et al.
ATCC strains of Lactic Acid Bacteria

Lactobacillus acidophilus (ATCC 314)
and Lactobacillus casei (ATCC 393)
strains were purchased from ATCC and
also evaluated for the production of butter
flavour.
Recovery of butter oil

Butter oil was extracted from fermented
butter fat samples for the sensory
evaluation, fatty acid analysis and
dertermination of diacetyl and acetoin in
the fermented samples.
Pasteurization and sterilization of butter

fat
The recommended protocol by the
International Dairy Federation (Juffs and
Deeth, 2007) was used for the
pasteurization of butter. Sterilization of
butter fat was by autoclaving at 121 C at
15 psi, for 15 minutes.
Microbial fermentation of butter fat by

Solid Substrate Fermentation (SSF)
Ten ml of overnight culture in MRS broth
with the isolated LAB bacteria was
prepared individually. Six g of butter fat
each in glass Perti plates were autoclaved
(121 C, 15 psi, 15 minutes). The melted
butter in the petri dishes was gently
swirled and cooled. Individual petri dishes
were then inoculated with 2 ml of
overnight culture of the Lactic acid
bacteria. As a control, an uninoculated
petri dish with only the sterilized butter fat
was used. At fixed time intervals (24th, 48th
and 72th hour) samples were drawn from
the petri dishes and evaluated by sensory
evaluation. A total of 10 evaluators were
selected to provide descriptions of the
aroma from the samples such as odourless,
sweet, stale, sour, buttery or alcoholic.
Butter oil samples were then extracted
from the samples after scoring of the
aroma description. The samples were
stored in the cold room for further
analysis.
Standardization of butter oil extraction

methods
To standardize the butter oil extraction
method, three different protocols were
studied (Table 1). Based on the total
amount of oil extracted, the percentage
(%) of recovery was calculated based on
the following formula:
% recovery = Amount of pure product
recovered (g) x 100 / Amount of crude
material used (g)
Table 1: Different methods for the
extraction of butter oil from butter fat.
Method Conditions
1
a) Melting in a water bath at
40 C (5C) for 10 minutes.
b) Centrifugation at 800 rpm
for 3 minutes
(Chongcharoenyanon et al.,
2012).
2
a) Melting in a water bath at
50 C (5C) for 8 minutes.
b) Centrifugation at 3,700 rpm
for 12 minutes(Krause and
Gibson, 2008).
3
a) Melting in hot air oven at 70
C (5C) for 15 minutes.
b) Centrifugation at 3,500 rpm
for 5 minutes (Miura et al.,
2004).
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Supplementation of butter fat with

various carbohydrate sources
An experiment was conducted to
determine whether supplementation of
butter fat with various carbohydrates
sources such as galactose, lactose and skim
milk may enhance butter flavour
production
upon
solid
substrate
fermentation.
Initial
efforts
were
conducted in petri dishes where 6 g of
butter was weighed and supplemented with
10% (w/w) of galactose, lactose and skim
milk, individually. The supplemented
butter fat was then sterilized and uniformly
spread the butter fat in the petri dish. After
cooling, each Petri dish was inoculated
with 2 ml of overnight cultures of the
44

selected lactic acid bacteria and incubated
at 37C. One petri dish was designated as
control. Sensory evaluation of samples
was performed at fixed time intervals (24th,
48th and 72th hour) as reported previously.
Scale up studies of Solid Substrate
Fermentation (SSF)
Scales up studies were performed with
increased quantities of butter fat. In order
to increase the surface area for solid
substrate fermentation, experiments were
carried out with various amounts of butter
fat taken in conical flasks (30 g in 250 ml
conical flask, 60 g in 500 ml conical flask
and 160 g in 1000 ml conical flask).
Overnight
cultures
were
prepared
individually and the volume of overnight
inoculum used was 10 ml for 30 g, 20 ml
for 60 g and 40 ml for 160 g substrates,
respectively.
These
cultures
were
incubated at 37C under anoxic conditions.
At specified time intervals of 24th, 48th and
72th hour, samples were acquired for
sensory evaluation by 10 random
volunteers.
Fermentation (SSF) with 10% galactose
Scale up studies was carried out with 30 g,
60 g and 160 g of butter fat, supplemented
with 10% of galactose individually. The
flasks were sterilized and inoculated with
overnight inoculum respectively. They
were incubated and samples were drawn
and tested as previously.
Sensory evaluation of butter oil samples
from different fermented butter samples
that were obtained from the fermented
butter samples were performed as per the
methods by Anvoh et al. (2009). A 5-point
hedonic scale was utilized where 1
represents dislike the most, 3 represents
neither like nor dislike and 5 represents
like the most. Sensory scores were
evaluated based on preferences of odour
by 120 students of Biotechnology from the
Faculty of Applied Sciences, AIMST
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Nadaraj et al.
University. Prior to sampling, volunteers
were first requested of their health status
and free of any respiratory illnesses.
During inhalation of aroma, volunteers
were required to inhale the samples and
suspend breathing for 2-3 seconds and
mark their preferences in the flavour
survey form provided. Coffee powder was
provided as a neutralizer to eliminate
traces of previous aromas. Samples
indicating high preferences were selected
for further analysis by GC-MS (Gas
chromatography-mass spectrometry).
GC-MS analysis of butter oil for acetoin,
diacetyl and fatty acid
Volatile compounds such as acetoin and
diacetyl are generally analysed by GC-MS
(Gokce et al., 2014). Extraction of diacetyl
and acetoin from the treated butter oil was
performed. Chromatographic analyses of
the treated and control butter samples for
aroma compounds and fatty acid
components were performed using a split
less injector system gas chromatograph
coupled with a mass spectrometer
(SHIMADZU
GCMS-QP2010).
The
carrier gas used was ultra-pure helium with
a flow rate of 1.0 mL/min. The injection
port was worked at 250C in split less
mode coupled with 1 minute split less
time. A 1 l injection volume was applied
for each sample analysis and the syringe
was washed with hexane upon completion
of injection. Separation was performed
using a DB-WAX (60 m x 0.25 mm x 0.15
m) capillary column with a 0.15 m
stationary film. The oven temperature
programme was set as follows: initial
temperature 40C, increased by 7C min-1
to 200C and held for 1 minute. Mass
spectrometric parameters were set as
follows: electron impact ionization with 70
eV energy and 250C ion source. The
aromatic compounds and fatty acid
profiles were detected and quantified
based on their retention time and peak
areas on the chromatogram respectively.
45

Butter powder formulation with
maltodextrin
Maltodextrin was used as a carrier
material, as it is edible, slightly sweet, and
low in cost (Wandrey et al., 2010). For the
production of butter oil powder, the
extracted butter oil was mixed with
maltodextrin with different anti-caking
agents and the entire mixture was then
treated with liquid nitrogen and mixed
evenly using a mixer. The addition of
liquid nitrogen was to imitate freeze drying
conditions. Formulation of butter powder
from carrier material (maltodextrin) and
active material (butter oil) was performed
individually by using 50 g of maltodextrin
powder and varying amounts of butter oil
(5 g, 10 g, 15 g, 20 g, 25 g and 30 g). The
maltodextrin powder was added into a
plastic container, and butter oil of the
concerned weight was poured into the
container. Simultaneously, the mixture
was added with liquid nitrogen
(approximately 100 ml) and manually
stirred. The prepared powder was then
stored in air-tight glass containers and kept
at room temperature away from any light
source. Anti-caking agents are required in
the formulation of powdered food or drug
preparations to either stop or postpone
occurrences of caking (Lipasek et al.,
2011). For this, 50 g of the prepared butter
powder was placed in a plastic container
and supplemented individually with 2%,
4% and 6% (w/w) anti-caking agent that
comprised sodium chloride, sodium
silicate, sodium bicarbonate, bentonite,
mannitol and potato starch. The mixture
was manually stirred with intermittent
shaking to enable even dispersion of the
anti-caking agent and the powder. The
prepared powder was then stored in a glass
container that was sealed and stored in a
dry area.
Over the years lactic acid bacteria (LAB)
have been used in various fermented food
preparations. The benefits conferred by
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Nadaraj et al.
LAB are lactic acid production which
results in the improvement of flavour,
aroma, keeping quality and enhancement
in nutritional content (Halsz, 2009).
Butter is an important flavouring additive
and creates the distinctive aroma and taste
in bakery, dairy and other food products.
Diacetyl and acetoin are two important
constituents in butter fat that contribute to
unique butter flavour. Natural butter
flavour is expensive, hence artificial butter
flavour compounds are chemically
synthesized
from
petroleum-derived
precursors. Synthetic
flavours
are
produced in bulk at low cost and it is a
mixture of racemic compounds. The
prolonged exposure to synthetic flavour
compounds to the line workers was
reported harmful by National Institute for
Occupational Safety and Health (NIOSH)
(Kreiss, 2007).
Biotechnological approaches in producing
butter flavour compounds are being
investigated as a replacement to chemicalbased processes (Longo and Sanromn,
2006). This study was aimed at producing
butter flavour concentrate from butterfat
by a microbial process. The investigation
included isolating LAB strains as well as
Lactobacillus acidophilus (ATCC 314)
and Lactobacillus casei (ATCC 393) in the
fermentation of butter fat by solid substrate
fermentation (SSF). After fermentation,
removal of solids by centrifugation and
recovery of butter oil from the treated
butter was performed. The butter oil was
then subjected to sensory evaluation and
analysed for butter flavour compounds by
GC-MS.
Isolation of LAB from various samples
Raw milk, soured milk, cheese, unsalted
butter, salted butter, yoghurt and grass,
soil, and water samples from cattle grazing
areas were niches of LAB strains. A total
of ten colonies were isolated from the
different samples.
46

Pasteurization and sterilization of butter
fat
Two
different
methods
namely
pasteurization and sterilization of butter fat
were
evaluated.
Compared
to
pasteurization (63C5C for 30 minutes),
the sterilization of butter fat samples was
better. Hence, sterilization was used
subsequently.
Recovery of butter oil
Butter oil was recovered for sensory
evaluation, detection and quantification of
diacetyl, acetoin and fatty acids in the
fermented
butter
samples
which
constituted the major butter flavour
compounds. Three different methods were
adopted for extraction of butter oil from 6
g of butter fat (Figure 2).
Percentage recovery of butter oil by
different methods
On comparative analysis, it was found that
the percentage recovery of oil was high
(69.67%), when the butter fat was melted
at 50 C for 8 minutes and centrifuged at
3,700 rpm for 12 minutes (Table 2). The
oil recovery was recorded low in the other
two methods. However, the clarity of
butter oil was found to be good in the
second method when compared to methods
1 and 3. This may be attributed to high
centrifugal forces for efficient separation
of denser materials from lighter
counterparts (Anlauf, 2007).
Solid Substrate Fermentation (SSF) of
butter fat in glass petri dishes (Figure 3)
and sensory evaluation
After 24 hours of incubation, there was a
significant improvement in the flavour
content in the fermented samples with CFS
and CFG strains individually (Table 3).
Whereas, with prolonged incubation the
off flavour production was recorded.
Butter flavour production is based on
multi-step reactions which involve
lipolytic, proteolytic and glycolytic
reactions (Smit et al., 2005). It was
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Nadaraj et al.
reported that diacetyl may also contribute
to the formation of undesirable, offflavours as observed in spirits manufacture
(Krogerus and Gibson, 2013). The
fermented butter fat samples were
subjected to sensory evaluation by ten
volunteers.
Figure 2: Picture showing the recovery of

butter oil by using three methods.
Table 2: Percentage recovery of butter oil
by different methods.
Butter oil recovery (g)
M* Trial Trial Trial Average Recovery
%
1
2
3
1 4.14 4.11 4.12
4.12
68.67
2 4.17 4.19 4.18
4.18
69.67
3 3.79 3.81 3.77
3.79
63.17
Values represent the mean of three
replicates; *Method 1: Melting of butter
fat at 40 C (5C) for 10 minutes and
centrifugation at 800 rpm for 3 minutes
(Chongcharoenyanon et al., 2012);
Method 2: Melting of butter fat at 50 C
(5C) for 8 minutes and centrifugation at
3,700 rpm for 12 minutes (Krause et al.,
2008); Method 3: Melting of butter fat at
70 C (5C) for 15 minutes and
centrifugation at 3,500 rpm for 5 minutes
(Miura et al., 2004).
Sensory evaluation of SSF (Solid
Substrate Fermentation) samples with
various concentrations of butter fat
The fermented butter fat samples were
recorded with preferred sweet and buttery
flavour after 24 hours of incubation with
both the isolated strains (CFS and CFG) in
Table 4. The increase in butter fat quantity
47
Nadaraj et al.
Figure 3: Solid substrate fermentation of butter fat in glass petri dishes; CFS: bacterial strain
isolated from cattle field soil sample.CFG: bacterial strain isolated from cattle field grass
sample.
(esters, methylketones, lactones, etc.)
formation (Smit et al., 2005). Among the 2
Table 3: Sensory evaluation of SSF (Solid
strains tested with 160 g butter fat, CFS
Substrate Fermentation) fermented butter
strain was recorded with increased flavour
samples.
production.
Solid
Sensory evaluation
Supplementation of butter fat with
substrate
CFS
CFG
various carbohydrate sources
fermentation
strain
strain
Lactic acid bacteria are able to ferment
(SSF)
various hexose sugars, from simple
(hour)
(galactose) to complex (lactose) sugars but
24
Buttery
Sweet
is species-dependant (Halsz, 2009).
48
Sour,
Sour,
Galactose moieties present with lactose
alcoholic alcoholic
molecules in milk when metabolized, may
72
Stale,
Stale,
lead to intense aroma production
odourless odourless
(Hugenholtz et al., 2002). In order to

Scores based on the aroma description by
determine if addition of lactose, skim milk
10 volunteers; CFS: bacterial strain
and galactose would enhance the butter
isolated from cattle field soil sample;
flavour production, solid substrate
CFG: bacterial strain isolated from cattle
fermentation was carried with both the
field grass sample.
strains. The butter fat (6 g) was
supplemented with 10 % (w/w) substrate
in the medium may have facilitated the
individually and fermented with CFS and
volunteers in scoring more for sweet and
CFG strains. Table 5 summarizes sensory
buttery flavours. The increased amounts of
evaluation results of the experiment by
raw material (fats and milk sugars) are the
intermediates for flavour compounds
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48
Nadaraj et al.
Table 4: Sensory evaluation of SSF samples with varying concentrations of butter fat*.
Solid substrate
fermentation
(SSF)
(hour)
24
Butter fat 30 g
CFS
CFG
Sensory evaluation
Butter fat 60 g
CFS
CFG
Butter fat 160 g

CFS
CFG
Sweet,
Sweet
Sweet,
Sweet
Sweet,
Sweet
buttery
buttery
buttery
48
Sour,
Sour,
Sour,
Sour,
Sour,
Sour,
alcoholic alcoholic alcoholic alcoholic alcoholic alcoholic
72
Rancid
Rancid
Rancid
Rancid
Rancid
Rancid
*Scoring based the aroma descriptions by 10 volunteers; CFS: bacterial strain isolated from
cattle field soil sample; CFG: bacterial strain isolated from cattle field grass sample.
Table 5: Sensory evaluation of SSF samples fermented with CFS and CFG strains
supplemented with carbohydrate sources
Solid substrate
fermentation
(SSF) (hour)
24
Sensory evaluation
CFS strain
CFG strain
Galactose Lactose Skim Milk Galactose Lactose Skim Milk
Sweet,
Sour
Milky
Sweet
Sour
Milky
buttery
48
Sour
Sour
Milky
Sour
Sour
Milky
72
Stale
Sour
Sour
Stale
Sour
Sour
* Scoring based the aroma descriptions by 10 volunteers; CFS: bacterial strain isolated from
undergraduate students (Figure 4). The
sensory evaluation of the samples by
students is depicted in Table 6.
A sweet, butter flavour production was
enhanced with both the CFS and CFG
strains in 24 hours of incubation. Upon
prolonged incubation (48 and 72 hours),
stale and sour odour were recorded. The
results
indicated
that
butter
fat
supplemented with 10 % galactose and
fermented specifically with the CFS strain
was effective in producing butter oil with
intense butter flavour. Butter fat
supplemented with skim milk recorded a
more pronounced milk flavour, compared
to butter flavour. Therefore, scale up
studies were carried out with increased
amounts of butter fat (30 g, 60 g and 160
g) supplemented with 10 % galactose
individually.
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Fermentation (SSF) with galactose
Scale up studies were performed with
increased butter fat (30 g, 60 g and 160 g)
supplemented with 10% galactose
individually. Figure 5, indicates the scale
up studies of 160 g butter fat supplemented
with 16 g (10%) galactose. After
fermentation, the butter oil was extracted
and subjected to sensory evaluation. Based
on the aroma description (sour, sweet,
buttery, milky, stale and rancid). Scoring
was done by 10 volunteers. Table 6,
indicates the sensory evaluation results of
the fermented sample.
Sensory evaluation of butter fat samples
supplemented with galactose at scale up
studies
Based on the sensory evaluation, the butter
fat samples fermented with strains CFS
49
Nadaraj et al.
Figure 4: Sensory evaluation carried out by undergraduate students.

Table 6: Sensory evaluation of butter fat samples supplemented with 10 % galactose in scale
up studies at 30 g, 60 g and 160 g.
Solid substrate
Sensory evaluation
fermentation (SSF)
30 g butter +
60 g butter +
160 g butter +
(hour)
3 g galactose
6 g galactose
16 g galactose
CFS
CFG
CFS
CFG
CFS
CFG
strain
strain
strain
strain
strain
strain
24
Sweet,
Sweet
Sweet,
Sweet
Sweet,
Sweet
buttery
buttery
buttery
48
Sour,
Sour,
Sour,
Sour,
Sour,
Sour,
alcoholic alcoholic alcoholic alcoholic alcoholic alcoholic
72
Rancid
Rancid
Rancid
Stale,
Rancid
Sour,
odourless
alcoholic
* Scoring based the aroma descriptions by 10 volunteers; CFS: bacterial strain isolated from
and CFG individually were scored sweet
and buttery within 24 hours of incubation
(Table 6). Extended incubation period (48
and 72 hours) resulted in formation of
undesired, foul (stale or rancid or
alcoholic)
odours.
Therefore,
the
experiment was repeated and after 24
hours
of
incubation
period,
the
fermentation was arrested by freezing the
samples at -20C. The respective samples
were thawed at room temperature. Butter
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
oil was extracted and subjected to sensory

evaluation.
by 120 students
Compared to the control sample (BF), all
other samples were preferred (Figure 6).
Most individuals (38%) did not approve of
sample BF with 53% of the volunteers
being unsure of their preference. The least
amount (8.3%) of evaluators prefer this
50
Nadaraj et al.
Figure 5: Scale up studies of 160 g butter fat supplemented with 16 g (10%) galactose; 1:
BF=Untreated butter fat (160 g) (control); 2: BF+Gal+CFS=Butter fat (160 g) supplemented
with galactose (16 g) and fermented with CFS strain; 3: BF+Gal+CFG=Butter fat (160 g)
supplemented with galactose (16 g) and fermented with CFG strain.
sample on an overall basis. In comparative
analysis of unsupplemented butter fat
fermented with isolated LAB, sample
BF+CFS was more preferred (45%) by
students as compared to sample BF+CFG
(19.2%). For supplemented butter fat with
galactose and fermented with isolated
LAB, a large number of evaluators
(56.7%) preferred sample BF+Gal+CFS as
compared to sample BF+Gal+CFG (45%).
Hence,
samples
BF+CFS
and
BF+CFS+Gal were selected for further
GC-MS analysis of constituents with
sample BF serving as the control.
Analysis of butter oil volatile constituents
It was inferred that the butter fat samples
fermented with CFS strain either with or
without galactose was most preferred
based on the sensory analysis of five
different samples by 120 volunteers.
Therefore, butter oil samples extracted
from BF+CFS (butter fat fermented with
strain CFS), BF+Gal+CFS (butter fat
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supplemented
with
galactose
and
fermented with strain CFS) and from BF
(untreated butter as control) were analysed
for diacetyl and acetoin content by GC-MS
method. The results were tabulated.
Comparative analysis of diacetyl and
acetoin content in the unfermented and
fermented butter fat samples with CFS
strain
Acetoin content was highest (1321.2 ppm)
in the butter fat sample fermented with
CFS strain (BF+CFS). In contrast,
untreated butter fat (BF) reported lowest
amount of acetoin (211.5 ppm) amongst all
3 samples (Figure 7). With regards to
diacetyl content, butter fat supplemented
with galactose and fermented with CFS
strain had the highest concentration (511.4
ppm). Lowest content of diacetyl was
found to be from untreated butter fat
(161.7 ppm). Unlike acetoin, diacetyl
strongly contributes to the buttery aroma
(Fuquay et al., 2011). Only in cohesion
51
Nadaraj et al.
Figure 6: Sensory evaluation of butter oil samples by 120 participants; BFS: Butter Fat
Sample; BF: untreated butter; BF+CFS: butter fermented with strain CFS; BF+CFG: butter
fermented with strain CFG; BF+CFS+Gal: butter supplemented with galactose and fermented
with strain CFS; BF+CFG+Gal: butter supplemented with galactose and fermented with
strain CFG.
with diacetyl, does acetoin impart strong,
pleasant, mild, overall buttery aroma in
addition to toning down diacetyl roughness
(Bai et al, 2014). The results of diacetyl
and acetoin analysis tallies with the
preference of volunteers who preferred
supplemented butter fat and fermented
with CFS strain the most (56.7%).
Untreated butter fat was the least preferred
(8.3%) and is reflected by the lowest
concentrations of diacetyl and acetoin
content.
Butter powder formulation
Butter powder formulation was made with
maltodextrin and to enhance its flow
properties anti-caking agents (bentonite,
sodium silicate, sodium chloride, potato
starch and mannitol) were evaluated
(Figure 8). The samples were subjected to
sensory evaluation. The recovered butter
oil was then formulated to powder form by
addition of a carrier material and antiISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
caking agent. This was performed to

enhance flow capability and storage
property of the butter powder.
Standardization of butter oil and
maltodextrin ratio for butter concentrate
powder formulation
It was observed that the use of 25 g butter
oil resulted in the formation of minor
clumping with maltodextrin (Table 7).
Although lower amounts of butter oil did
not result in clumping, desired intensity of
butter aroma was not imparted.
Maltodextrin is a well-known carrier
material used for various preparations. It is
digestible, lacks artificial colouring or
flavour, low in cost and miscible in liquids
(Zuidam and Shimoni, 2010). In addition,
its good binding and high viscosity
capabilities enables spray or freeze drying
of active ingredients to be made into
powders easily (Akhilesh et al., 2012).
From the various anti-caking agents tried,
52
Nadaraj et al.
Figure 7: A comparative analysis of diacetyl and acetoin content in the unfermented and
fermented butter fat samples with CFS strain; BF: untreated butter; BF+CFS: butter
fermented with strain CFS; BF+Gal+CFS: butter supplemented with galactose and fermented
with strain CFS.
Figure 8: Butter powder formulation with different concentrations of butter oil in

maltodextrin.
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53

Table 7: Standardization of butter oil and
maltodextrin ratio for butter concentrate
powder formulation.
Maltodz Butter
Quality attributes
(g)
oil (g)
Texture
Aroma
50
5
No clumps
Faint
50
10
No clumps
Faint
50
15
No clumps
Decent
50
20
No clumps
Decent
50
25
Less clumps Strong
50
30
Grainy clumps Strong
zMaltodextrin
Nadaraj et al.
method for optimum recovery of butter oil
from the butter fat samples was
standardized, which resulted in 69.67 %
recovery of butter oil. Upon preliminary
screening of the LAB isolates, the CFS
and CFG isolates were selected based on
the sensory evaluation by 10 volunteers.
none are able to enhance the flow rate of

the formulated powder at various
concentrations (2%, 4% and 6% w/w).
Such findings are in contrast to reports by
(Ganesan et al., 2008) who stated
application of 2% anticaking agent is
adequate for enhancing powder flow rates.
A possible reason for failing to achieve the
desired powder texture might be due to
structural collapse of the sample. This may
be attributed to decreased product
molecular porosity and overall volume, as
a result of improper sample drying and
storage (Bhadra et al., 2011).
The solid substrate fermentation condition

by the two strains produced sweet and
buttery notes within 24 hours. In the final
phase, a scale up study of butter fat
supplemented with 10% galactose was
carried out by SSF. A large scale sensory
evaluation was performed with 120
volunteers. The elevated concentration of
both diacetyl and acetoin was most
preferred (56.7%). The formulation of
butter powder with maltodextrin was
investigated for its flow properties.
Overall, the results obtained from this
study will pave way for the future
investigations for the development of a
microbial process for the production of
butter flavour concentrate from butter fat
with lactic acid bacteria via solid substrate
fermentation.
CONCLUSION
REFERENCES
The demand for natural butter flavours are

driven by growing consumer awareness.
This has lead to the development of
biotechnological processes wherein Lactic
acid bacteria (LAB) widely recognized as
GRAS (generally regarded as safe) and
used for enhancement flavour in fermented
food. This research work addressed the
application of lactic acid bacteria in
obtaining high yields of desirable
enantiomeric butter flavour compounds
exclusively from butter fat by solid state
fermentation. In the first phase, the work
focused on the isolation of lactic acid
bacteria (LAB) strains from various dairy
and environmental samples and screened
for the fermentation of butter fat. The
pasteurization of unsalted butter fat was
ineffective in killing the native microbes.
Hence sterilization was adopted. The
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Akhilesh, D., Faishal, G. and Kamath,

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56

Res. Highl. 4Bs (2016), P57-66
Reconfigurable Filter Bank for Accurate Spectral Decomposition of EEG

Signals
Biju K. S.1, *, Hareeshkumar M.2, Girishkumar C.3, Jibukumar M. G.1
1
Electronics Engineering Division, School of Engineering, Cochin University of Science &

Technology, Kochi, 682022, India. 2Electronics and Communication Engineering Department,
Government Engineering College, Bartonhill, Thiruvananthapuram, 695035, India.
3
Faculty of Engineering & Computer Technology, AIMST University, Malaysia;
*corresponding author, e-mail: bijukarunnya@gmail.com
ABSTRACT
Aim: Electroencephalography (EEG) signals contain vital information which is extremely
helpful for studying the functionalities and disorders of brain. For detailed analysis, the spectral
decomposition of EEG signals are split into different EEG rhythms. Since the different EEG
rhythms are of non-uniform bandwidth, existing techniques results in inaccurate decomposition
and which in turn inaccurate results. The reconfigurable filter bank proposed can replace the
existing methods for an efficient and accurate spectral decomposition of EEG signals.
Methodology and results: The structure of the reconfigurable filter bank (RFB) includes a
uniform filter bank followed by frequency response masking (FRM) filters. The uses of FRM
filters provide a sharp transition bandwidth which optimizes the design. The first masking filter
for delta band was designed such that it extracts the 0.5-4 Hz band. The second masking filter for
theta band was of extracts 4-8 Hz. The masking filter for alpha band extracted about 8-13 Hz.
The beta band was extracted using the masking filter applied to the sub filter of fourth stage
extract greater than 14Hz. Analysis of RFB magnitude spectrum of both healthy EEG and
seizure EEG signal is carried out. Conclusion: The proposed reconfigurable filter bank is based
on frequency response masking technique and it provides an accurate extraction of EEG
rhythms. The spectra of each band are very much clear that the extracted rhythms have much less
components from the adjacent spectra.
Keywords: Frequency response masking; Reconfigurable filter banks; Spectral decomposition.
INTRODUCTION
Brain is the most complex part in the human
body and therefore studying and analysis of
the same for understanding the features,
functioning and artifacts are difficult as
compared to the other organs and or parts in
the body. Brain waves are analyzed for the
study of functioning and diagnosis of brain
disorders. A number of techniques have
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been in use for recording these brain signals.

Electroencephalogram (EEG) recording is
one of the most prominent among them
(Teplan, 2002).
The brain reacts differently at different
stages of time; hence, the brain signals will
be different accordingly. Brain signals
consist of different frequency components
termed as EEG rhythms. For a proper
57
Spectral Decomposition of EEG Signals

analysis, these EEG rhythms have to be
extracted
separately.
For
spectral
decomposition and analysis lots of
techniques like time-frequency analysis have
been introduced earlier (Shayan et al.,
2014). The various time-frequency analysis
techniques in practice include Short Time
Fourier Transform (STFT), Wavelet
Transform (WT), Wavelet Packet Transform
(WPT), Filter Banks, Moving Average
filtering, Auto Regressive analysis, Auto
Regressive Moving Average model (Saeid
and Chambers, 2007). The STFT, WT,
WPT,
Filter
Banks
etc.
enhance
decomposition of signals into sub-bands and
simultaneous analysis of the various
(wanted) spectral components.
Spectral analysis is performed on the signals
to extract various key-information (Bhagwat
and Vinod, 2013). For the spectral analysis
lots and lots of transform techniques and
their variants have been proposed and are
being used through decades. Since, in
particular, EEG is a non-stationary signal,
transforms like DFT, FFT etc. cannot
describe it completely, and some transform
technique localized in time and frequency as
well is needed (Bhagwat and Vinod, 2013).
The wavelet transform technique is in wide
practice for analysis of EEG signal for
decomposition into different bands of equal
widths (Bhushan, 2013; Rafiee et al., 2011).
Wavelet Packets has been found better in the
analysis of biological signals. At higher
frequencies WT fails to localize time with
required accuracy. Discrimination of
frequency is sacrificed at higher frequencies
for localization of time. WPT generalizes
the time-frequency analysis of WT. Filter
Bank is also a candidate of sub-band
decomposition of EEG signals (Alfred,
1999; Chen et al., 2014; Darak et al., 2011).
In all these cases the sub band bandwidths
are fixed. This leads to an inaccurate
decomposition and analysis and in turn
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Biju et al.
inaccurate results. Use of Reconfigurable
Filter Banks helps solving this issue.
Various methods have been proposed for
achieving the reconfigurability in the cutoff
frequency and bandwidths of filters. One
approach is to implement a variable cut off
digital filter in which the cut off frequency
could be controlled through a single
parameter which uses the principle of
frequency transformation of linear phase
FIR filters (Oppenheim et al., 1976). In
fractional delay method, each unit in delay
operator in fixed coefficient FIR filter is
replaced with second order FIR fractional
delay structure (Sasikumar et al., 2013).
Cut-off frequency is changed by changing
the FD value which in turn changes the
bandwidth. The frequency response masking
(FRM) technique comprises the complete
finest transition-band filter using many wide
transition-band sub-filters (Lim, 1986; Lim
and Boroujeny, 1992). In the modified
frequency transformation technique the
fixed-coefficient low-pass sub filter in the
first stage of a Fast Filter bank (FFB) is
replaced by a modified second-order
frequency transformation (MFT) based low
pass variable digital filter (Filipe et al.,
2007).
In this paper the design of a reconfigurable
filter bank for EEG spectral decomposition
is proposed. Here a uniform filter bank is
used as a prototype filter bank and FRM
Technique is applied for achieving
reconfigurability in sub band bandwidths.
METHODOLOGY
The brain cells transfer the information by
means of biochemical reactions across small
spaces which are termed as synapses. The
nerve cells can be considered as a dipole
with its own particular orientation as well as
polarity. Such dipoles with identical polarity
will receive similar inputs and they add up
58

together resulting in potentials. These
signals are termed as brain waves and when
recorded
using
Electroencephalogram
referred to as EEG signals (Alfred, 1999).
EEG rhythms are different phenomena or
events in the EEG. The commonly used
terms for EEG frequency () bands are as
follows (Saeid and Chambers, 2007).
delta () 0.5 f < 4

theta () 4 f < 8
alpha () 8 f 13Hz
beta() 13Hz f
In general, EEG signals do not reveal
information about any abnormalities or
disorders of brain. Time-frequency analysis
is required to extract such information, if
any,
from
EEG
signals.
Spectral
decomposition of EEG signals into EEG
rhythms have been always a challenge since
the EEG rhythms are of low frequency and
accurate decomposition is very difficult
(Shayan et al., 2014). The non-uniform band
widths of the sub bands also add up to the
complexity in spectral decomposition. Lots
of methods like Wavelet Transform,
Wavelet Packet transform, Filter Banks, etc.
have been used for the same spectrum
(Alfred, 1999; Shayan et al., 2014). The
analysis using WT and WPT decomposes
the finite energy signal in successive stages
each with different scaling factor, Thus it
gives the information in both time and
frequency domains. But at higher
frequencies discrimination of frequencies is
sacrificed for localization of time (Alfred,
1999). Wavelet Packet Transform mitigates
this problem. But, the bandwidth of packets
is multiples of two; hence, it is suitable only
for uniform sub band decomposition. The
existing methods have not succeeded in nonuniform sub band decomposition of EEG
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Biju et al.
signals. Since each rhythm of EEG
represents the activity of brain in different
stages of activity accurate spectral
decomposition is of great importance (Saeid
and Chambers, 2007). So far no work has
been done on the design of a method for
accurate sub band decomposition. The
proposed reconfigurable filter bank is the
first attempt of its kind in the scenario of
EEG signal processing.
In the proposed Reconfigurable Filter Bank,
the center frequency and bandwidth of the
sub filters can be varied according to the
requirements. The design requirements
include sub band filters with sharp transition
bands and considerably large stop band
attenuation.
Proposed design
The proposed RFB consists mainly of two
stages: i) A perfect reconstruction Uniform
Filter Bank and ii) Frequency Response
Masking based masking filters. In section
3.1, the design and response of Uniform
Filter Bank is described. In the following
section the principle of Frequency Response
Masking and the design steps of FRM
masking filters is discussed.
Design of perfect reconstruction 16
channel filter bank
The filter banks comprise two stages,
analysis filter banks and synthesis filter
banks filter banks (Vaidyanathan, 1993).
Each stage consists of low pass, band pass
and high pass filters. In analysis filters the
input finite energy signal is applied to M
filters which will be decomposed into M
uniform width sub-bands. The resulting
signals are sub-sampled by a factor N to
avoid redundancies. At the synthesis filter
bank stage, up sampling is done to recover
the sampling rate of input signal. The up
sampled signals are applied to synthesis
filters which will compose the original
signal. If number of filter stages equals the
A.
59
Biju et al.
decimation factor it is referred to as critical

sub-sampling, since above that factor perfect
reconstruction is not possible. The perfect
reconstruction filter bank returns the original
input signal with time shift and amplitude
scaling (Vaidyanathan, 1990).
Let c(n) be the input signal and r(n)
represents the response, here the signals are
related as
R0(z2) = 0.5 [A0(z) C(z) + A0(-z) C(-z)] (1)
R1(z2) = 0.5 [A1(z) C(z) + A1(-z) C(-z)] (2)
C'(z) = [R0 (z2) S0(z) + R1(z2) S1(z)]
(3)
Combining these equations,

input-output relation is given by
the
C'(z) = 0.5[A0(z)S0(z) +A1(z)S1(z)]C(z) +

0.5[A0(-z)S0(z)+A1(-z) S1(z)]C(-z) (4)
Here A(z) represents the analysis filter bank
frequency response and S(z) represents the
synthesis filter bank frequency response.
The first term and the second term represent
transmission of the c(n) through the filters
and the aliasing component at the output of
the filter bank respectively. Perfect
reconstruction is achieved if the transfer
function for the aliasing component is zero.
Design of masking filters
The reconfigurable Filter Bank was obtained
as a result of combining the uniform filter
bank with masking filters generated using
the Frequency Response Masking technique
(Mahesh and Vinod, 2011). The masking
filters of desired bandwidths are applied at
the output of the uniform filter bank sub
filters at the desired stages to extract the
desired bands. In the masking filter method
the spectral analysis of complementary pair
filters are masked by the two suitable
masking filters. So the desired output is the
B.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
combination of the masking filters. This

method has very scanty coefficients and
very low computation (Lim, 1986).
Initially a low pass fir filter is selected
which is termed as the modal filter with
transfer function Ha(z). Now each delay of
this filter is replaced by M delays to get a
new filter with transfer function Hb(z) =
Ha(zM). If Hb(z) is masked with another filter
Hc(z) well get a new impulse response
Hd(z) with transition width 1/M times that of
Ha(z). This method is used to obtain a sharp
transition band filter using Frequency
response masking. The consequences of the
masking technique is the transition width
reduce the by a factor of M for every M
delay. Which affect width of the pass band
reduced by M factor (Sumedh et al., 2013;
Lim and Lian, 1993). Hence, it is suitable
only for a narrow-band design. Since our
application requires narrow band filters only
FRM technique is optimum for the
achieving reconfigurability in Filter banks
for non-uniform sub-band decomposition of
EEG signals.
Proposed method
As initial stage, a 16-channel perfect
reconstruction filter bank was designed. For
this initially, a two channel perfect
reconstruction filter bank was designed. The
order chosen was 90. This was chosen as the
prototype filter bank for designing the Mchannel Uniform Filter Bank. Then, the tree
type 16 channel filter bank was developed.
Here, each filter impulse response is
convolved with the interpolated impulse
response of the filters in previous stage to
get the new impulse response.
C.
Let and are the low pass filter and

the complementary filter of the 2 channel
perfect reconstruction model filter. Let
and are represent their interpolated
versions respectively. Then the sub-band
filters in the (i+1)th stage of the M stage
60

filter bank are given by the following design
equations.
Biju et al.
( + 1)= () * ()
( + 1) = ()* ( + 1)
(5)
(6)
single hardware structure can be used for

mapping these sub-filters. Hence, just one
filter structure is needed and the number of
adders and multipliers can be reduced. This
reduces the complexity in design.
( + 1) = () ( + 1)
(7)
( + 1) = () ()
(8)
The EEG signals were adopted from CHBMIT scalp EEG database prepared and
hosted by Children's Hospital Boston (CHB)
and the Massachusetts Institute of
Technology (MIT). The signals are in the
European data format (.edf), which is a
standard file format used for the storage and
exchange of medical time series. The
sampling frequency is 256 Hz and the file
includes signals from 16 channels. Another
source of database was that from
Department of Epileptology, University of
Bonn.
The response shows minimum overlap in

complementary bands and also the filter
bank has got fairly sharp transition band.
The response of the 16-channel perfect
reconstruction filter bank is shown in the
following Figure 1. As the next stage
masking filters were designed using FRM
technique. This helps in perfect extraction of
EEG Rhythms. The cut off frequency and in
turn bandwidth of the required masking
filters were reconfigured by adjusting the
input pass band and stop band frequencies
without changing the order.
Since the designed sub-filters are identical
The procedure for simulation of the

reconfigurable filter bank as follows. The
Figure 1: Magnitude response of 16-channel uniform filter bank.

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61
Biju et al.
input signals read in the .edf format were

applied to pre-processing which includes
low pass filtering for restricting to 64 Hz
followed. The signal was then applied to a
notch filter for removing power line
components. Pre-processed signal is applied
to uniform filter bank. The uniform filter
bank designed here is a 16-channel perfect
reconstruction filter bank which provides
accurate decomposition into bands of width
4 Hz each. Masking filters are applied to
sub filters for extracting the desired
Rhythms. Masking filters are designed
according to the requirements. The first
masking filter for delta band was designed
such that it extracts the 0.5-4 Hz band. The
second masking filter for Theta band was of
bandwidth 4 Hz. The masking filter for
Alpha band was of frequency width 5 Hz
and was applied to the sub filter of third
stage. The Beta band was extracted using the
masking filter applied to the subfilter of
fourth stage of uniform filter bank. The
output response and spectrum of uniform
Input Signal
500
0
-500
5000
10000
15000
10000
15000
10000
15000
10000
15000
10000
15000
Delta Band
500
0
-500
5000
Theta Band
50
0
-50
5000
Alpha Band
20
0
-20
5000
Beta Band
50
0
-50
5000
Figure 2: Response of Uniform Filter Bank.

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2
1
0
2
1
0
4000
2000
0
40
20
0
4000
2000
0
Input Signal
x 10
0
5
5
x 10
10
15
20
25
Delta Band
30
35
40
45
10
15
20
25
Theta Band
30
35
40
45
10
15
20
25
Alpha Band
30
35
40
45
10
15
20
25
Beta Band
30
35
40
45
10
15
20
30
35
40
45
25
Figure3: Magnitude spectrum of Uniform

Filter Bank response.
Filter Bank is shown in the Figure 2 and
Figure 3 respectively. From the waveforms
it is clear that there is an overlap in the
spectra; hence, the obtained result is
inaccurate.
The
adjacent
spectral
components are present in the desired
spectrum which will affect the accurate
diagnosis of diseases. Use of Reconfigurable
Filter Bank helps to mitigate this problem.
The proposed RFB provides accurate
extraction of EEG rhythms with high
resolution. The waveforms in the Figure 4
and Figure 5 are magnitude spectrum of
healthy EEG signal and the abnormal
(seizure) EEG signal respectively.
From the spectra it is clear that the extracted
rhythms have much less components form
adjacent spectra. Hence, it will be easier to
analyze different bands separately and to
determine in which band abnormality
occurs.
This
makes
the
spectral
decomposition analysis for EEG signals and
62
Biju et al.
to detect the seizure activity which is

dominating in the sub-bands.
provides considerably narrow transition

band width without higher orders. Hence,
the circuit complexity is less. Since FRM
filters are FIR based, the design retains all
the advantages of FIR filters including
guaranteed stability, low coefficient
sensitivity, free of phase distortion etc.
The main advantage of using FRM filters for

reconfiguring bandwidth is that no hardware
overhead is required for changing the
bandwidth and centre frequency. Only thing
required is the change in coefficients. Also it
Input EEG Spectrum
x 10
1
0
10
15
delta band
20
25
30
10
15
theta band
20
25
30
10
15
alpha band
20
25
30
10
15
beta band
20
25
30
10
15
20
25
30
x 10
1
0
10
5
0
0
4
x 10
1
0
10000
5000
0
Figure 4: RFB Magnitude Spectrum of healthy EEG signal.

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Biju et al.
Figure 5: RFB Magnitude Spectrum of seizure EEG signal.

CONCLUSION
In this paper design of a reconfigurable filter
bank for efficient and accurate spectral
decomposition of EEG signals is proposed.
The existing techniques employed for
spectral decomposition such as Wavelet
Transform, Wavelet Packet transform and
DFT Filter bank could only extract bands of
uniform bandwidth. The proposed RFB
based on FRM technique provides accurate
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
extraction of EEG Rhythms. Use of

reconfigurable filter bank is the first attempt
in EEG rhythm extraction. A perfect
reconstruction 2-channel filter bank was
used as modal filter bank and an M-channel
filter bank was designed using this modal
filter bank. To mitigate the problem of
varied bandwidth EEG spectral extraction,
FRM based filters were applied as masking
filters. Use of FRM technique for the
generation of masking filters has provided
64

sharp transition width narrow band low pass
and band pass filters to be used for
extracting desired bands. Since the sub
filters are identical they can be mapped into
a single hardware structure, it reduces the
complexity of hardware implementation.
Since the reconfigurability is achieved
without a change in hardware the technique
is found to be optimum for EEG rhythm
extraction.
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and
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filter design based on fractional
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Based on Frequency Response
Masking
for
Non-uniform
Channelization in Software Radio
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Mersereau, R. (1976). Variable
cutoff linear phase digital filters.
Circuits and Systems 23(4), 199
203.
Rafiee, J. Rafiee, M. A. Prause, N. and
Shoan, M. P. (2011). Wavelet basis
functions in biomedical signal
65

processing. Expert Systems with
Applications 38(5), 6190-6201.
Saeid, S. and Chambers, J. A. (2007).
EEG Signal Processing, John Wiley
& Sons Ltd. pp.51-75.
Sasikumar, G. VudiS. M. and Rittwika,
G. (2013). Analysis and simulation
of brain signal data by EEG signal
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technique
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MATLAB. International Journal of
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2771-76.
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reconfigurable channel filter based
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P. (2013). A low complexity
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66

Res. Highl. 4Bs (2016), P67-76
Coenzyme Q10 Dietary Supplementation during Antitubercular Therapy

Prevents Renal Damage in Rats
Udhaya Lavinya B. and Evan Prince Sabina*
School of Biosciences and Technology, VIT University, Vellore-632014, Tamilnadu, India;
*corresponding author, e-mail: eps674@gmail.com
ABSTRACT
Aim: Antitubercular therapy leads to the development of acute renal injury (ARI) in some
individuals. Though isoniazid (INH) has been evidenced as an ARI inducing drug, mounting
evidences from several studies identify rifampicin (RIF) as the most common ARI inducing
antitubercular drug. Current study was carried out to evaluate the nephroprotective effect of
the oral supplementation of coenzyme Q10 in INH and RIF treated Wistar albino rats.
Methodology and results: Rats were administered with INH and RIF (50 mg/kg b.w.
each/day) for 28 days. The effect of concomitant treatment with coenzyme Q10 (10 mg/kg
b.w./day) on INH and RIF-induced renal injury was evaluated by estimating the serum levels
of renal functional markers such as creatinine, urea, uric acid and acid phosphatase. In
addition, the antioxidant profile, levels of non-enzymic antioxidants and lipid peroxidation
were assessed in renal homogenates of experimental rats. Histological studies were also
performed. The standard hepatoprotective drug silymarin (25 mg/kg b.w./day) was used for
the purpose of comparison. The tested parameters of the coenzyme Q10 treated INH and RIFinduced rats were compared with that of the normal control rats and silymarin-treated INH
and RIF-induced rats. Coenzyme Q10 significantly reduced the elevated levels of serum renal
functional markers in INH and RIF-administered rats. Also, the food supplement was able to
restore near normal the antioxidant status and noted to prevent renal damage in experimental
rats. Conclusion, significance and impact of study: Current study reveals the potential of
coenzyme Q10 in minimizing the renal injury due to antitubercular therapy. Hence, CoQ10
supplementation would be useful to patients on antitubercular regimen.
Keywords: Acute renal injury; Coenzyme Q10; Isoniazid; Rifampicin.
INTRODUCTION
Oxidative stress plays a major role in the
induction and progression of renal failure
both acute and chronic. Several conditions
like hypertension, diabetes, infection,
obstruction in the urinary tract,
autoimmune disorders such as lupus
erythematosus, genetic disorders such as
polycystic kidney disease, drugs such as
antibiotics, diuretics and anti-inflammatory
drugs induce oxidative stress in renal
tissues (Schattner et al., 2000; Crispn et
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
al., 2008; Chevalier et al., 2010). Several

medications are known inducers of acute
interstitial nephritis which includes
commonly used non-steroidal antiinflammatory drugs (NSAIDS), interferon
and antibiotics such as penicillins,
cephalosporins and anti-tubercular drugs
such as rifampicin (Rossert, 2001). Studies
exploring the sub-cellular mechanisms of
renal injury causing AIN have been carried
out recently (Mitchell et al., 1977; Servais
et al., 2007; Chevalier et al., 2010). It has
67
Coenzyme Q10 Dietary Supplementation

been proven that interstitial inflammation
in renal tissue might be due to the
predominant infiltration of local B-cells,
eosinophils and mononuclear leucocytes in
the interstitium (Heller et al., 2007). In
addition, the development of antirifampicin antibodies occurs rarely in
patients on antitubercular therapy. Studies
have shown that these rifampicindependent
antibodies
cause
acute
haemolysis and renal failure (van der
Meulen et al., 2009; Beebe et al., 2015).
Several case studies have reported the
occurrence of acute interstitial tubulopathy
in pulmonary tuberculosis patients on
antituberculosis regimen (Muthukumar et
al., 2002; Rosati et al., 2013). Druginduced ARI is a major adverse effect due
to
rifampicin
though
uncommon.
Isoniazid (INH) and rifampicin (RIF) coadministration causes injury in the
hepatocytes leading to hepatotoxicity
(Baskaran and Sabina, 2015).
Coenzyme Q10 is a fat soluble vitaminlike compound with significant antioxidant
potential. It plays key role in
mitochondrial bioenergetics. Though the
compound is synthesised within the body,
there are certain conditions that lead to low
levels of coenzyme Q10 (CoQ10) such as
vitamin B deficiency, use of statins for
hypercholesterolemia,
old
age,
cardiovascular disorders and oxidative
stress (Gempel et al., 2007; Quinzii et al.,
2007; Quinzii and Hirano, 2011).
Recently, it has been found that this
antioxidant also plays significant role in
the regulation and alteration of genes
involving cell signalling and metabolic
pathways (Groneberg et al., 2005). Our
previous study showed significant
protective effects of CoQ10 against INH
and RIF induced hepatotoxicity (Baskaran
and Sabina, 2015).
Current study was an attempt to evaluate
the occurrence of renal injury due to the
co-administration of INH and RIF and the
role of CoQ10 as a nephroprotective agent
in Wistar albino rats.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Udhaya and Sabina

Chemicals and drugs
Synthetic coenzyme Q10 was purchased
from Sigma Aldrich, India. INH and RIF
were purchased from Lupin Ltd.,
Aurangabad, India and the standard
hepatoprotective drug silymarin from
Quality Pharmaceuticals Ltd., India. INH
and RIF were dissolved in normal saline
while silymarin was dissolved in sterile
distilled water. Coenzyme Q10 was
dissolved in 0.2 ml corn oil. All the other
chemicals and reagents used were of
analytical grade procured from SD Fine
Chemicals Pvt. Ltd., Mumbai, India.
Animals
30 female Wistar albino rats of body
weight 143.2612.81 g were procured
from the Animal House, VIT University,
Vellore, Tamilnadu. The rats were divided
into 5 groups and treated as follows for 28
days: group I was normal control; group II
was treated with INH and RIF (50 mg/kg
b.w. each/day); group III was INH and
RIF-induced rats co-administered with
coenzyme Q10 (10 mg/kg b.w./day); group
IV was INH and RIF-induced rats coadministered with silymarin (25 mg/kg
b.w./day); group V was treated with
coenzyme Q10 (10 mg/kg b.w./day) alone.
The animals were sacrificed after the study
duration using ether anaesthesia. Blood
and kidneys were procured for further
analysis. Renal homogenates were
prepared using 5 % phosphate buffered
saline (PBS).
Estimation of serum markers of renal
function
Serum levels of renal functional markers
such as creatinine, urea, uric acid and acid
phosphatase were
estimated using
commercial diagnostic kits obtained from
AutoSpan Diagnostics Ltd., India.
Assessment of antioxidant profile
Renal homogenates were used to assess the
activities of superoxide dismutase
68

(Marklund and Marklund, 1974), catalase
(Sinha, 1972), glutathione peroxidase
(Rotruck et al., 1973) and Glutathione-Stransferase (Habig et al., 1974); levels of
total reduced glutathione (Moron et al.,
1979) and lipid peroxidation (Ohkawa et
al., 1979).
Histopathological analysis
A portion of the kidneys were fixed in 10
% formalin after thorough washing with
ice-cold 5 % PBS. The tissues were then
dehydrated with descending grades of
isopropanol. After treating with xylene, the
tissues were embedded in molten paraffin
wax and tissue sections (5 m) were cut.
The sections
were
stained with
hematoxylin and eosin and examined
microscopically for pathological changes.
Udhaya and Sabina

Effect of coenzyme Q10 on serum
markers of renal function in INH and
RIF induced rats
INH and RIF induced rats showed
significant increase in the levels of urea,
creatinine, uric acid and acid phosphatase
while concomitant administration of
coenzyme Q10 caused significant (P<0.05)
reduction in the elevated levels of the
aforementioned parameters (Figures 1-4).
This particular effect of coenzyme Q10 in
minimizing INH and RIF-induced changes
in serum markers of renal function was
comparable with that of silymarin treated
rats. The oxidative stress parted by the
toxic metabolites of isoniazid on renal
tissue might lead to oxidative cellular
damage. In addition, evidence from
literature suggest that the generation of
anti-rifampicin
antibodies
causes
Figure 1: Effect of coenzyme Q10 on serum urea in INH and RIF induced rats. Each value
represents the Meansd of six rats. Comparisons were made as follows: a-group I vs. groups
II, III, IV, V; b-group II vs. group III, IV, V. c-group III vs. groups IV, V; d-groups IV vs.
group V. The symbol represents statistical significance at *(p<0.05>. Statistical analysis was
calculated by one-way ANOVA followed by the student Newman-keuls test.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
69

formation of drug-antibody immune
complexes which result in glomerular
endotheliosis and cell damage thereby
leading to tubular injury and reduced renal
function (Muthukumar et al., 2002). Acute
tubular necrosis and interstitial nephritis
Udhaya and Sabina

are adverse effects of RIF reported in
tuberculosis patients (van der Meulen et
al., 2009). A case study has reported the
evidence of acute renal failure (ARF) in a
38 year old tuberculosis patient (Beebe et
al.,
2015).
Figure 2: Effect of coenzyme Q10 on serum creatinine in INH and RIF induced rats. Each
value represents the Meansd of six rats. Comparisons were made as follows: a-group I vs.
groups II,III,IV,V; b-group II vs. group III,IV,V. c-group III vs. groups IV,V; d-groups IV vs.
group V. The symbol represents statistical significance at *(p<0.05>. Statistical analysis was
calculated by one-way ANOVA followed by the student Newman-keuls test.
Figure
3:
Effect
of
coenzyme
Q10 on serum
uric acid in
INH and RIF
induced rats.
Each value represents the Meansd of six rats. Comparisons were made as follows: a-group I
vs. groups II,III,IV,V; b-group II vs. group III,IV,V. c-group III vs. groups IV,V; d-groups IV
vs. group V. The symbol represents statistical significance at *(p<0.05>. Statistical analysis
was calculated by one-way ANOVA followed by the student Newman-keuls test.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
70
Udhaya and Sabina
Figure 4: Effect of coenzyme Q10 on serum acid phosphatase in INH and RIF induced rats.
Effect of coenzyme Q10 on antioxidant
profile in INH and RIF induced rats
There was significant (P<0.05) reduction
in the activities of antioxidant enzymes
such as superoxide dismutase, catalase,
glutathione peroxidase and glutathione-Stransferase in renal homogenates of INH
and RIF induced rats (Table 1). Also, there
was significant (P<0.05) reduction in the
levels of reduced glutathione and
significant (P<0.05) increase in the levels
of lipid peroxidation on treatment with
INH and RIF. Co-administration of
coenzyme Q10 was able to restore these
parameters to near normal levels which
were compared with that of silymarin
treated INH and RIF induced rats. The
reduction in the activities of the enzymic
and non-enzymic antioxidants and elevated
lipid peroxidation levels reflect the extent
of oxidative stress in the INH and RIF
treated rats. It has been proven that
mitochondrial membrane damage induces
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
DNA fragmentation and apoptosis in

organs that are vulnerable to oxidative
damage. The mechanism that underlies
mitochondrial membrane damage is
oxidative stress exerted due to the
production of ROS.
Effect of coenzyme Q10 on renal tissues
in INH and RIF induced rats
ARI is an uncommon and less known
adverse
effect
arising
due
to
antituberculosis regimen which includes
rifampicin (Topping et al., 2012). Case
studies in elderly population have shown
evidence of ARF (De Vriese et al., 1998;
Chang et al., 2014). There are also
mounting evidences on the occurrence of
ARI in patients undergoing intermittent or
interrupted administration of rifampicin
giving rise to the development of antirifampicin antibodies (Pereira et al., 1991;
Rosati et al., 2013). Histopathological
examination of renal tissue architecture in
71
Udhaya and Sabina
the current study reveals damage to

glomerular cells and karyolysis in the INH
and RIF treated rats. Apart from ARI
caused by rifampicin, there could have
been significant damage caused due to
oxidative stress and depletion of cellular
antioxidants parted by the toxic
metabolites of isoniazid (Chowdhury et
al., 2006). Mitochondrial membrane
potential (MMP) collapse causes apoptosis
(Ly et al., 2003). It has been shown that

CoQ10 prevents MMP collapse by
preventing mitochondrial depolarization
and hence may possess antiapoptotic
activity (Papucci et al., 2003). This
particular activity of CoQ10 along with its
antioxidant and free radical scavenging
activities might have prevented tubular
injury.
Table 1: Effect of concomitant administration of coenzyme Q10 on antioxidant parameters in

renal tissue homogenates of INH and RIF induced rats.
Parameters
SOD
(units/min/mg
protein)
Catalase
(units/min/mg
protein)
Glutathione
peroxidase (g
of GSH
utilized/min/
mg protein)
Reduced
glutathione
(nmol/mg
protein)
Glutathione-Stransferase
(nmol of CDNBGSH conjugate
formed/min/mg
protein)
TBARS
(mM/TBARS/100
g of wet tissue)
Group 1
(Control)
Group 2
(INH+RIF 50
mg/kg b.w.)
Group 3
(INH+RIF &
CoQ10 10mg/kg
b.w.)
Group 4
(INH+RIF &
silymarin 25
mg/kg b.w.)
Group 5 (CoQ10
10 mg/kg b.w.)
180.143.42
95.043.40a*
165.112.29a*b*
168.055.21a*b*
186.033.84b*c*d*
60.142.85
35.122.84a*
55.042.00b*
53.052.28a*b*c*
64.323.13b*c*
38.082.28
18.022.28a*
32.071.42a*b*
32.643.49a*b*
37.051.42b*c*
46.141.45
28.042.00a*
42.044.00b*
41.142.02b*
47.092.01b*d*
20.091.43
11.1421.45a*
16.001.41b*
16.513.78b*
20.012.00b*
0.800.20
1.800.14a*
1.000.14b*
1.100.14b*
0.700.14b*d*
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
72
Udhaya and Sabina
Figure 5: Effect of CoQ10 on the renal histology of INH and RIF treated rats. Kidney
histopathology (haematoxylin and eosin staining): A) normal control rats showing normal
histology of glomeruli and renal tubules; B) INH and RIF treated rats showing normal to
decreased cellularity of glomerulus, renal tubules showing cell swelling with increase in
eosinophilia of the cytoplasm with karyolysis in few of the cells; C) CoQ10 supplemented
INH and RIF treated rats showing normal histoarchitecture of renal tissue being maintained
D) silymarin administered INH and RIF treated rats showing normal morphology of
glomeruli and tubules E) CoQ10 alone treated rats showing normal renal tissue histology.
CONCLUSION
Current study shows that CoQ10 was able
to restore normal antioxidant status in INH
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
and RIF treated rats. The determination of

serum renal function markers and kidney
histopathological alterations reveal that
CoQ10 minimizes damage to renal tissue
73

due to INH and RIF. Free radical
scavenging property of CoQ10 might have
reduced the extent of damage to cellular
membranes and macromolecules thereby
rendering protection against oxidative
stress. In addition, the anti-apoptotic
property of CoQ10 might have minimized
cell death due to mitochondrial membrane
depolarization thereby reducing injury to
tubular interstitium which might have
aided
the
maintenance
of
renal
histoarchitecture. Further studies would
explore the molecular mechanisms of the
effect of CoQ10 in INH and RIF induced
renal injury.
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76

Res. Highl. 4Bs (2016),P77-83
Safe Water as the Key to Food Safety and Global Health

Md. Latiful Bari*
Center for advanced Research in Sciences, University of Dhaka, Dhaka-1000, Bangladesh;
*corresponding author, e-mail: latiful@du.ac.bd
ABSTRACT
Access to clean water is the inborn rights for everyone in the world and is a prerequisite for safe
food production and health improvement. However, till today, water scarcity or lack of safe
drinking water is one of the world's leading problems affecting more than 1.1 billion people
globally, meaning that one in every six people lacks access to safe drinking water. By 2025, 1.8
billion people will be living in countries or regions with absolute water scarcity. The clean water
scarcity usually exists in developing countries due to 1) lack of capacity, 2) community capacity
and engagement, 3) technological capacity, 4) institutional capacity and 5) inadequate financial
support. The new global nancing initiative within the water and sanitation sector is highly
required before being advocating food safety issues in these countries. In addition, moral, civil,
political and economic investment needs to bring adequate sanitation for the global population to
improve health and welfare.
Keywords: Accessibility; Food safety; Global health; Health improvement; Safe water.
CHALLENGES RELATED TO WATER

Access to safe drinking water is the
fundamental need for healthy human life and
water has been shown linked to the
development of all areas including health,
nature,
urbanization,
industrialization,
energy production, food security, equality
and
other
aspects
of
community
development (Colwell et al., 2003; Huq et
al., 2010). In all these areas, not only water
but also clean, pure, safe and quality of
water are required to ensure proper
development. According to United Nations
(UN), to meet up daily necessities like
drinking, cooking, and personal hygiene,
approximately 20 to 40 liters of clean water
is required by each individual. In todays
world, scarcity of safe water is a burning
issue; especially in developing countries
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
where nearly 1.0 billion people lack fresh

water for daily uses (Aho et al., 2012).
Although, approximately 71% of earths
surface is covered with water and more than
97% of the earths water is salty sea water,
while 2% is stored in glaciers, ice caps, and
snowy mountain ranges (Yongabi, 2010);
thus, less than 1.0% of the earths water is
surface water, of lakes, rivers, streams,
ponds (0.022%) and groundwater (0.397%)
available for humans for their daily needs
(Yongabi, 2010). However, all these
accessible surface water is not necessarily
safe for drinking, and depending on the
location and sources, clean water is exposed
to a variety of contaminants, many arising
from human and animal wastes, debris from
homes, chemicals from industries, fertilizers
and pesticides from agricultural lands, and
77
Safe Water as the Key to Health

environmental pollutants as well as other
contributing factors (Figure 1) (Zeenath et
al., 2015). In addition, insanitary practices
of people have greatly contributed to the
deterioration of the quality of surface water
sources (Zaman et al, 2013). On the other
hand, groundwater is the most important
source of water supply in most of the
countries including Bangladesh.
Bari
programme (2014), about 28 million
Bangladeshis, or just over 20% of the
population, are living in harsh conditions in
receiving clean water, and safe clean water
supply situation is going to become worst in
coming decades to meet the need of
predicted 200 million people by 2020 (Silvia
et al., 2011).
CHALLENGE
RELATED
CLIMATE CHANGE
Figure 1: Important drivers that impacted

health.
Physically groundwater is clear, colorless
with little or no suspended solids and has a
relatively
constant
temperature.
Groundwater is also free from diseaseproducing micro-organisms which are
normally present in large numbers in surface
waters (Bari and Yeasmin, 2014). However,
the groundwater for drinking purposes has
become a problem because of: 1) the
presence of arsenic, 2) excessive dissolved
iron, 3) salinity in the shallow aquifers in the
coastal areas, 4) lowering of groundwater
level (water table), and 5) rock/stony layers
in hilly areas (USEPA, 2015). Among these
problems arsenic in groundwater has
become a great concern for water supply in
Bangladesh. According to a study by the
World Bank's water and sanitation
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
TO
Climate change can impact human health

and wellbeing through multiple avenues and
at varying scales. Effects of climate change
could include more frequent and intense
rainfall events, leading to increased overland
and shallow sub surface ow which can
mobilize pathogens and other contaminants.
Increased frequency and magnitude of ood
events impacts not only availability of clean
water, but chemical storage and sewage
facilities, compromising quality. Links
between weather events and waterborne
illness have been identied as water
temperatures,
increased
precipitation
intensity and longer periods of low ows
will exacerbate many forms of water
pollution, with impacts on ecosystems and
human health as shown in Figure 2.
The clean water scarcity usually exists in
developing countries due to: 1) lack of
capacity, 2) community capacity and
engagement, 3) technological capacity, 4)
institutional capacity, and 5) inadequate
financial support. New global nancing
initiative within the water and sanitation
sector is highly required before being
advocating food safety issues in these
countries (UNU-INWEH, 2010). As the
accessibility of clean drinking water is
directly related to, poverty, social and
economic development, and human health;
thus, the benets of water and sanitation
program can be made in terms of improved
78
Bari
Figure 2: Interrelationships between major global environmental changes (Source: McMichael

et al., 2003).
contamination, 2) finding more multistate
well-being of people and communities,
outbreaks, 3) new and emerging bacteria,
reduction in public health costs, and
toxins and antibiotic resistance, 4)
catalysis for local economic growth. Such
unexpected sources of foodborne illness,
benets accrue in perpetuity and can
such as ice cream and raw sprouted nut
potentially improve food safety and human
butter etc. The food safety challenges exist
health.
in both developed and developing countries
and differ by region, due to differences in
income level, diets, local conditions, and
CHALLENGES RELATED TO FOOD
government infrastructures. Figure 3 shows
SAFETY
number of foodborne cases per year in
The battle against foodborne diseases is
percentage of population in different
facing new challenges due to the
countries. There are four broad uses of water
globalization of the food market, climate
in every food production: 1) primary
change and changing patterns of human
production (e.g. farming), 2) cleaning and
consumption as fresh and minimally
sanitation, 3) as an ingredient or component
processed foods are currently preferred (Bari
of an ingredient, and 4) processing
and Ukuku, 2016). Furthermore, food safety
operations (e.g. heating, refrigeration etc.).
challenges will continue to arise in
unpredictable ways, largely due to: 1)
Adequate safe water supplies directly affect
changes in the environment leading to food
the safety of food; therefore, food handlers
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
79
Bari
Figure: 3: Foodborne cases per year in percentages of population in different countries.

should follow good-sense of practices when
considering the source, treatment and
intended use of water in food production to
ensure the quality and safety of the food
products.
Food safety concerns in developing
countries typically include: 1) the
inappropriate use of agricultural chemicals,
2) the use of untreated or partially treated
wastewater, 3) the use of sewage or animal
manure on crops, 4) the absence of food
inspection, including meat inspection, 4) a
lack of infrastructure, such as adequate
refrigeration, and finally 5) poor hygiene,
including a lack of clean water supplies.
However, food safety concerns in developed
countries are mainly associated with: 1)
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
changes in animal husbandry practices, 2)

changes in agronomic process, 3) changes in
lifestyle and consumer demands, 4) increase
in susceptible populations, 5) increased
travel, and 6) increasing international food
trade etc., (Bari and Ukuku, 2016). Thus, it
appears that each developing countries
limitations are more basic in nature, which
are recoverable than the developed countries
limitation
that
are
non-recoverable.
Therefore, greater emphasis on capacity
building needs to be built internally to a
country
and
through
South-South
collaborations and partnerships, rather than
depending on consultants from developed
countries. Capacity is a exible concept and
encompasses the public sector, academia;
community based organizations, private
80
Bari
sector, which ranges from the individual to

institutions to society as a whole.
CREATING FOOD SAFETY CULTURE
Peoples attitudes, choices and behaviors are
some of the most important factors
influencing the overall safety of a food. A
culture of food safety required to be built on
a set of shared values that operators and
their staff follow to produce and provide
food in the safest manner (Figure 4).
Maintaining a food safety culture means that
operators and staff know the risks associated
with the products or meals they produce,
know why managing the risks is important,
and effectively manage those risks in a
demonstrable way. In an organization with a
good food safety culture, individuals are
expected to enact practices that represent the
shared value system and point out where
others may fail. By using a variety of tools,
consequences and incentives, businesses can
demonstrate to their staff and customers that
they are aware of current food safety issues,
that they can learn from others mistakes,
and that food safety is important within the
organization (Jack et al., 2012). Thus,
creating a culture of food safety requires
application of the best science with the best
management and communication systems,
including compelling, rapid, relevant,
reliable and repeated food safety messages
using multiple media.
Training employees on food safety practices
has been shown to be one of the most
important programs that food service
establishments can implement. However,
numerous study reports suggested that
traditional approaches used to educate and
train employees (such as Serve Safe) may
not be particularly effective and new
behavior-based approaches that include food
safety education as part of the culture of the
organization needs to be developed.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Figure 4: Building a food safety culture.
CONCLUSION
It is very clear that water-related diseases
are responsible for a significant proportion
of the global health burden. On the other
hand, it is equally clear that adequate
sanitation measures have not been managed
to keep up with population growth.
Sanitation is not only critical for dignity and
health but it is the most basic form of source
water protection also. This realization is not
new, and focus on clean water alone does
not necessarily result in improved access to
sanitation. Thus, each gap needs to be taken
into account and adequate investment in
terms of education, capacity and financing
to be done to improve the food safety and
sanitation status. A strong moral, civil, and
political will is essential to bring in an
adequate sanitation for the global population
to improve health and welfare.
REFERENCES
Aho, I. M. and Lagasi, J. E. (2012). A new
water treatment system using
Moringa oleifera seed. American
Journal of Scientific and Industrial
Research Science Hu 3 (6), 487492.
81

Colwell, R. R., Huq, A., Islam, M. S.,
Aziz, K. M. A., Yunus, M., Khan,
N. H., Mahmud, A., Sack, R. B.,
Nair, G. B., Chakraborty, J., Sack,
D. A. and Russek-Cohen, E.
(2003). Reduction of cholera in
Bangladeshi villages by simple
filtration. Proceedings of the
National Academy of Sciences 100,
1051-1055.
Bari M. L. and D. O Ukuku (2016).
Foodborne Pathogen and Food
Safety (Eds) Md. Latiful Bari and
Dike O Ukuku, Published by CRC
Press ( Taylor and Francis Group),
6000 Broken Sound Parkway NW,
Suite 300, Boca Raton, FL 33487,
USA. pp 1-37.
Bari, M.L. and Yeasmin, S., 2014. Water
Quality
Assessment:
Modern
Microbiological Techniques. In:
Batt, C.A., Tortorello, M.L. (Eds.),
Encyclopedia of Food Microbiology,
vol 3. Elsevier Ltd, Academic Press,
pp. 755765.
Huq, A., Yunus, M., Sohel, S.S., Bhuiya,
A., Emch, M., Luby, S. P., RussekCohen, E., Nair, G. B., Sack, R. B.
and Colwell, R. R. (2010). Simple
sari cloth filtration of water is
sustainable and continues to protect
villagers from cholera in Matlab,
Bangladesh. mBio 1(1), 1 e0003410.
Bari
Githeko, A., Scheraga, J.D. and
Woodrow, A. (Eds.). (2003).
Climate Change and Human Health:
Risks and Responses. WHO, WMO
and
UNEP.
Available
from:
http://www.who.int/globalchange/pu
blications/climchange.pdf (Accessed
June 9, 2016)
Silvia, B., Elena, B., Sara, B., Osvaldo, C.,
Franca, P. and Elisabetta, C.
(2011). Development of a PCR
protocol for the detection of
Escherichia coli O157:H7 and
Salmonella spp. in surface water.
Environmental
Monitoring
and
Assessment 177, 493503.
UNU-INWEH (2010). Sanitation as a Key
to Global Health: Voices from the
Field. United Nations University
Institute for Water, Environment and
Health. pp 2-45.
USEPA.
(2015).
Drinking
Water
Contaminants,
http://www.epa.gov/dwstandards
regulation (Accessed on April 19,
2016).
[WHO] World Health Organization.
(2001). Water Quality: Guidelines,
Standards and Health. Chapter 6
and 13. Edited by L. Fewtrell and J.
Bartram. Published by IWA
Publishing, London, UK. ISBN 1
900222 28 0.
Jack, A., Neal, M. B. and Henroid, D.

(2012).
Assessing
Factors
Contributing to Food Safety Culture
in Retail Food Establishments. Food
Protection Trends 32(8), 468476.
Yongabi, K. A. (2010). Biocoagulants for

water and wastewater purification: A
review. International Review of
Chemical Engineering 2(3), 444458.
McMichael, A.J., Campbell-Lendrum,

D.H., Corvolan, C., Ebi, K.L.,
Zaman, S., Yeasmin, S., Inatsu, Y.,

Ananchaipattana, C. and Bari, M.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
82

L. (2013). Low-Cost Sustainable
Technologies for the Production of
Clean Drinking WaterA Review.
Journal of Environmental Protection
5, 42-53.
Zeenath,
F.,
Md.
Harunur,
R.,
Chowdhury, M. A. Z., Alam, Md.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Bari
K., Rahman, Md. A., Uddin, M. A.
and Bari, M.L. (2015). Assessment
of Heavy Metals, Minerals and
Pesticides in different Branded
Drinking
Bottled
Water
of
Bangladesh and their impact on
human health. Online publication (25
Jan 2015).
83

Res. Highl. 4Bs (2016),P84-89
Short Communication
The Kratom Plant [Mitragyna speciosa (Korth.)] Paradox: Beneficial or
Detrimental?
Low J.W.1, Leela A.*1 and Bhore S.J.2
1
Faculty of Medicine, AIMST University, Bedong-Semeling Road, 08100 Bedong, Kedah,

Malaysia. 2Department of Biotechnology, Faculty of Applied Sciences, AIMST University,
Bedong-Semeling Road, 08100 Bedong, Kedah, Malaysia; *corresponding author, e-mail:
drleelaa@yahoo.com, Ph No: +604-429 8063
The aim of this study was to assess the level of knowledge and practices of some
selected/vulnerable residents from Tanjung Dawai village (Kedah, Malaysia) about Kratom
plant [Mitragyna speciosa (Korth.)]. Several drug addicts from Tanjung Dawai village as well
as from adjacent areas come to specific places of Tanjung Dawai village on a regular basis to
buy Kratom drinks and or products. We have visited this village to collect the first-hand primary
information about traditional use of Kratom plant. We selected some residents from Tanjung
Dawai village as well as some individuals those have Kratom plant in their backyard and
interviewed them. We found that some consume it intermittently or daily for strength and energy.
They felt they could work better after consuming Kratom. Some subjects highlighted that small
amounts of Kratom boost their strength and energy, while others claimed that Kratom is useful
for lowering blood pressure, treating diarrhea, and as anxiolytic, antidiabetic. However, now
Kratom usage is causing concern as it emerges as a potential drug of abuse. Although Kratom
has been associated with drug abuse and as a dangerous drug in recent years; it possesses certain
beneficial properties as claimed by the villagers. The systematic pharmacological study needs to
be done to understand the beneficial properties of this plant. Hence, further research is required
to explore the potential applications of this plant.
Keywords: Drugs; Health; Kratom, Malaysia; Mitragyna speciose; Tanjung Dawai.
Kratom plant [Mitragyna speciosa (Korth.)]

is indigenous to Thailand and Southeast
Asia. This plant is also known as Biak or
Ketum in Malaysia. It is a member of the
Rubiaceae family, and grows in tropical and
subtropical regions of Asia (Puff et al.,
2005; Hassan et al., 2013). It is well
distributed in Southeast Asia, especially
Malaysia in general and the northern region
bordering Thailand in particular (Hassan et
al., 2013; Suwanlert et al., 1975).
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Kratom leaves (Figure 1) are known to

produce complex stimulant and opioid-like
analgesic effects (Adkins et al., 2011; Babu
et al., 2008). In Malaysia, Kratom has been
used to starve off fatigue by enhancing
tolerance for hard work and to manage pain,
diarrhea, and opioid withdrawal (Hassan et
al., 2013). Recently, Kratom has become
widely available in the Malaysia, as people
are allowed to keep the plants for
individuals personal use. In Malaysia,
commercial planting of this plant is banned
84
Kratom Plant: Beneficial or Detrimental?
Low et al.
Figure 1: Morphological features of a leaf, inflorescence and fruit of Kratom plant [Mitragyna
speciosa (Korth.)] collected at Tanjung Dawai, Kedah, Malaysia. A) Morphology of a leaf; B) a
snap of an inflorescence, and C) morphology of an immature fruit.
and its cultivation is considered illegal.
Analyses of medical literature indicate that
individuals in Malaysia, especially in the
north peninsular Malaysia are increasingly
using Kratom.
Kratom contains pharmacologically active
constituents, most notably mitragynine and
7-hydroxymitragynine. Kratom possesses
dose-dependent pharmacological effects,
with stimulant effects in lower doses and
opiate-like effects in higher doses (Babu et
al., 2008). Thus, the potential for
youngsters addiction and adverse health
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
consequences are becoming an important

issue for health authorities of Malaysia.
There were studies performed to observe the
effect of mitragynine several years ago
(Grewal, 1932; Macko et al., 1981). The
acute toxicities studies in rates include
increasing blood pressure, hepatotoxicity,
and nephrotoxicity (Harizal et al., 2010).
The recent research findings of Kamal et al.
(2012) suggest that in very high doses,
Kratom did not cause death and or any
significant toxicity. In humans, its adverse
effects include dry mouth, changes in
85

urination, nausea, vomiting, anorexia,
weight loss, constipation, nystagmus, tremor
and seizures (Babu et al., 2008; Boyer et al.,
2008; Ulbricht et al., 2013; Nelsen et al.,
2010; Trakulsrichai et al., 2013). Primary
hypothyroidism (Sheleg and Collins, 2011)
and intrahepatic cholestasis (Kapp et al.,
2011) are also reported in several cases
related to Kratom. For chronic toxicity,
impairment of cognitive behavioral function
is found in mice fed with mitragynine for a
long period (Apryani et al., 2010). However,
in a study of chronic Kratom administration
showed that it does not significantly impair
social functioning of users in a natural
context in Malaysia (Singh et al., 2014). In
humans,
anorexia,
weight
loss,
hyperpigmentation over skin and cheeks on
face, and psychosis are also described in
chronic abusers (Babu et al., 2008).
Kratom is well known and consumed as a
norm in the northern peninsular region of
Malaysia. Based on prior reports which
suggest that several drug addicts and some
villagers from Tanjung Dawai village as
well as youngsters from adjacent areas come
here [at Tanjung Dawai village] on a
regular basis for Kratom drinks and or
products. We visited the village to collect
primary information. We carefully selected
some residents from Tanjung Dawai
village and also those grew them in their
backyard and interviewed them. An
interview with the village head was also
carried out and comments were noted.
The information we received from villagers
suggest that the majority of Kratom users
are adult males, mostly those are manual
laborers. Addicts consume Kratom drinks to
enhance their physical endurance, boost
their energy, productivity, elicit euphoric
effects, and to improve their sexual
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Low et al.
performance. Some of them use it as a
replacement for expensive drugs if they do
not have the cash to buy drugs.
From interviewed villagers, we gathered that
Kratom is also used as a folk medicine by to
treat several diseases or as an opioid
alternative by chewing fresh leaves and
brewing as tea or concoction. Some villagers
claim that Kratom is useful for lowering the
blood pressure, treating diarrhea, and as
anxiolytic and antidiabetic.
Previously, Kratom was not considered to be
a drug of abuse, but as a supplement for
better health; because, it is known to help in
lowering the blood pressure, as an
antidiarrheal, anxiolytic, antidiabetic and
antinociceptive (Hassan et al., 2013;
Suwanlert, 1975). Villagers strongly
believe that Kratom is a powerful
antidiarrheal property. An antidiarrheal
property of plant was confirmed when there
was an outbreak of food poisoning after a
wedding reception, where majority of those
who had consumed Kratom drink did not
suffer from food poisoning while others who
did not were affected.
Currently, Kratom is causing concern as it
appears as a potential drug of abuse (Hassan
et al., 2013). It was noted by Boyer et al.
(2008) and Vicknasingam et al. (2010) that
Kratom is often used as opioid replacement
to alleviate opioid withdrawal symptoms
and it is an easily accessible and serves as an
economical alternative to other opiodreplacement medications. Kratom is often
used as a drug of abuse alone or in
combination with other substances, has been
shown to cause dependence and withdrawal
symptoms following repeated consumption
(Hassan et al., 2013; Babu et al., 2008;
Vicknasingam et al., 2010; McWhirter and
86

Morris, 2010; Saingam et al., 2013;
Saingam et al., 2014; Singh et al., 2014).
However, based on the animal studies,
Kratom is only slightly toxic (Macko et al.,
1981).
In
mice,
repeated
7hydroxymitragynine administrations elicit
tolerance, cross-tolerance to morphine, and
naloxone-precipitated withdrawal symptoms
(Matsumoto et al., 2005). Few poisoning
cases related to Kratom consumption has
been reported in the past (Kapp et al., 2011;
Neerman et al., 2012; Tungtananuwat and
Lawanprasert, 2010). However, people
including youngsters are using it; hence, its
(Kratom) usage is causing concern as it
emerges as a potential drug of abuse. Most
recently, Malaysian police have seized 2.8
tonne of Ketum powder worth MYR
235,000 which also indicates that Kratom
(Ketum) is smuggled for the international
market (Star, 2016).
In a conclusion, although Kratom has been
associated with drug abuse and as a
dangerous drug in recent years, it possesses
certain beneficial properties. Currently, we
do not have enough scientific information to
weigh the health benefits of this medicinally
important
plant.
The
systematic
pharmacological study needs to be done in
order to understand the beneficial properties
of this plant. Hence, further research is
required to explore the potential applications
of this plant.
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Low et al.
Apryani, E., Hidayat, M.T., Moklas, M.A.,
Fakurazi, S. and Idayu, N.F.
(2010). Effects of mitragynine from
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E.W. (2008). Opioid receptors and
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Boyer, E.W., Babu, K.M., Adkins, J.E.,
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Tharakan, J.K. and Abdullah, J.
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(2012). Acute toxicity study of
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(1981). Some observations on the
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Saingam, D., Assanangkornchai, S.,

Geater, A.F. and Balthip, Q. (2013).
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Matsumoto, K., Horie, S., Takayama, H.,

Ishikawa, H., Aimi, N., Ponglux, D.,
Murayama, T. and Watanabe. K.
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Saingam, D., Assanangkornchai, S.,

Geater, A.F. and Lerkiatbundit, S.
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McWhirter, L. and Morris, S. (2010). A

case report of inpatient detoxification
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coincidence
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addiction
to
Kratom and severe primary
hypothyroidism. J
Addict
Med. 5(4):300301.
Neerman, M.F., Frost, R.E. and Deking, J.

(2012). A drug fatality involving
Kratom. J Forensic Sci. 58(S1),
S278-S279.
Singh, D., Mller, C.P. and Vicknasingam,

B.K. (2014). Kratom (Mitragyna
speciosa) dependence, withdrawal
symptoms and craving in regular
users. Drug Alcohol Depend. 139,
132137.
Nelsen, J.L., Lapoint, J., Hodgman, M.J.

and Aldous, K.M. (2010). Seizure
and coma following Kratom
(Mitragynina
speciosa
Korth)
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Star (Newspaper), March 11 2016: Cops

smash ketum plants.
88

Suwanlert S. (1975). A study of Kratom
eaters in Thailand. Bull Narc. 27(3),
2127.
Trakulsrichai, S., Tongpo, A., Sriapha, C.,
et al. (2103). Kratom abuse in
Ramathibodi
Poison
Center,
Thailand: A five-year experience. J
Psychoactive Drugs. 45(5), 404408.
Tungtananuwat, W. and Lawanprasert, S.
(2010). Fatal 4x100: Homemade
Kratom juice cocktail. J Health Res.
24(1), 43-7.
Low et al.
Windsor, R.C. (2013). An evidencebased systematic review of Kratom
(Mitragyna speciosa) by the Natural
Standard Research Collaboration. J
Diet Suppl. 10(2), 152170.
Vicknasingam, B., Narayanan, S., Beng,
G.T. and Mansor, S.M. (2010). The
informal use of ketum (Mitragyna
speciosa) for opioid withdrawal in
the northern states of peninsular
Malaysia and implications for drug
substitution therapy. Int J Drug
Policy. 21(4), 283288.
Ulbricht, C., Costa, D., Dao, J., Isaac, R.,

LeBlanc, Y.C., Rhoades, J.,
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
89

Abstracts (Keynote and Plenary Talks)

Moving the Regional Biotechnology and Bioeconomy Forward
YBhg. Dato' Prof. Dr. Asma Binti Ismail*
Director General, Department of Higher Education Ministry of Higher Education Malaysia;
*corresponding author, e-mail: asma@mohe.gov.my, / asmainformm@yahoo.com, Ph No:
+603 88706381.
Abstract: Asian countries are blessed with plenty of natural resources that can be used
systematically in boosting regions bioeconomy significantly. In line with the international
agenda of the sustainable development, each Asian country can and should take initiatives to
boost the bioeconomical growth. In fact, many Asian countries are aggressively taking steps
to grab the opportunities to enhance their biotechnology sectors in order to boost the national
GDP. By realizing the applications of bio-based innovative technologies and its economic
benefits to the society, Malaysia has taken several initiatives including Bioeconomy
Transformation Programme (BTP). In fact, Malaysia is the 2nd in Asia and 1st in Southeast
Asia to announce a bioeconomy initiative as platform for bioeconomy development. The
Organisation for Economic Co-operation and Development (OECD) estimates clearly suggest
that bioeconomy is going to contribute a global average of 2.7% to GDP by 2030. It appears
that bioeconomy is the way forward to address regional and global sustainability challenges.
In Asia and beyond, we need to focus on renewable resources (energy, chemical and material
products), enhance agriculture-based industry (production of high-value end products using
innovative technologies), use innovative healthcare products and services (cheaper and more
accessible). The coherent and supportive policy framework will be the key for Asian
countries for the development of their Bioeconomy. In my keynote address, I will highlight
my perspectives on the opportunities and challenges in boosting bioeconomy in Asia region
and beyond.
Keywords: Asia; Bioeconomy; Biotechnology; Health Care; Renewable Resources.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
90

Nanotechnology Oriented Biosensors and Biomedical Application

Tamiya E.*
Nanobio Engineering and Biosensor Lab. Department of Applied Physics,
Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871,
Japan; *corresponding author, e-mail: tamiya@ap.eng.osaka-u.ac.jp
Abstract: Nanostructured metals have been studied for the localized surface plasmon
resonance (LSPR) and electrochemical biosensors. Photonic plasmon spectra are caused by
the refractive index variations that result from the binding of molecules to the metal
nanostructures. There are optically detectable parameters in biophotonics and biosensor
devices. We have studied several types of nanostructures, (1) gold-capped nanostructure
connecting with the core of silica nanoparticle capped by deposited gold film, (2) golddeposited porous anodic alumina layer chip, (3) gold nanoparticles onto silicon oxide /silicon
interferrometric multilayer and (4) nano-pillar structures by nanoimprinted polymer materials
and sputtering a thin gold layer on them. The bio-sensing of these nanostructures have been
examined by monitoring the biomolecular interactions in various flexible formats. Antibodyantigen and DNA hybridization reactions were performed to detect various biomarkers, with
the detection limit of picogram levels. The multi array format was constructed by a core-shell
structured nanoparticle layer, which provided 3001000 spots on the sensing surface. A
microfluidic biochip based on PDMS was useful for real-time analysis, rapid detection. DNA
amplification process, and monoclonal antibody production from hybridoma cell library can
be monitored. Electrochemistry measurements connecting to LSPR chips were successfully
exploited in a simultaneous detectable scheme. The binding of melittin to lipid membrane
was measured using localized surface plasmon resonance, and the permeability of the lipid
membrane was then assessed electrochemically as a function of melittin with the purpose of
seeking a novel, sensitive detection system for peptide toxins. These nanoporous structures
were transferred to the cyclo-olefin polymer film surface from the porous mold by a thermal
nanoimprinting process. A plasmonic substrate was fabricated by sputtering a thin layer of
gold onto this nanopillar polymer structure and the refractive index response in a variety of
media was evaluated. Finally, the biosensing capacity of this novel plasmonic substrate was
verified by analysis of human immunoglobulin With the advantages of mass production with
consistent reproducibility stemming from the nanoimprint fabrication process, our goldcapped polymeric pillars are ready for the transition from academic interest into
commercialization systems for practical use in diagnostic applications. Surface Enhanced
Raman Scattering (SERS) was also discussed with gold and silver nanoparticles interacting
with bio-molecules. Gold nanoparticles were successfully delivered into single cells.
Spatiotemporal measurements of SERS fingerprints suggested the dynamic molecular
interactions and transformations taking place at different locations with time in
cardiomyocytes.
Keywords: Antibody; Biosensors; DNA; Innovation; Nanotechnology; PCR.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
91

Current Progress in Cholera Diagnostics

Chan Yean Yean1 and Manickam Ravichandran*2
1
Department of Medical Microbiology and Parasitology School of Medical Sciences, Health

Campus, Universiti Sains Malaysia, Kelantan, Malaysia. 2AIMST University, Bedong,
Semeling, Kedah, Malaysia; *corresponding author, e-mail: ravichandran@aimst.edu.my,
Ph. No.:+60 4 429 8103.
Abstract: Cholera is a diarrhoeal disease caused by a Vibrio cholerae. The diagnosis of this
disease is by the conventional culture method and more recently by various PCR based
molecular diagnostic tests. The main limitation of these tools is that it requires skilled
personnel and its reagents have to be transported in cold storage. To overcome these problem,
our research team have developed simple platform technologies i.e., a). thermostabilization of
PCR reagents and b). biosensor method. A thermostabilized PCR kit was developed for the
detection of V. cholerae. This kit detects all serogroups (O1, O139 and non O1, non O139)
based on lolB gene and rfb gene, biotypes (classical and EI Tor) identification that is coded
by tcpA gene, virulence genes namely ace, zot and ctxA genes and tetA antibiotic
(tetracycline) resistant determinant. The kit can be performed by simply adding water and
bacterial lysate to the kit. This kit does not require multiple pipetting steps and cold chain for
the transport. The second series of platform technology developed by our research team is
called biosensors. It is an enzyme-based amperometric electrochemical biosensor assay to
detect PCR product on a screen-printed carbon electrode (SPCE). The methods we have
developed are multiplex genosensor and magentosensor. The main advantage of these
methods is that it can detect PCR product without utilizing agarose gel electrophoresis. Hence
a combination of cold chain free formulation of molecular diagnostic test with field adaptable
biosensor detection method will be very useful for the detection of cholera in resourcelimited settings.
Keywords: Biosensors; Cholera; Diagnostics; DNA; PCR; Vibrio cholera.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
92

Supercomputing in Biotechnology: Making Sense of Big Data

Bent Petersen*
Center for Biological Sequence Analysis, Institute of Systems Biology, Technical University
of Denmark, Dk-2800 Kongens Lyngby, Denmark; *corresponding author, e-mail:
bent@cbs.dtu.dk, Ph No: +45 20 84 74 92.
Abstract: Currently, genomic and metagenomic experiments are generating massive amounts
of data. Due to modern day technological advances, it has become increasingly affordable to
produce sequencing data, which becomes very cost effective even when compared to storage,
management, and analytical expenses. Simultaneously, the importance of biotechnology
towards the production of safer, healthier and a sustainable food source is constantly
increasing. How can the abundance of sequencing data benefit a food product? In this talk, I
will briefly touch upon two projects that exemplify this point of view. In the first project,
genomics was the game changer towards establishing a modern fermentation process for
carmine production a widely used natural red dye found in pastries, confections, cosmetics
and a plethora of other products. In the other project, metagenomics played a key role
towards the identification of new bacteria and yeast species that can be utilized as starter
cultures in wine and silage production. Both projects faced the modern challenge of
accumulating massive amounts of data where state-of-the-art supercomputers were crucial in
order to deliver the anticipated results. I will also give a short introduction to other exciting
projects where big data and supercomputers played a central role.
Keywords: Big data; ; Biotechnology; Genomics; Metagenomics; Supercomputers.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
93

Molecular Approaches to Fundamental Studies on Biomarkers and

Development of Sustainable Rapid Nano-biodiagnostics to Enteric Diseases
for Low Resources Settings
Phua K. K* and Asma Ismail
Enteric Diseases Research Cluster, Institute for Research in Molecular Medicine
(INFORMM), Health Campus, Universiti Sains Malaysia (USM), 16150 Kubang Kerian,
Kelantan, Malaysia; *corresponding author, e-mail: kkphua7@gmail.com
Abstract: The Enteric Diseases Research Cluster is a multi-disciplinary project that harness
the expertise from various fields of fundamental research and applied sciences to embark on
Translational Research to develop innovative diagnostics for enteric diseases in low resources
settings. By means of computer modelling, molecular docking and X-ray crystallographic
techniques, coupled with various technology platforms, including non-PCR and lateral flow
technolgy, sustainable diagnostics that fulfill WHOs ASSURED criteria were developed and
evaluated. Talents from Engineering, Chemistry and Physics enabled indigeous materials and
reagents, such as nano-gold particles, recombinant enzymes, nitrocellulose membrane and
synthetic peptides to be manufactured, which enabled these diagnostics to be produced at
competitive prices for developing countries. Partnerships forged with foreign research
institutions, such as the National Synchrotron Radiation Research Center, Taiwan; Sanger
Institute, United Kingdom; King Saud University, enable high-impact factor publications to
be enable Universiti Sains Malaysia (USM) to remain relevant and referred. With a budget of
RM4.8 million, and a total of 10 principle investigators; 58 publications in citation-indexed
journals, 66 cumulative Impact Factor and 107 citations; a book on Sustainable Diagnostics
for Low Resource Areas; 3 patent filings; 22 awards; over 70 conference presentations; and
provided opportunities for 1 Post-doctoral trainee, 25 Masters and Doctoral postgraduate
students. But perhaps our most significant achievement is in providing impactful solutions
for the community and local government agencies to help reduce the burden of Enteric Fever
in the state of Kelantan. Industrial engagement, with manufacturing companies, such as
Reszon Diagnostics and Malaysian Biotechnology corporation, will help realise the RMK10
agenda, ie. to provide health and wealth for the nation.
Keywords: Biomarkers; Diagnostics, DNA; Health; PCR; Protein.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
94

Bio-Applications of Innovative Nano-materials

T. Theivasanthi*
International Research Center, Kalasalingam University, Krishnankoil 626126, India;
*corresponding author, e-mail: ttheivasanthi@gmail.com, Ph No: +91-4563-258084 / +919524818862, 9344643384.
Abstract: This lecture will deliver nanoscience & nanotechnology based research activities.
Sensitive nanomaterials like graphene, graphite, carbon nanotubes and metal nanoparticles
are used to prepare electrochemical biosensors. Graphene has exquisite sensitivity to
environmental changes. This property, combined with other characteristics such as optical
transparency and excellent electrical / electronic properties provides the bio/inorganic
interface required for sensors. Recent, innovations such as Worlds first plants materials
based superparamagnetic particles named Santhi Particles and superparamagnetic plants
materials like turmeric (Curcuma longa) & coconut shell (Cocos nucifera) are useful in
cancer hyperthermia which will be discussed. Superparamagnetism improves the accuracy of
spintronic sensors because a small sensed field is sufficient to order the spins in a
superparamagnetic material. Such improved and accurate sensors are useful in various
biomedical applications. Vegetable powder (Abelmoschusesculentus) for diabetes,
nanoparticles for treatment of cancer, diabetes, psoriasis and Worlds first plants materials
Nanoparticles (Andrographispaniculata) for EBOLA, DENGUE, HIV & H1N1 virus
infections, nanostructured / smart materials for Bio-sensors (like Amaranthus), Surface
Enhanced Bio-materials, Bio-nanomaterials, Bio-polymers will also be discussed. This work
also deals with societal impacted innovative research works like agricultural products like
nanofertilisers (produced from Jack fruit seeds) etc. Various Technologies / Advanced
Materials like low cost / mass production of Graphene, polymers, Synthesis, characterizations
of metal nanopowders, metal oxide nanopowders, polymer-metal nanocomposites and their
novel biotechnological applications will be explored. Large surface area, high surfacearea
tovolume ratio and compatibility with flexible substrates of these materials make them as
unique candidate for various applications. Surface Plasmon Resonance (SPR) of nanometallic surfaces is the incoming light results in a collective oscillation of the electrons at the
metals surface. It has many promising applications which can be exploited to transmit
optical signals, to interact with bio-molecules. Influences of nanomaterials in applications
like sensor, imaging and biomedicine will be discussed. In addition, Research Motivation
lecture will be presented to motivate students/ researchers to concentrate more on / towards
research.
Keywords: Biosensors; EBOLA; H1N1; HIV; Innovation; Nanomaterial; Technology.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
95

Aptasensors: Bench to Bedside and Beyond

Subash C.B. Gopinath*
Institute of Nano Electronic Engineering (INEE), Universiti Malaysia Perlis (UniMAP),
01000 Kangar, Perlis, Malaysia; *corresponding author, e-mail: subash@unimap.edu.my,
Ph No: (+60) 125565881.
Abstract: Aptamers are single-stranded nucleic acids, so-called artificial antibodies,
identified from the randomized combinatorial library against the target by the process called
SELEX (Systematic Evolution of Ligands by EXponential enrichment). Target can have
any sizes from small molecules to the whole cell, attests the versatility of aptamers to bind a
wide range of targets. Aptamers showed properties that are analogous to antibodies with high
specificity and affinity to their target molecules. Aptamers have several advantages over
antibodies, such as they are easy to prepare, cheaper, have no batch variations, are easy to
modify, stable and most importantly, non-immunogenic. Because of these positive
characteristics, aptamers are incorporated in different fields, and most attractive in
applications involving therapeutics and diagnoses (theranostics). Aptamer-mediated
biosensors (Aptasensors) have been attested with high-performance with different
interdisciplinary sciences, making roads from laboratory to Industry and bridging the gaps.
With either aptamers alone or complementing with antibodies, several high sensitive,
portable sensors have been demonstrated for use in bedside analysis. Moreover, aptamers
are more amenable to chemical modifications, making them capable of utilization with the
most developed sensors. The development of more sensitive aptasensors could be useful and
important for medical diagnosis, identification of pathogens for the quality control of
consumable items, and surveillance of emerging diseases. In fact, aptasensors have already
shown efficacy in the detection of life threatening diseases caused by early stage of viral and
bacterial infections. Further, success in aptasensor development was driven in most cases by
bottom-up and top-down approaches with inter- and multi-disciplinary strategies.
Commercialization evidences a complete platform for aptasensor research and development.
Current researches are expanding towards high-throughput screening and drug discovery
process for next-generation sensing, using the aptamer as the probe. In this talk, will discuss
about progression in the development of aptasensors, as bench to bedside and beyond.
Keywords: Aptamer; Aptasensor; Bedside analysis; Diagnosis; SELEX.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
96

Recent Progress in the Production of Biodegradable Plastics from Palm Oil

in Malaysia
Sudesh K.*
School of Biological Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia;
*corresponding author, e-mail: ksudesh@usm.my, Ph No: (+6)04-6534367
Abstract: Background: Palm oil is known as an essential vegetable oil commodity. It is a

renewable resource which provides a wide array of applications. Studies have shown that
palm oil is an efficient feedstock for the production of biodegradable plastics, namely
polyhydroxyalkanoate (PHA) via microbial fermentation. PHA is accumulated as water
insoluble storage polyester in the cell cytoplasm of bacteria. For the past three decades, PHA
has been the subject of intense investigation due to its thermoplastic properties as well as
being biodegradable and biocompatible.Therefore, PHA has a high potential in substituting
some petrochemical plastics as a renewable and sustainable material. Bacterial strains of
Cupriavidusnecator H16 are reported to be cultivated to more than 100 g/L dry cell weight
containing up to 80 wt% bioplastics using palm oil or used cooking oil as the carbon source.
However, successful commercialization of PHA is currently hindered by its high cost
compared to existing polymers in the market. Recovery and purification process of PHA from
bacterial cells are mainly responsible for costly production of PHA. Methods: A novel
biological extraction method has been developed by feeding freeze-dried cells containing
PHA granules to animal models. The resulting whitish fecal matter was collected and
subjected to GC, GPC, DSC, TGA, rheology and protein analyses. Results: GC analysis
revealed that the fecal pellets contained up to more than 90 wt% of PHA. Further increase to
95% PHA can be achieved by washing treatments with detergents. DSC, GPC and TGA
analyses proved that the PHA granules recovered using this method had similar properties
when compared to PHA extracted with chloroform. However, there was obvious differences
in rheological properties between the PHA obtained from both methods. Biologically
extracted PHA granules contained traces of proteins, which may be the reason for differences
in rheological properties. Conclusion: This simple biological extraction method is scalable
and can be incorporated into some existing animal production systems.
Keywords: Biodegradable; Biological extraction; Palm oil; Polyhydroxyalkanoate.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
97

Recent Advances in Biosensors Based on Enzyme Inhibition

Aziz Amine*
Faculty of Sciences and Techniques, Hassan II University of Casablanca, Morocco;
*corresponding author, e-mail: azizamine@yahoo.fr
Abstract: Background: Biosensors based on enzyme inhibition represent a cost-effective

device for fast screening of inhibitors. They could be used as alarm systems for
environmental monitoring and as a complementary technique to traditional methods. In the
present conference we would like to underpin the recent advances in biosensors based on
enzyme inhibition field, focusing on:
the investigation of a new theoretical approach in order to easily understand the type of
inhibition and calculate the kinetic parameters;
the evaluation of the performances of the biosensor in the case of reversible and irreversible
inhibition, in terms of time of analysis, detection limit, matrix effect
the use of nanomaterials;
the development of biosensors based on enzyme inhibition embedded in labs on a chip;
The applications of biosensors based on enzyme inhibition in real samples.
Methods: In the case of reversible inhibition, the most frequent procedure is based on the
measurement of enzyme activity in absence (I0) and in presence of inhibitor (I1). The
decrease of enzymatic activity can be evaluated by measuring the degree of inhibition as
follows: ((I0 I1)/I0) 100. After inhibition, the biosensor is washed with buffer, and the
enzyme activity is again evaluated. If the enzyme restores the initial activity, then the
inhibition is reversible, otherwise the inhibition is irreversible. Furthermore, in the case of
irrreversible inhibition, an incubation time is required. Results: Experimental results of
enzymatic inhibition by pesticides aflatoxins, cyanide, sulfide, methylmercury, nerve agents,
and various other drugs and toxins will be presented. These inhibitors were usually detected
at levels of ppb. Conclusion: The diagnosis of type of inhibition greatly improves sensitivity
by a judicious choice of the enzyme concentration and incubation time. The use of
nanomaterials in the preparation of the inhibitive biosensors improves some analytical
features including sensitivity and stability.
Keywords: Analytical applications; Enzymatic biosensors; Inhibition; Nanomaterials.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
98

Genomics of the Endangered Orang Asli: Disease Susceptibility and

Sustainability
Mohd Zaki Salleh* and LRGS Orang Asli team
Integrative Pharmacogenomics Institute (iPROMISE , Universiti Teknologi MARA, 42300
Puncak Alam, Selangor, Malaysia; *corresponding author; e-mail:
zakisalleh@puncakalam.uitm.edu.my, Ph No: (+603) 3258 4652
Abstract: Different groups of indigenous people had populated Southeast Asian regions via
different waves of migration. As such, the indigenous people have been the subjects for
studies (1) to track patterns of migration of modern human; (2) to understand the
mechanisms of evolution and natural selection; and (3) evolution and mechanism of
diseases. Despite the developmental programmes runned by the government, most of the
Orang Asli in Malaysia are still plagued with negative pressures in both the socio economics and health aspects. Some of the sub-tribes of the Orang Asli such as the
Kanaq, Che Wong and Lanoh are left with less than 500 surviving individuals. Factors
such as capital domain and human resourse domain that may contribute to these
observations are continuously being researched by many. To understand the problems
better, we integrated the omics and anthropological approaches in a multipronged
research in order tofind solutions to improve the situation.Orang Asli from different
geographical areas in Peninsular were recruited. Medical examinations, biochemical and
metabolomics analysis were conducted. The DNAs of the Orang Asli were sequenced using
second generation technologies. Only reads with a Q score of 30 and above (>Q30) were
included in the analysis.The variants were uncovered using the GATK Best Practices
workflow for variant discovery. The genomics structures of the Orang Asli were successfully
mapped. Genetic variants, both existing and novel ones were detected. Genetic variants that
predispose them to higher risks towards diseases were determined. Correlations of the
genomics risks with the biochemical profiles and metabotypes provide an in depth
understanding on the disease susceptibilities of the Orang Asli. This study also provide a
wealth of data on the whole genome sequences of the Orang Asli which can be continuously
mined by researchers for advancement of knowledge. There is still much to be done with the
metadata generated to protect and enhance the health and welfare of the Orang Asli
communities in the country, especially those in the minority groups. The assistance provided
should gear towards improving control over preventable diseases and physical conditions that
cause the population to be endangered.
Keywords: DNA; Genetics; Genomics; Medication; Orang asli; Sustainability.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
99

Highly Sensitive Detection of DNA Hybridization and Immunoassay Based

on Nanomaterials
Werasak Surareungchai*
King Mongkuts University of Technology Thonburi, Bangkhuntien, Bangkok 10150,
Thailand; *corresponding author, e-mail: werasak.sur@kmutt.ac.th
Abstract: High specific and sensitive detections can be achieved via labeling techniques in
DNA hybridization and antibody-antigen interaction. Labels based on nanoscale materials
open a new opportunity over the traditional methods - in terms of greater reporting signal per
binding event. We have been able to lower the limit of detection of DNA hybridization and
Ab-Ag binding from fM to aM, and fg, respectively. In addition, some possibilities on highthroughput simultaneous assays have been attempted and reported. The talk will describe
some our approaches engineered either electrochemical or optical labels using nanomaterials
such as carbon nanotubes, graphene, and metal nanoparticles. Last, the talk will also present
some real foodborne pathogen applications.
Keywords: Biodiagnotics; Biosensors; DNA; Immunoassay; Nanomaterial.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
100

Fungal Secondary Metabolites - A Pharmaceutical Chemist Perspective

J.-F. F. Weber*
Microbial Metabolites Laboratory, Atta-ur-Rahman Institute for Natural Product Discovery,
Aras 9, Bangunan FF3 Fakulti Farmasi, Universiti Teknologi MARA, Kampus Puncak Alam,
42300 Bandar Puncak Alam, Selangor, Malaysia; *corresponding author, e-mail:
jffweber@puncakalam.uitm.edu.my
Abstract: Drug discovery (DD) by the big pharmas went down to a dangerous low level
that could be explained by a general natural tendency of staying confined into ones comfort
zone. DD from natural products (NP) is and shall remain a primary source of inspiration for
pharmaceutical chemists, even though the challenges are nowadays much greater than they
used to be. DD is akin to a number game: how many compounds should a good library
include and how fast can we get that library? Microorganisms are the main suppliers of new
drug leads. Yet, DD from microbial NPs is hampered by two issues: the low available
biomass and the variable phenotypic expression of biosynthetic genes. At Atta-ur-Rahman
Institute for Natural Product Discovery, UiTM, we have developed a unique protocol that we
named MECSUS (Microtiter plate, Elicitors in Combination, Solid phase extraction, UHPLC,
Statistical analysis) aimed at addressing the above issues and amenable to automation. Some
aspect of this protocol will be presented together with some results accumulated along the
development path.
Keywords: Bioactive
Phytochemicals.
compounds;
Biodiagnotics;
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Biosynthesis;
Drugs;
Fungi;
101

Foot-and-Mouth Disease: Current Scenario in Asia and Bangladesh

M Anwar Hossain*
Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh;
*corresponding author, e-mail: hossaina@univdhaka.edu
Abstract: Background: Foot-and-mouth disease virus is a positive stand RNA virus

(FMDV, family Picornaviridae, genus Aphthovirus) causes an acute vesicular disease of
bovid wild and domesticated ruminants. Foot-and-mouth disease virus (FMDV) comprises of
7 antigenically distinct serotypes (Type O, A, Asia1, C and SAT1-3) that do not provide
cross-protection against one another. FMD is a pandemic disease accounting for a global loss
of 6.5-21 billion USD per annum. Methods: Categorizing, estimation and demography of
FMD occurrence in Asia continent is analyzed. Comparative genomic and phylogeography of
the FMDV in Asia is discussed. Results: Three serotypes of FMDV are circulating in Asian
territory including mainland Southeast Asia, South Asia and Middle East with predominance
of type O, whereas Serotype A and Asia1 are found to be confined in certain geographical
region. Cattle is the most susceptible toward FMD, whereas Pig serves as mixing vessel that
may boosts the emergence and re-emergence episode of several lineages/genotypes. Whole
Genome and phylogeography analysis revealed that transboundary movement of FMDVs are
responsible for spreading of this disease in Asian regions. In 2013-2015, Saudi Arabia
experienced the emergence of Ind-2001 lineage under Middle East South Asia (ME-SA)
topotype of FMDV type O and Genotype VII of FMDV type A which are normally endemic
in the Indian subcontinent. Intrusion of type SAT1-3 in Arabian Peninsula occurs due to
transboundary animal movement from FMDV enzootic African countries. Conclusion:
Transboundary movement of FMDV, inappropriate vaccination and inadequate awareness are
the main reason of FMD spread in most of the Asian Countries.
Keywords: Asia; Bangaladesh; Foot-and-mouth disease; RNA; Serotypes; Virus.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
102

Abstracts (Oral Presentations)

Paper-based visual detection of Salmonella bacteria using Isothermal DNA
amplification and magnetic beads aggregation
Wei, S.X. Sharmili Roy S., Rahman, I.A., and Ahmed, M.U.*
Biosensors and biotechnology laboratory, Chemical Science programme, Faculty of Science,
Universiti Brunei Darussalam; *corresponding author, e-mail: minhaz.ahmed@ubd.edu.bn,
Ph. No.: +673 888 4752
Abstract: Background: The aim of this study was to develop a sequence-specific and simple
method for detection of Salmonella bacteria using isothermal loop mediated amplification
(LAMP) and MB-mediated aggregation. Salmonella is a food-borne bacterial that can affect
human and animals alike. According to World Health Organization (WHO), the disease,
salmonellosis, affects tens of millions of people worldwide every year which can cause
hundred thousand deaths. Following LAMP amplification, the magnetic beads (MBs) were
added to the LAMP products. This produced aggregates that showed up as dark spots on
paper surface. By contrast, when there was the absence of LAMP products, stable
aggregation did not form. This method can detect bacteria DNA at picogram level in 25 th
minutes. Methods: Salmonella bacteria was used here as a model organism.The magnetic
beads were used as an alternative method for detection of DNA. Whatmanpapers were used
as a sample carrier for the aggregation of magnetic beads with DNA. In the presence of a
magnetic field, the MBs form aggregates (LAMP-MBs) with the long DNA strands produced
by the LAMP process, and these aggregates are stable when the magnetic field is removed.
By contrast, LAMP-MBs with short strands of DNA will lose their aggregated form and be
resuspended in the solution when the magnetic field is removed.The aggregation of LAMPMBs complex was measured and analysed using ImageJ software. Isothermal amplicons were
also tested concurrently with agarose gel electrophoresis after staining with DNA
intercalators. Results: Using this method,1 pg/L of Salmonella DNA was detected on paper
for the first time. On the other hand, this faster and sophisticated instrument free protocol
allowed LAMP products detection starting from 20th min of amplification.The LAMP-MBs
took around 5 minutes to detect on to the paper which is obviously efficient compared with
other conventional methods. For example, our result shows better resolution compared to
agarose gel electrophoresis. For the selectivity test, Listeria innocua, Staphyloccous aureus,
Bacillus subtilis microorganisms were also tested and found no cross-reactivity with these
organisms. Conclusion: It can be concluded that LAMP- MBs technique was a sensitive,
specific, cost-effective and time efficient method. It only took a total of 25 minutes to obtain
the result which can be detected visually. This technique could potentially be employed in
health and environmental screening with the development of paper based microdevices.
Keywords: Magnetic beads; Loop-mediated amplification (LAMP); Paper; Salmonella.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
103

Development of a Reverse Hybridization Assay (RHA) for Simultaneous

Identification of Salmonella Serotypes Causing Enteric Fever
Carlos S., Huda H. A., Goay Y. X., Phua K. K.*
Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia
(USM), 11800 Penang, Malaysia; *corresponding author, e-mail: kkphua7@gmail.com
Abstract: Background: Enteric fever is a fatal systemic infection caused by four humanadapted pathogens, Salmonella Typhi and Salmonella Paratyphi A, B and C. Multiplex PCR
has increased the molecular detection capacity for diagnosis of enteric fever as multiple DNA
targets can be detected in a single test. This study reports the development of a reverse
hybridization assay (RHA) for detection of the 4 Salmonella serotypes using multiplex PCR.
Interpretation of RHA results was easy as the appearance of black dot(s) on the test strip,
visualized using naked eyes and without the use of any carcinogenic chemicals or specialized
equipment. Unlike other PCR-based diagnostic assays, this assay can differentiate several
Salmonella serotypes more effectively in terms of cost and time. Methods: A multiplex PCR
assay with biotinylated primers specific for S. Typhi, S. Paratyphi A, B and C, and a PanSalmonella gene (invA) as PCR amplification control was developed and optimized. The
RHA strip was developed with a blue line to mark the orientation of the strip, and five DNA
probes which capture specific biotinylated-PCR products for the 4 Salmonella serotypes and
1 Pan-Salmonella control. 125 strains of S. Typhi, S. Paratyphi A, B or C, and 42 Salmonella
and non-Salmonella bacteria were tested using the RHA test. Results: The assay was found
to be 100% specific (42/42) and 100% sensitive (125/125). Conclusion: A highly sensitive
and specific RHA has been developed and is now ready for clinical field trials to ascertain its
diagnostic sensitivity and specificity.
Keywords: Enteric fever; Diagnosis; Multiplex PCR; Reverse hybridization assay;
Salmonella.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
104

Decrypting the Evolutionary Path of Antimicrobial Resistance of

Acinetobacter baumannii via Next-Gen Sequencing
Mohamad Izwan Ismail, Lee Lian Shien, Teh Lay Kek, and Mohd Zaki Salleh*
Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA (UiTM)
Puncak Alam, Bandar Puncak Alam, Selangor, Malaysia; *corresponding author, e-mail:
zakisalleh.mzs@gmail.com, Ph No: +603-3258 4685
Abstract: Background: Ever since the conception of the first antimicrobial, the threat of
resistance has become an ongoing problem. Despite the copious amounts of research on the
matter, antimicrobial resistance has yet to be eradicated. Despite the discovery of the key
mutations in pathogens in response to exposure to antimicrobials, the step-by-step process of
this adaptation has yet to be clearly understood. Common nosocomial pathogens such as A.
baumannii have been widely reported to develop such resistances at an alarming rate, adding
pressure to the need to understand their adaptive evolution process. Here, we aim to generate
four drug-resistant variants of a susceptible A. baumannii strain and track its mutation
development against ciprofloxacin, erythromycin, meropenem and imipenem. Methods: A.
baumannii was isolated from the blood of a septicemic patient hospitalized at a local hospital.
After confirmation of antimicrobial susceptibility toward the four target drugs, the isolate was
cultured and divided into four subcultures and subjected to daily exposure to ciprofloxacin,
erythromycin, meropenem and imipenem, separately. The antimicrobial concentrations were
steadily increased by two-folds until MIC-level, and high-level resistances were acquired.
The whole-genome of each isogenic variant and the susceptible parent were then sequenced
using the Illumina GAIIx sequencer. Variant analysis of the sequencing output was done
using CLC Bio, and primers were designed using PerlPrimer. PCR, gel electrophoresis and
amplicon sequencing were carried out on the selected samples in each timeline of the
isogenic variants. Results: Each isogenic variant was confirmed to carry different
combinations of mutations; AbRC (gyrA and yihG), AbRE (bvgS, srrA, ftsI and ribonuclease
I), AbRM (epsL, mexB and atpD), and AbRI (ftsI and acrB). The following mutation timeline
analysis revealed variations in early-, middle-, and late-stages of the resistance induction,
leading to a final stable resistance. Furthermore, isogenic variant AbRI was identified to carry
two mutations in a single ftsI gene, occurring at two varying time points throughout the
resistance induction process. Conclusion: The chronology of mutations was suggested to be
influenced by both the duration and concentration of antimicrobials that the A. baumannii is
exposed to. As such, these two parameters need to be taken into account when tackling the
issue of antimicrobial resistance.
Keywords: Acinetobacter baumannii; Antimicrobial; Resistance; Sequencing; Timeline;
Whole-genome.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
105

Isoluminol-functionalized gold nanoparticles and graphene oxide

nanoribbons composite for development of enzyme-based
electrochemiluminescence biosensors
Nur Syakimah Ismail1* and Eiichi Tamiya2
1
School of Microelectronic Engineering, Kampus Pauh Putra, Universiti Malaysia Perlis,

02600 Arau, Perlis, Malaysia; 2Department of Applied Physics, Graduate School of
Engineering, Osaka University, 2-1Yamadaoka, Suita, 565-0871 Osaka, Japan;
*corresponding author, e-mail: syakimah@unimap.edu.my
Abstract: Background: Electrochemiluminescence (ECL) has become an important

detection method due to its simplicity, rapidity and high sensitivity. N-(aminobutyl) -N(ethylisoluminol) (ABEI) is luminol derivatives, which widely use luminophore to generate
ECL emission. Enhanced ECL emission can be obtained by using gold nanoparticles
(AuNPs) that facilitate the generation of oxidative radicals and fast electron transfer due to
catalytic surface effects. Previously, we have synthesized AuNPs coated with ABEI (ABEIAuNP) that demonstrated ECL intensity enhancement by graphene oxide nanoribbon
(GONR) as functional supporting matrix on disposable screen printed electrode (SPE).
Herein, we utilized ABEI-AuNP-GONR/SPE in development of enzyme-based ECL
biosensors. Methods: GONRs and ABEI-AuNPs were synthesized through the longitudinal
unzipping of MWCNTs and seed growth method, respectively. SPE was used for all
electrochemical measurements that contained a three-electrode system; working electrode,
counter electrodes, and the Ag/AgCl reference electrode. GONRs (1 mg/mL) were mixed
with as-prepared ABEI-AuNP in the ratio of 1:3 before casted on SPE working electrode and
dried at room temperature overnight. All electrochemical measurements were performed with
a USB-powered handheld potentiostat BDTminiSTAT100 while ECL intensities were
recorded by Hamamtsu Photon Detection Unit. Results: Firstly, glucose oxidase enzymes
catalyzed production of hydrogen peroxide that subsequently turns to superoxide radicals to
promote ECL enhancement. Secondly, urease enzyme used to catalyze the decomposition of
urea into ammonia, which consequently increases the solution pH. Therefore, ECL intensity
increases proportionally with urea concentration. Conclusion: We have successfully
developed ECL glucose and urea sensors by using ABEI-AuNP-GONR/SPE. They provide
sensitive detection paving the way for future development of portable ECL biosensors.
Keywords: Electrochemiluminescence; Gold nanoparticle; Graphene oxide nanoribbon;
Luminol.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
106

Disposable Screen-Printed Electrodes Modified With Nanoparticles for

Sucrose Sensor
Detpisuttitham W.a, Rijiravanich P.b,c*, and Surareungchai W.d
a
Pilot Plant Development and Training Institute, King Mongkut's University of Technology
Thonburi, Bang Khun Thian, Bangkok 10150 Thailand; bBiochemical Engineering and Pilot
Plant Research and Development Unit, King Mongkut's University of Technology Thonburi,
Bang Khun Thian, Bangkok 10150, Thailand; cNational Center for Genetic Engineering and
Biotechnology, NSTDA, Khlong Luang, Pathum Thani 12120, Thailand; dSchool of
Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bang
Khun Thian, Bangkok 10150, Thailand; *corresponding author, e-mail:
patsamon.rij@biotec.or.th, Ph No: +662-4707475.
Abstract: Background: The measurement of sucrose is important not only in industrial

analysis but also in clinical analysis. Sucrose detection can be accomplished by detecting its
oxidation at high pH, with no need for complex and time consuming sample preparation.
However the oxidation products can gradually passivate the electrode surface, causing a loss
of analyte signal. Methods: Here, the disposable screen-printed carbon electrodes modified
with Au-SiO2 nanoparticles (Au-SiO2/SPEC) were developed for amplifying electrochemical
signal in nonenzymatic electrochemical sucrose sensors. Each Au-SiO2 nanoparticle consists
of an amine functionalized silica particle (SiO2), coated sequentially with polyelectrolyte and
gold nanoparticles. After casting Au-SiO2 on the surface of the carbon working electrode, the
electrochemical characterization of Au-SiO2/SPCE was investigated by voltammetry and
amperometry. Results: Au-SiO2/SPEC Provide highly catalytic oxidation peak of sucrose at
potential above 50mV in a NaOH electrolyte. Then the amount of Au-SiO2 casting,
concentration of electrolyte, and potential were optimized to establish high and reliable
responses for sucrose detection. The characterization of this sensor have also been described.
Conclusion: Our disposable Au-SiO2/SPEC was successfully fabricated for sucrose sensor
determination. This sucrose sensor can be directed towards applications in biofuel and food
industries and clinical fields.
Keywords: Electrochemical sucrose sensor; Gold nanoparticles (AuNPs); Screen printed
carbon electrodes.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
107

Analysis of Chalcone-Flavanone Isomerase (CHI) Gene cDNA Isolated

from American oil-palm (Elaeis oleifera) Mesocarp Tissue cDNA Library
Yu Chuen Leow1, Amelia Kassim2, Subhash J Bhore1, 2 *
1
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong

Semeling Road, Semeling 08100, Kedah, Malaysia.
2
Department of Molecular Biology, Melaka Institute of Biotechnology, Lot 7, Melaka
International Trade Centre City, 75450 Ayer Keroh, Melaka, Malaysia; *corresponding
author, e-mail: subhash@aimst.edu.my / subhashbhore@gmail.com
Abstract: Background: The nutritional quality of the American oil-palm (Elaeis oleifera)
mesocarp oil is superior to that of African oil-palm (Elaeis guineensis Jacq. Tenera)
mesocarp oil. Therefore, it is important to identify the genetic features for its superior value.
This could be achieved through characterization of oil-palm genes. Hence, we constructed a
cDNA library and generated expressed sequence tags (ESTs) from the mesocarp tissue of the
American oil-palm. The primary analysis turned our attention to the Elaeis oleifera chalconeflavanone isomerase (EoCHI) enzyme encoding cDNA. This EoCHI is an important enzyme.
Therefore, to understand more about it this study was undertaken. The objective of this study
was to elucidate EoCHI gene cDNA sequence and to predict the three-dimensional (3D)
structure of deduced protein. Methods: Positive and negative strands of the EoCHI cDNA
clone were sequenced using M13 forward and M13 reverse primers to elucidate the
nucleotide sequence. The EoCHI cDNA and deduced protein sequence was analysed for their
basic features using online bioinformatics tools. Sequence comparison was carried out using
bl2seq program, and tree-view program was used to construct a phylogenetic tree. The
secondary structures and 3D structure of EoCHI protein were predicted by using the PHYRE
automatic fold recognition server. Results: The sequencing results analysis showed
that EoCHI cDNA is 942 bp in length. Its open reading frame (ORF) encodes for a protein
that contains 234 amino acids. The analysis results showed the presence of chalcone
superfamily domain in the protein sequence. The multiple sequence alignment of selected
CHI amino acid sequences from other plant species with E. oleifera CHI using ClustalW and
its phylogenetic analysis suggest that CHI from Elaeis guineensis and Phoenix dactylifera are
the most closely related with EoCHI. Conclusion: The annotation of EoCHI showed that 942
bp long EoCHI cDNA encodes for 234 amino acid long protein that contains conserved
domains required for biological functions of CHI. The predicted deduced EoCHI protein's 3D
structure is predicted. However, further study is required to validate the predicted structure.
Keywords: African oil-palm; American oil-palm; Chalcone-flavanone isomerase; Edible oil;
Palm oil
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
108

Non-Protein coding RNA genes as novel diagnostic markers to detect

pathogenic bacteria
Suresh C.V.*
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling,
Bedong 08100, Kedah, Malaysia; *corresponding author, e-mail:
cvsureshgupta@gmail.com, Ph No: 044298000-8165
Abstract: Background: Small non protein-coding RNAs (npcRNAs) have emerged as the
important regulators of many cellular pathways in bacteria. The specificity of the npcRNA
varies from species to phylum level. High accuracy in the detection of specific pathogen is
very important in clinical diagnosis. We used npcRNA genes as diagnostic markers for
various pathogenic bacteria detection. Methods: We used various computational tools to
identify specificity of the npcRNAgenes in Salmonella typhi, Vibrio cholerae, Shigella
flexneria and Staphylococcus aureus. Using specific primers, we performed PCR to amplify
the target npcRNA genes from aforementioned bacteria. Specificity of the target was checked
using various Gram positive and Gram negative bacteria. Sensitivity was recorded with
serially diluted genomicDNA and bacteria separately. Results: The results confirmed that the
genes StyR-36, VrrA, CssrB and Sau-02 are very specific to Salmonella typhi, Vibrio
cholerae, Shigella flexneri and Staphylococcus aureus respectively. The sensitivity limit of
detection using genomic DNA of these bacteria attained was in the range of 30fg to 10pg.
The sensitivity of detection using whole bacteria obtained was in the range of 7-15 CFU/ml.
Conclusion: This investigation reveals that npcRNA genes serve as excellent molecular
biomarkers for the effective diagnosis of bacterial infections. The results of the present study
support the use of npcRNA genes in other molecular-based diagnostic methods, such as
nucleic acid sequence-based amplification (NASBA). Also, npcRNAs are acquiescent for use
in real-time detection of live bacterial species via RT-qPCR diagnostics.
Keywords: Pathogenic bacteria; Diagnostic marker; Non-protein coding RNA; Specificity
and sensitivity.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
109

Herbal Based Stabilizers of Native and Misfolded State of Nuclear Corepressor (N-CoR)
Matiullah Khan1*, Thunga Pandurangan2, Sridevi Visvanathan3, Sam Annie Jeyachristy3,
Mahes Maheswaram4
1
Department of Pathology, 2Surgery and Biochemistry3, Faculty of Medicine: Department of

Biotechnology4, Faculty of Applied Sciences, AIMST University, Semeling, Bedong 08100,
Kedah, Malaysia; *corresponding author: Matiullah Khan, e-mail: khanmmd@gmail.com,
Ph No: +60125855284
Abstract: Background: Nuclear receptor co-repressor (N-CoR) is a generic co-repressor

essential for the suppression of cellular self-renewal potentials during cell maturation.
Finding from our lab suggest that heterogeneous oncogenic insults can induce nearly
homogenous change in N-CoR conformation through phosphorylation, which ultimately
leads to its misfolding and premature loss in wide variety of human cancers. The misfolded
N-CoR acquires pleotropic properties due to the highly transitional nature of its misfolded
state and triggers malignant growth and transformation through a variety of unique "loss and
gain of function mechanisms. The aim of this project was to investigate the role of
genistein and curcumin, two pleotropic medicinal compounds linked to the regulation of
protein misfolding pathway, on the conformation and function of native and misfolded NCoR protein as well as fate of tumour cells harbouring the misfolded N-CoR protein.
Methods: We analysed the fate of tumour cells along with the conformational dynamics of
native and misfolded N-CoR protein in tumour cells treated with genistein and curcumin, the
herbal based stabilizers of native and misfolded state of N-CoR protein. Results: Genistein
promoted differentiation of tumour cells through the stabilization of native N-CoR state while
curcumin promoted apoptosis by stabilizing the misfolded state of N-CoR. Conclusion: Our
finding illustrate how small molecule mediated stabilization of native and misfolded N-CoR
state could be exploited for the design and development of highly selective anti-cancer
therapeutic approach.
Keywords: Curcumin; Genistein; Misfolded protein; N-CoR.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
110

Hepatoprotective effect of methanol extract of Polygonum minus leaves in

carbon tetrachloride-induced liver damage in rats
Manoontri T, Joe L.S, Tanusha S. B. M, Parasuraman S, *Christapher P.V
Faculty of Pharmacy, AIMST University, Semeling 08100, Malaysia; *corresponding author,
e-mail: christapher@aimst.edu.my, Ph No: +60 44298000 (extn: 1029)
Abstract: Background: Hepatotoxicity, damage to liver cells caused by various chemicals

and drugs, is a serious health risk. Treatment options are inadequate and novel cost effective
and low toxic drugs are need of the hour. Polygonum minus (Family: Polygonaceae) is a
commonly available food additive plant in Malaysia. Different parts of this plant are used in
the Malay folk medicine; to relive pain, as health supplement and to get rid of dandruff.
Pharmacological studies and clinical trials have reported its various effects including
antioxidant, anti-inflammatory, analgesic, antiulcer, sexual well-being and cognitive
improvement functions. The present study evaluates the hepatoprotective potential of
methanol extract of leaves of P. minus on chemical-induced liver injury. Method: P. minus
leaves were shade dried, powdered, and macerated in methanol for 5 days, filtered and
evaporated to obtain dry methanol extract (MEPM). The standard drug and MEPM treated
groups of animals were administered with sylimarin (50 mg/kg) or MEPM (100 mg/kg or 200
mg/kg) for 14 days. All the animals were administered carbon tetrachloride (1:1 v/v in olive
oil, 2 ml/kg, intraperitoneally; on day 6, 9 and 12) except normal control group to induce
liver toxicity. Body weight, biochemical parameters and histopathology studies were
conducted. Results: Body weight of animals did not show significant variation in any treated
group, compared with normal control, but was markedly decreased in carbon tetrachloride
control group. The biochemical analysis showed that the higher dose of MEPM exhibited
significant hepatoprotective activity. The histopathology study also confirmed the
hepatoprotective effect. Conclusion: Methanol extract of P. minus possesses significant
hepatoprotective activity and the activity is increased with dose. More detailed studies are
required to establish its hepatoprotective efficiency.
Keywords: Carbon tetrachloride (CCl4); Hepatoprotective effect; Polygonum minus;
Sylimarin.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
111

Molluscicidal Effect of Poly herbal Extracts on Golden Apple Snail,

Pomacea maculata
Guruswamy Prabhakaran1*, Thambirajah, J.J2, Subhash Janardhan Bhore1 and Manikam
Ravichandran1
1
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling

Campus, 08100 Bedong, Kedah Darul Aman, Malaysia.
2
Faculty of Business Management, AIMST University, Semeling Campus, 08100 Bedong,
Kedah Darul Aman, Malaysia; *corresponding author, e-mail: prabhakaran@aimst.edu.my
Abstract: Background: The Golden Apple Snail (GAS), Pomacea maculata, is a major rice
pest in Southeast Asia. The cost of synthetic molluscicides, their toxicity to non-target
organisms and buildup of snail resistance have given new impetus to study on plant
molluscicides. Most research efforts have focused on individual plant extract for
molluscicidal properties, and are not proved entirely effective. Selective consortium of potent
molluscicidal compounds from various plants might be an effective alternative. Six different
plants extracts viz; Nerium indicum, Azadirachta indica, Nicotiana tabacum, Piper nigrum,
Pongamia pinnata and Zingiber officinale were evaluated individually as well as in selective
combinations on golden apple snails mortality in laboratory conditions. Methods: The dried
and coarse plant material (100 g) was macerated in 1000 ml of 95% ethanol at room
temperature (25 2C) for 3 days. The filtrate was concentrated to dryness with a rotary
evaporator at 60 C. Individual extracts were then combined at 1:1 to 1:1:1 ratio by w/w and
diluted to different concentrations with distilled water. Groups of 10 juvenile snails were
placed in plastic containers with 1000 ml of plant extract suspension and set in triplicate at
room temperature. The number of dead snails was recorded after 24 hours, 48 hours and after
72 hours. Results: Combining extracts of two or three plant extracts of Nerium indicum,
Nicotiana tabacum, Piper nigrum and Azadirachta indica cause 73 to 96 % mortality of
GAS. The poly extracts were more effective at Lethal Concentration (LC90) of 177 to 191
mg/l on GAS in comparison to individual extracts. The synergistic effect of the combined
extracts resulted in around 45 % reduction in LC90 values of the individual extracts.
Conclusion: This study demonstrated that combined crude extracts of Nerium indicum,
Nicotiana tabacum, Piper nigrum and Azadirachta indica are effective for sustainable
control of GAS.
Keywords: Azadirachta indica; Combined plant extracts; Golden apple snail; Nerium
indicum, Nicotiana tabacum; Piper nigrum; Plant molluscicides; Pomacea maculate.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
112

Design and Characterization in Time of an On-off DNA Biosensor

Guajardo-Yvenes C. F.1,4*, Issaragkul P.2,4, Sae-Tang C.2,4 and Surareungchai W.3,4
1
Pilot Plant, Development and Training Institute

2
Department of Chemistry, Faculty of Science
3
School of Bioresources and Technology
4
King Mongkut's University of Technology Thonbury, Bangkok, Thailand; *corresponding
author, e-mail: cristian.gua@kmutt.ac.th, Ph. No: (+66) 2470 7562
Abstract: Background: Naked-eye detection and detection time are important

characteristics for biosensors. Also their ease-of-use is improved if the output is clearly off
in absence and clearly on in presence of analyte. In this research an on-off DNA biosensor
was designed and characterized in time. This biosensor produces a fixed readable output, for
small or large concentrations of analyte, by means of a catalytic amplification mechanism.
Methods: The catalytic amplification was based on DNA strand displacement and toehold
exchange (seesaw gate motif). Here an analyte strand displaces the output from a doublestranded gate:output complex, later a fuel strand displaces and releases the analyte from the
gate:analyte complex formed previously, completing a catalytic cycle. Simulations were
performed to get approximate detection times. Finally, preliminary experiments were
conducted using fluorescent reporter probes, in order to measure detection times. Results:
Simulations indicate that detection times are in the order of hours for low nM range of
analyte, whereas they can go down to minutes for hundreds of nM. In both cases the output
signal reached the same readable level. Likewise, experiments indicate that detection times
decrease with increasing concentration of analyte, however it is not as evident as in the
simulations. The fluorescent output reached only similar readable levels for different
concentrations of analyte. Conclusion: Characterization through simulations and experiments
show that readable outputs can be obtained, even when analyte concentration decreases.
Detection times are also reduced as analyte concentration increases, however there is clear
difference between detection times obtained from simulations and experiments.
Keywords: Chemical reaction network; DNA Biosensor; DNA strand displacement; Toehold
exchange.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
113

Optimization of PCR for Rapid Detection of CTX-M Gene in ESBL

Producing Klebsiella pneumoniae Clinical Isolates
Rasheeda B1*, Neelam Z1, Imran A2, Shagufta N1
1
2
Department of Biotechnology, Lahore College for Women University Lahore, Pakistan.

Quality Operational Laboratory, University of Veterinary and Animal Sciences Lahore,
Pakistan; *corresponding author, e-mail: rashidasbs@yahoo.com, Ph No: 099203024680187
Abstract: Background: The emergence of drug resistance bacteria are the burning issue of
modern medical science associated with the infectious diseases. The detection of antibacterial
resistant genes is important for successful treatment against infectious disease by using the
effective antibiotics. Among many methods the PCR is most precise and acceptable
procedure for the detection of different antibacterial genes. This study was designed to check
the antibiotic resistance pattern and frequency of CTX-M in Extended spectrum beta
lactamase producing Klebsiella pneumoniae clinical isolates from different region of Punjab,
Pakistan. Methods: Two hundred isolates of Klebsiella pneumoniae isolates were obtained
from different clinical samples. Blood and Mac-Conkey Agar were used to isolate and
identify bacterial microorganisms while Muller Hinton Agar was used to evaluate the
antimicrobial susceptibility against different antibiotics as per CLSI 2012 guidelines. ESBL
producing bacteria were screened by Double Disk Synergy and Combination Disk test. For
the molecular detection of the resistant gene (CTX-M) PCR was performed. Results: 49% of
the total isolates showed beta lactamase activity and resistant to multiple antibiotic including
Cephalosporin, Aztreonam, Sulphamethoxazole/Trimethoprim, Ciprofloxan, Doxycyclin.
Imipenam and Amikacin were observed to be least resistant in ESBL producing isolates as
13% and 12% respectively. CTX-M gene was detected in 94% of ESBL isolates.
Conclusions: Based on the finding of this study it is suggested that prevalence of CTX-M
gene (95%) is very high among ESBL producing isolates. Therefore PCR based method may
help clinicians for rapid detection and treatment of patients by choosing right medication
against the resistant bacteria as early as possible.
Keywords: Antibiotic resistance; CTX-M; ESBL bacteria; Klebsiella pneumonia.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
114

Umami Tasting Detection Based Electrochemical Sensor

Promphan R.a, Chaiboon T.b, Lertanantawong B.b,* and Surareungchai W.c
a
Department of Chemical Engineering, King Mongkut's University of Technology Thonburi,

ThungKhru, Bangkok 10140, Thailand.
b
Pilot Plant Development and Training Institute,King Mongkut's University of Technology
Thonburi, Bang KhunThian, Bangkok 10150, Thailand.
c
School of Bioresources and Technology, King Mongkut's University of Technology
Thonburi, Bang KhunThian, Bangkok 10150, Thailand;
*corresponding author, e-mail: benchaporn.ler@kmutt.ac.th, Ph No: +662-4707475
Abstract: Background: Umami taste is elicited from small amino acid, mainly glutamic
acid. Glutamic acid can be found in a variety of natural materials such as plant, meat and
vegetable root. Umami is the fifth flavor which has a characteristic taste and as a stimulant
the appetite. Methods: In this work, we developed an electrochemical glutamate sensorbased
enzymatic assay. Glutamate Dehydrogenase (GLDH) is aspecific receptor for glutamic acid
sample. The enzymatic reaction of GLDH and glutamic acid in the presence of NAD+at
carbon screen printed electrode (CSPE) produces the product and NADH.NADH was
undergo oxidation at the CSPE and the current was detected by differential pulse
voltammetric technique (DPV). Results: The optimal amount of enzyme and NADH suitable
for the measurement of glutamate are at 50 units of the enzyme GLDH with 6 mM NAD+ in
0.1 M phosphate buffer in the presence of 0.1 M potassium chloride, respectively. The
oxidation peak potential of NADH was found at 0.50 0.66 V vs.Ag/AgCl. The linearity of
this umami sensor is in the range of 0.1 mM to 1000mM and the limit of detection is at
0.61mM. Conclusion: We have reported a method to detect umami by combining an
electrochemical technique with enzymatic assay. Our sensors are simple with high specificity
and sensitivity so we believe that the glutamic acid sensor may be a good alternative for
testing umami in food sample.
Keywords: Electrochemistry; Enzyme biosensors; Glutamate dehydrogenase; Umami
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
115

Detection of Salmonella enterica serovar Typhi Form Water Samples and

Its Association with Geographical Clustering of Enteric Fever
Ahmad Filza Ismail2, Mohd Irwan Maarof2, Salwani Mohd Harish1, Phua Kia Kien1, Fauziah
Mohd Nor3, Hani Mat Hussin4, Ismail Aziah1*
1
Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia,

Penang, Malaysia; 2Department of Community Medicine, School of Medical Sciences, Health
Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia;
3
Communicable Disease Control Unit, Kelantan State Health Department, 15200 Kota
Bharu, Kelantan Malaysia; 4Public Health Laboratory, 16450 Kota Bharu Kelantan,
Malaysia; *corresponding author, e-mail: aziahismail@usm.my, Ph No: +60 9 767 2426
Abstract: Background: Typhoid fever infection caused by Salmonella enterica serovar

Typhi remains as a significant cause of morbidity and mortality of children and adults in
underdeveloped and developing countries. The aim of the study is to detect the presence of S.
Typhi in water samples and the spatial distribution of enteric fever cases and its water source
positivity. Methods: A cross sectional study was conducted among all positive enteric fever
cases from January 2012 to December 2013 in five districts (Pasir Puteh, Bachok, Kota
Bharu, Pasir Mas and Tumpat) in Kelantan. The isolation of S. Typhi and polymerase chain
reaction were carried out to detect the bacteria in water as well as stool samples of food
handlers. Genotyping was carried out using PFGE and genome sequencing from two S. Typhi
were performed from the isolates from human and water samples. All data of coordinate of
cases and positive water samples were analyzed using ArcGIS 10.2 and ArcMap 10.2 GIS
software. Results: The data showed 72 confirmed enteric fever cases in five districts in
Kelantan from January 2012 to December 2013. Eight positive water samples were detected
by culture method and PCR. The point pattern analysis showed that the distribution of enteric
fever cases was clustered with the NNI value less than 1 (NNI = 0.59; z score = - 6.61: CI =
99%) whereas for positive water sample, it was random with NNI more than 1 (NNI = 1.042;
z score = 0.228). Six of the cases (8.3%) lived within 500 meters, 19 (26.4%) within 1000
meters and 59 (81.9%) within 3000 meter from the river. All eight (100%) of positive water
sample sources were within 3000 meter from river. There was one positive water source that
has 17 (23.6%) of positive enteric fever cases lived within 500 meters around it, 17 (23.6%)
within 1000 meters and 23 (31.9%) within 3000 meters. Genotyping of S. Typhi of showed
similar pattern when analyzed using PFGE. Next generation sequencing showed both S.
Typhi are highly homology. Conclusion: Enteric fever in five districts in Kelantan was
distributed in cluster form. Majority of the cases stayed near the river and near the source of
positive water sample area. It is suggested that there is a correlation between positivity of
water samples and typhoid cases.
Keywords: Geospatial analysis; S. enterica subsp. enterica ser. Typhi; Typhoid.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
116

The Fabrication of Membrane-Based Pneumatic Microvalves in

Microfluidic System
Suppaso L.1, Boonpakdee D.1, Ngamchana S.2*, Guajardo-Yvenes C. F.3 and Surareungchai
W.4
1
Department of physics, Faculty of Science, KMUTT, Bangkok 10150 Thailand.

Biochemical Engineering and Pilot Plant Research and Development Unit, National Center
for Genetic Engineering and Biotechnology at King Mongkuts University of Technology
Thonburi (KMUTT), Bangkok 10150 Thailand.
3
Pilot Plant Development and Training Institute, KMUTT, Bangkok 10150 Thailand.
4
School of Bioresources and Technology, KMUTT, Bangkok 10150 Thailand;
*corresponding author, e-mail: sirimarn.nga@kmutt.ac.th, Ph No: (+66) 24707561
Abstract: Background: Membrane-based pneumatic microvalves have been fabricated to

control the flow of a solution in microfluidic channels. Methods: Two PDMS layers on glass
substrate were fabricated by soft lithography technique. The top layer of PDMS was designed
to bear the flow channels for a solution. The bottom layer corresponded to pneumatic
channels for microvalve control. The PDMS membrane was placed in between these two
layers to form the microvalves. The air pressure, which was generated from solenoid valves,
has been controlled by LABVIEW in order to close and open microvalves automatically. To
get accurate thickness of channel and membrane, the optimization of spin-coater speed was
performed for a positive photoresist (AZP4620), a negative photoresist (SU8-50) and PDMS
coatings on silicon substrate. Results: After the microfluidic channel with membrane-based
pneumatic microvalves was fabricated, the flow control of solutions has been demonstrated in
an automated and programmable fashion. The thickness profile for speeds within the range of
1000 4000 rpm was obtained for AZP4620 and SU8-50, and ranged between at 16.67
6.25 m and 172.42 31.08 m respectively. The thickness profile of PDMS for speeds
within the range of 500 6000 rpm, and different curing temperatures 60, 80 and 100 C,
was obtained in order to fabricate films with thickness in the micrometer range. Conclusion:
The fabrication of this prototype of membrane-based pneumatic microvalves and their
automatic control by software programming could be demonstrated. This will be applied for
sensing and microfluidic manipulation in the future.
Keywords: Microvalve; Microfluidics; Membrane-based pneumatic microvalves; PDMS
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
117

Role of Outermember Proteins (OMP) and Lipopolysaccharides (LPS) in

Antibody Response Against Pasteurella multocida type B-2 in Bovines
Imran A1*, Anika K2, Jawad N2, Waseem S3, Rasheeda B4
1
Quality Operational Laboratory (QOL), University of Veterinary and Animal Sciences

(UVAS) Lahore Pakistan. 2Department of Microbiology, University of Veterinary and Animal
Sciences (UVAS) Lahore, Pakistan. 3Institute of Biochemistry and Biotechnology (IBBT),
University of Veterinary and Animal Sciences (UVAS) Lahore, Pakistan. 4Department of
Biotechnology, Lahore College for Women University (LCWU) Lahore, Pakistan;
*corresponding author, e-mail: imran.altaf@uvas.edu.pk, Ph No: 0092-3074464628
Abstract: Background: The activation of cellular and humoral immunity depend upon
nature of antigens. Complex proteins like bacterial outer membrane proteins (OMP) usually
successfully activate both wings whereas antigens like bacterial lipopolysaccharides (LPS)
usually elicit T-independent immunityi.e. homural immunity without the activation of cellular
immune wing. Hemorrhagic septicemia is highly contagious bacterial disease of bovine cause
by gram negative bacteria Pasteurella multocida (PM). Both LPS and OMP play important
role in the pathogenic potential of PM. The present study was under taken to evaluate the
comparative immunologic behavior of both the important molecules of pasteurella multocida
alone and in combination in bovine calves in field conditions. Methods: Pasteurella
multocida was isolated, purified and identified from an outbreak by mean of culture and
biochemical methods. The pathogenicity of the confirmed isolates was done in rabbits on the
principles of Kochspostulates. For vaccine preparation dry mass was estimated by filter
method and vaccine was alum gel precipitated Complement fixation test (CFT) was used for
the antibody against Outer membrane protein (OMP) and LPS separately. Results: The
results showed that the antibody titer against OMP and LPS in whole culture vaccine is
significantly higher than the respective tested vaccines.These results concluded that OMP no
doubt is an active T-dependent immunogenic molecule but it immunogensity increases many
times when combined with LPS in whole culture vaccine. Conclusion: Lipopolysaccharides
(LPS) in combination with outer membrane proteins (OMP) synergistically boost up the
humoral immune response in vaccinated animal.
Keywords: Complement fixation test (CFT); Lipopolysaccharides (LPS); Outer membrane
proteins (OMP); Pasteurella multocida (PM).
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
118

Exploration of Novel Endophytic Bacterial Isolates for Their Antioxidant

and Pro-oxidant Properties
Monowar T.1,2, *, Md. Sayedur Rahman2, Bhore S. J.2
1
Unit of Microbiology, Faculty of Medicine, AIMST University, 08100 Bedong, Kedah Darul
Aman, Malaysia. 2Department of Biotechnology, Faculty of Applied Sciences, AIMST
University, 08100 Bedong, Kedah Darul Aman, Malaysia; *corresponding author, e-mail:
tahminamonowar@aimst.edu.my, Ph No.: +6044293006.
Abstract: Background: Endophytes are known to produce various secondary bioactive

compounds which may be used therapeutically as antimicrobial, antiviral, anticancer,
antioxidants, antidiabetic, and immunosuppressant agents. The objective of this study was to
evaluate antioxidant properties of five novel endophytic bacterial strains which were isolated
in our laboratory. Methods: Five endophytic bacterial isolates namely, Acinetobacter
baumannii, Bacillus subtilis, Enterobacter hormaechei, Klebsiella pneumoniae and Pantoea
ananatis were cultivated in nutrient broth separately at 37C, 180 rpm for 24 hours. Crude
extracts were prepared from the broth using three different solvents such as chloroform,
diethyl ether and ethyl acetate. Total antioxidant capacity (TAC), total phenolic content
(TPC), total flavonoid content (TFC), 2,2-diphenylpicrylhydrazyl (DPPH) radical scavenging
activity and metal chelating assay were performed in triplicates. Results: TAC, TPC and
TFC were found in the range of 175.717.05 to 761.326.01 g Ascorbic Acid
Equivalent/mg extract, 254.445.36 to 1451.6727.54 g Gallic Acid Equivalent/mg extract,
and 12.229.62 to 615.0030.05 g Rutin Equivalent/mg extract, respectively. Crude
extracts of A. baumannii, B. subtilis and E. hormaechei exhibited pro-oxidant properties at
lower concentrations while those of the remaining two strains showed antioxidant properties
to some extent. Crude extracts of B. subtilis and P. ananatis were found as good metal
chelators. Conclusion: Crude extracts of K. pneumoniae and P. ananatis showed antioxidant
properties. But, crude extracts of A. baumannii, B. subtilis and E. hormaechei exhibited prooxidant properties at lower concentrations. Further studies in this respect are highly
warranted to explore their pharmacological activities based on their antioxidant and prooxidant properties.
Keywords: Antioxidants; Endophytic bacteria; DPPH; Metal chelating assay; Pro-oxidants;
Total antioxidant capacity.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
119

Sensitivity Analysis of Graphene Based Surface Plasmon Resonance

Biosensor
Toloue H.1,2*, Centeno A.1, Tamiya E.2, Kuwano N.1
1
Malaysia-Japan International Institute of Technology (MJIIT), University Teknologi

Malaysia, Kuala Lumpur, Malaysia; 2Department of Applied Physics, Graduate school of
Engineering, Osaka University, Osaka, Japan; *corresponding author, e-mail:
h.toloue@gmail.com, Ph No: (+60) 173002072.
Abstract: Background: In a conventional surface plasmon resonance sensor a thin metal

layer is sandwiched between two dielectrics. Noble metals such as gold (Au) or silver (Ag)
are used as the metallic films since they lead to SPR at visible light frequencies. The use of
both Au and Ag in a biosensor is limited because of their weak biomolecule adsorption,
restricting the sensitivity of the sensor. Functionalization of metal film with biomolecular
recognition elements (BRE) is a way to improve sensitivity for surface plasmon resonance
biosensor. Methods: In this paper, the effect of variation in thickness of graphene layer as a
BRE on reflection curve and sensitivity of surface plasmon resonance biosensor is
numerically presented. The method is based on transfer matrix for N-layer Fresnel equation
in Kretschmann configuration. The electromagnetic field in SPR condition analyzed using
COMSOL Multiphysics. Results: The change in the minimum reflection in regard to the
number of extra graphene layer on varied metal films is demonstrated. The result illustrated
by coating a graphene layer on metal surface, the sensitivity improved as compared to that of
conventional sensor. This is due to a better adsorption efficiency of graphene and greater
change in refractive index of sensing medium near the sensor surface after biomolecule
adsorption. Conclusion: The model suggested that graphene is a promising material as a
BRE to increase total sensitivity with the number of graphene layers used.
Keywords: Biosensor; COMSOL; Graphene; SPR.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
120

Ameliorative Effect of Curcumin on Olanzapine Induced Obesity in

Sprague Dawley Rats
Parasuraman. S.*, Khor. M. Z., Ee Wen. L.
Unit of Pharmacology, Faculty of Pharmacy, AIMST University, Bedong 08100, Malaysia;
*corresponding author, e-mail: parasuraman@aimst.edu.my,
Ph No: (+60) 103895096.
Abstract: Background: Antipsychotic drugs are very much essential in the treatment and
management of various mental illnesses such as schizophrenia and other psychoses. Although
they have many beneficial effects, they are also not devoid of serious side effects. The
management of antipsychotics (olanzapine) induced weight gain has very limited options.
The effect of natural food antioxidant on weight gain is known, but the effect of the same on
drug induced weight gain remains unclear. Hence the present study was planned to evaluate
the effect of curcumin on olanzapine induced obesity in rats. Methods: Sprague-Dawley
(SD) rats were used for experiments. The animals were divided into six different groups viz.,
normal control, olanzapine control, betahistine (10 mg/kg), curcumin 50, 100 and 200 mg/kg
treated groups. Except the normal control group, all other animals were administered with
olanzapine 4 mg/kg intraperitoneally to induce obesity. The drugs were administered once
daily, per oral for 28 days. During the experiment, body weight changes and behavior
alterations were monitored at regular intervals. At the end of the experiment, blood sample
were collected from all the experimental animals for biochemical analysis. Part of the liver
and kidney tissues were excised from the sacrificed animals and preserved in neutral formalin
for histopathological studies. Results: Curcumin showed a significant reduction in
olanzapine induced body weight gain on the rats and improved the locomotor effects. The
effect of curcumin on olanzapine induced body weight gain is not comparable with that of
betahistine. Olanzapine treated animals showed significant increase in levels of AST,
creatinine, TC, TG, LDL, HDL, VLDL and AD levels compared with control group, whereas
the animals treated with curcumin 200 mg/kg prevented the olanzapines induced changes in
biochemical parameters. In histopathological analysis, olanzapine treated animals showed
mild degeneration of hepatocytes in liver and moderate to severe tubular cell degeneration in
kidneys, whereas curcumin 200 mg/kg prevented the olanzapine inducted tubular cell
degeneration in kidneys. Conclusion: This study has shown metabolic alteration effect of
curcumin on olanzapine treated SD rats.
Keywords: Betahistine; Curcumin; Obesity; Olanzapine.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
121

Study of Nanoparticle-Modified Screen-Printed Electrodes for detection of

Sudan I contamination in chili
Phanthong C.1 and Khownarumit P.2*
1
for Genetic Engineering and Biotechnology at King Mongkuts University of Technology
Thonburi (KMUTT), Bangkok 10150, Thailand; e-mail: chatuporn.pha@biotec.or.th; 2Pilot
Plant Development and Training Institute, King Mongkuts University of Technology
Thonburi, Bangkok 10150, Thailand;
*corresponding author, e-mail: porntip.tas@kmutt.ac.th, Ph No:+662 470-7475.
Abstract: Background: Sudan I is a cancer-causing chemical used in chili and cosmetic to

give a strong color. Sudan I is a category 3 carcinogen previously, now banned, used to color
certain foodstuffs. Hence, the sensing of Sudan I is of interest. Methods: Screen-printed
electrodes (SPCEs) were modified by nanoparticles: graphene oxide (GO), silicon dioxide
(SiO2), and magnetic iron oxide (Fe3O4). The electrochemical characteristics of the SPCEs
were studied for the irreversible electrochemical oxidation of sudan I. Results: The standard
rate constants (ks(cm/s)) for the reaction were determined using linear sweep voltammetry.
The values of ks were found to be 0.009(0.00051), 0.01(0.00084), and 0.0006(0.00081)
cm/s; for GO/SPCE, SiO2/SCPE, and Fe3O4/SPCE, respectively. The total active area
(A(cm2)) was determined from the Anson equation using 20 M. Sudan I and assuming a
diffusion coefficient of 3.41 10-5 cm2/s. Surface coverages ( (mol/cm2)) were also
determined from the Anson equation, using 0.1 mM. potassium hexacyanoferrate (III) and
assuming a diffusion coefficient of 7.6 10-6 cm2/s. A was found to be 1.11 10-5, 5.12 106
, and 6.04 10-6 cm2 from which was found to be 5.43 10-6, 7.26 10-7, and 4.00 10-7
mol/cm2 for GO/SPcCE, SiO2/SPCE, and Fe3O4/SPCE, respectively. Calibration curves for
the analytical determination of Sudan I by the modified SPCEs are presented. Conclusion:
The GO/SPCE showed the highest A and values, which resulted in a higher sensitivity than
the SiO2/SPCE, and Fe3O4/SPCE.
Keyword: ; Active area; Graphene oxide; Magnetic iron oxide; Sudan I; Silicon dioxide;
Surface coverage.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
122

Bioengineering of Tacca integrifolia (Bat flower): Effects of Hormones on in

vitro Rooting and Production of Taccalonolides
Fatimah A.L.1, 2, Teh L.K.2, Asmah A.1 and Salleh M.Z.2,*
1
Faculty of Plantation and Agrotechnology, Universiti Teknologi MARA, Shah Alam,

Selangor, Malaysia;
2
Integrative Pharmacogenomics Institute (iPROMISE) , Universiti Teknologi MARA, 42300
Puncak Alam, Selangor, Malaysia; *corresponding author, e-mail:
zakisalleh.mzs@gmail.com / zakisalleh@puncakalam.uitm.edu.my, Ph No: (+603) 3258
4652.
Abstract: Background: Bat flower plant (Tacca integrifolia Ker Gawl) is a rare plant
species that is often collected for its medicinal value. However, its distribution is limited due
to poor germination of seed and short period of seed viability. The objective of this study was
to develop an in vitro rooting system for T. integrifolia from in vitro seedlings. Methods:
Murashige and Skoog (MS) basal medium was used for the growth of seedlings. Seeds were
sown on the MS basal medium whilst shoots from the in vitro germinated seedlings were
excised and cultured on MS medium containing three different hormones {1Naphthaleneacetic acid (NAA), Indole-3-butyric acid (IBA) and Indole-3-acetic acid (IAA)}
with concentrations of 0.25, 0.5, 1.0, 2.0 and 4.0 mg/L, respectively. After 12 weeks, the
characteristics of the newly produced roots were observed, measured and analysed
statistically. Metabolites produced from the treatments were profiled using LCMS Q-TOF.
Results: The MS basal medium produced plantlets with the highest number of leaves, shoots
and roots compared to the other three rooting hormones. However, MS basal medium
supplemented with 1.0 mg/L IBA produced the longest roots. Interestingly, Taccalonolides A
and B were detected in plantlets grown on MS basal medium. On the other hand,
Taccalonolides E, N and Z were detected in plantlets grown on MS medium supplemented
with 0.25 mg/L IAA, 1.0 mg/L NAA and 1.0 mg/L IBA, respectively. Conclusion: Tacca
integrifolia were successfully grown using tissue culture techniques at laboratory scale and
the Taccalonolides A, B, E, N and Z were detected using LCMS Q-TOF.
Keywords: Bioengineering; In vitro; MS medium; Tacca integrifolia; Taccalonolides.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
123

Isolation, characterization and potential application of bacteriophages for

phage therapy
Bhandare, S.G.; Barrow, P.A. and Atterbury, R.J.*
University of Nottingham, School of Veterinary Medicine and Science, Sutton Bonington
Campus, Sutton Bonington, Loughborough LE12 5RD, United Kingdom;
Faculty of Veterinary Medicine, Universiti Malaysia Kelantan, Locked Bag 36, Pengkalan
Chepa, 16100 Kota Bharu, Kelantan, Malaysia; e-mail: sudhakar@umk.edu.my, Ph No:
+609-7717325; *corresponding author, e-mail: robert.atterbury@nottingham.ac.uk
Abstract: Background: The recent surge in bacteriophage (bacterial viruses) research is

indicative of their huge potential utility for various applications which includes phage therapy
for bacterial pathogens, rapid detection of bacteria and for other biotechnological purposes.
The bacteriophages can be used as natural bio-control, bio-sanitation and bio-preservation
agents in the food processing environment, pre-harvest application in animals prior to
slaughter and in rapid diagnostics. Bacteriophages specific to Vibrio cholerae O1 were
characterized for potential application in phage therapy. Methods: Sewage samples were
collected on two occasions from Severn Trent Sewage Works, Raynesway, Derby, UK, a
local wastewater treatment plant. The protocol used was modified from Van Twest and
Kropinski, (2009). Two phages (2 and 3) were obtained from Public Health England
(PHE), London. The phages were characterized biologically (Lytic spectra and One step
growth curve); physically (Electron Microscopy) and Genomically (PFGE and Restriction
analysis). Results: Bacteriophages could not be isolated from the sewage samples from the
local wastewater treatment plant. The two phages obtained from PHE, London; 2 and 3
could lyse 4.3% and 62.6% of the total 91 Vibrio cholerae strains, respectively. The latent
period and burst size of 2 were 14 1.6 m and 06 01 PFU/cell; while that of 3 were 13
4.1 m and 54 26 PFU/cell, respectively. Electron microscopy revealed that 2 was of the
Myoviridae family while 3 was of the Siphoviridae family. Phage 2 had a genome of less
than 48.5 kb; while 3 had a larger genome of 114 kb. Restriction analysis could
differentiate these phages. Conclusions: The non-isolation of Vibrio cholerae O1 specific
phages from the UK environment is likely to be due to cholera being non-existent in that
country. The phages characterized have potential to be phage therapy candidates.
Keywords: Bacteriophages; Biocontrol; Genome; Phage therapy; Vibrio cholera.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
124

Eco-friendly Biosynthesis of Atrocarpus altilis Mediated Silver

Nanoparticles - Characterization and Evaluation of its Antimicrobial and
Antioxidant Potential
Ravichandran V.1*, Vasanthi S.2, Shalini S.1, Syed Adnan A.S.3and Haris R.4
1
Faculty of Pharmacy, AIMST University, Kedah, Malaysia; 2Faculty of Engineering,

University of Nottingham, Semenyih, Selangor, Malaysia; 3Faculty of Pharmacy, Universiti
Teknologi MARA, Selangor, Malaysia; 4SLT Institute of Pharmaceutical Sciences, Guru
Ghasidas University, Bilaspur, India; *corresponding author, e-mail:
sameshyaravi@gmail.com, Ph No: +60164581626
Abstract: Background: The biological entity is gaining significance in biosynthesis of silver

nanoparticles as a result of their potential applications in nanomedicine and material
engineering. Numerous approaches are used to prepare the nanoscale silver particles such as
electrochemical, sonochemical and microwave-assisted process, but many of these strategies
are having difficulty in purification, used hazardous chemicals and need high energy. To
toggle over these difficulties, recently the eco-friendly approaches are established by using
biological principles. In the present study we aimed to use plant materials for nanoparticles
synthesis; hence, it does not need any elaborate processes such as compound purification
steps and the microbial cell cultures maintenance. Methods: Silver nanoparticles (AgNPs)
were synthesized by biological reduction of silver nitrate with aqueous extract of Breadfruit
(Artocarpus altilis) leaves. Results: Synthesized colloidal BAgNPs were confirmed
spectrophotometrically at 432 nm and the various reaction conditions were optimized. The
SEM, TEM and DLS analysis confirmed that the average particle size of 34 nm, 38 nm, and
162.3 d.nm, respectively. Nature and presence of silver were confirmed by XRD and EDX.
Further, FT-IR spectra of the synthesized AgNPs authorized the presence of phenols, proteins
and flavonoids within the plant extract which can be accountable for the reduction and
stabilizing the nanoparticles by capping. The AgNPs showed moderate antimicrobial actions
than A. altilis leaf extract indicating its antimicrobial value. The DPPH assay results indicated
the good antioxidant activities of AgNPs. Conclusion: The present study revealed the ecofriendly biosynthesis of AgNPs and the safer use of it in the field of biomedicine, water
treatment/purification, and nanobiotechnology.
Keywords: Antimicrobial; Antioxidant; Artocarpus altilis; Biosynthesis; Colloid; Silver
nanoparticles.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
125

Mutiplex Isothermal Amplification for Detection of Melioidosis

Jilien Michelle Wong Tzelinga, Chan Yean Yeana,b*
a
Department of Microbiology & Parasitology, School of Medical Science, Universiti Sains

Malaysia, 16150 Kubang Kerian, Kelantan.
b
Institute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains
Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia; *corresponding author, e-mail:
jilienwong@hotmail.com / yeancyn@yahoo.com, Ph No: +609 7676258.
Abstract: Background: Burkholderia pseudomallei is a causative agent of melioidosis,

causing potentially fatal disease of humans and animals in the tropics. Laborious and time
consuming laboratory diagnostic may delay the treatment and results in high mortality rate.
This study was conducted to develop a multiplex isothermal amplification assay (MIA) for
rapid and sensitive identification of B. pseudomallei. An internal amplification control (IAC)
was included for assay reliability purpose. Methods: A duplex isothermal amplification assay
incorporating an IAC were performed with sets of loop-mediated isothermal amplification
(LAMP) primers, which were designed to specifically identify the conserved region of B.
pseudomallei. The sensitivity of the assay was performed by defining the limit of detection
(LOD) of the optimized multiplex LAMP assay with serially diluted B. pseudomallei
genomic DNA. Clinical isolates of B. pseudomallei, B. cepacia, B. thailandensis, and other
bacteria were used to evaluate the analytical specificity of the assay. Results: The multiplex
LAMP assay was found to be highly specific when tested with other similar bacteria. The
assay of serially diluted gave a limit of detection (LOD) of B. pseudomallei genomic DNA
100 fg/ul. Conclusion: This MIA assay demonstrating promising results which can be used
as method to detect melioidosis in near future for better and prompt therapeutic approach.
Keywords: Burkholderia pseudomallei; Melioidosis; Multiplex loop-mediated isothermal
amplification (M-LAMP); Rapid diagnostic.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
126

Effective Pulmonary Therapeutic Delivery via Surface Acoustic Waves

Nebulization and Phononic Crystal Structures
Mohd H. Ismail*
School of Microelectronic Engineering, Universiti Malaysia Perlis, Malaysia.
Division of Biomedical Engineering, School of Engineering, University of Glasgow, United
Kingdom; *corresponding author, e-mail: hafizismail@unimap.edu.my, Ph No:
+60172386048.
Abstract: Background: Effective pulmonary therapeutic delivery requires a device which

delivers sufficient medication to the site of action with minimal wastage. The effective
delivery of the medication to the targeted area in body depends crucially on the droplet size
distribution. A new platform which utilizes the surface acoustic waves (SAWs) and phononic
crystal (PnC) technologies has been developed to generate monodispersed droplet size within
the respirable fraction (between 1 m to 5 m). In this paper, the nebulized droplet size
distributions of deionized water and budesonide generated by commercialized nebulizers are
compared with the ones generated by the SAWs and PnC. Methods: The piezoelectric SAW
substrate and the silicon PnC superstrate were fabricated using the standard microelectronic
fabrication process. To perform nebulization of the deionized water and budesonide
suspension for inhalation, a high frequency electrical signal was supplied to the electrodes
using the signal generator and amplifier. The electrical signal generated mechanical
oscillations on the LiNbO3, which subsequently produced a surface acoustic wave. The mean
diameters of nebulized droplet sizes were measured using Malvern Spraytec. Results: A
higher respirable fraction of droplet has been successfully obtained from nebulization
performed on the PnC superstrate at low input power as compared to directly on SAW
substrate and the commercialized nebulizers. Conclusion: The efficient nebulization on
silicon PnC coupled to SAW propagating on a piezoelectric substrate has been successfully
demonstrated. The advancement of SAW technology offers opportunities for the
development of nebulizers as efficient and portable pulmonary therapeutic delivery platform.
Keywords: Phononic crystal structure; Pulmonary therapeutic delivery; Surface acoustic
waves nebulization.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
127

Development of a Novel Duplex PCR Assay for Specific Detection of

Salmonella enterica subspecies enterica serovar Typhi Based on SingleGene Target
Goay Y. X.a, Carlos S.a, Suresh V. C.b, Zaidah A. R.c, and Phua K. K.a*.
a
Malaysia (USM), 16150 Kubang Kerian, Kelantan, Malaysia;
b
Faculty of Applied Sciences, Asian Institute of Medicine, Science & Technology (AIMST),
Jalan Bedong-Semeling, 08100 Bedong, Kedah; cDepartment of Medical Microbiology and
Parasitology, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan,
Malaysia; *corresponding author, e-mail: kkphua7@gmail.com
Abstract: Background: Typhoid fever, caused by Salmonella enterica subspecies enterica

serovar Typhi (S. Typhi) remains major public health concern worldwide. Rapid and accurate
detection is essential for surveillance, treatment and control of the disease. In this study, a
duplex PCR assay, which is faster, more sensitive and specific than traditional biochemical
and serotyping method, was developed for diagnosis of typhoid fever. Methods: Primers
targeting STY0307 gene were designed and used in the PCR assay. Primers targeting 16S
rRNA gene was included to serve as an internal control. The PCR assay was optimized using
Taguchi method. The analytical sensitivity and specificity of the optimized PCR assay were
determined using DNA obtained from; 1) ATCC 7251 S. Typhi reference strain, 2) 38
different PFGE-typed S. Typhi local strains, and 3) 72 strains from other enteric pathogens.
The diagnostic sensitivity and specificity of the assay was further evaluated using a total of
120 human clinical specimens collected from Hospital Universiti Sains Malaysia (HUSM).
Results: The assay was found to be 100% sensitive and specific in detecting 39/39 S. Typhi
strains and 0/72 strains from other enteric pathogens. The assay limit of detection was as low
as 1.5 x 105 cfu/ml of bacteria count or 1.28 pg of purified DNA. The sensitivity of the PCR
assay using spiked stool samples was found to be 1.5 x 104 cfu/ml. Evaluation of the PCR
assay using 120 human clinical specimens showed 100% diagnostic specificity and
sensitivity. Conclusion: A highly sensitive and specific duplex PCR assay has been
developed using single-gene target, STY0307, for the detection of S. Typhi, and was found to
be suitable for diagnosis of typhoid fever in clinical settings.
Keywords: Diagnostic; Duplex PCR; S. Typhi; Typhoid fever; Single-gene target; STY0307.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
128

Assessment of Biodiesel Properties From the FAME Composition of a

Malaysian Rhodophyte (Kappaphycus sp.)
Md. Sayedur Rahman, and Kathiresan V. Sathasivam*
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, 08100
Bedong, Kedah Darul Aman, Malaysia; *corresponding author, e-mail:
skathir@aimst.edu.my
Abstract: Background: Biodiesel derived from renewable lipid feedstocks is largely used in
diesel engine. Third generation biodiesel is known to produce from oil of algal biomass and is
considered as a very promising fuel. The objective of the present study was to evaluate some
physico-chemical properties of biodiesel produced from a Malaysian Rhodophyte,
Kappaphycus sp. using its fatty acid methyl esters (FAMEs) composition. Methods: Lipid
was extracted from the dried Kappaphycus sp. collected from Sabah, Malaysia, which was
converted into FAMEs using base-catalyzed transesterification process. The FAMEs were
analyzed by GC-FID (Agilent 7890A, USA). Some characteristic biodiesel properties were
calculated from the FAMEs composition using reference standard mathematical models.
Results: Total lipid contents in the dried biomass of Kappaphycus sp. was found as 15.66
mg/g dw whereas biodiesel yield was recorded as 91.09% of lipid weight. The percentage of
saturated, mono- and poly-unsaturated fatty acids in FAMEs was recorded as 85.98, 8.39 and
5.63 wt.%, respectively. Comparative study showed that biodiesel produced from
Kappaphycus sp. is fairly better than those produced from some land plants as well as
microalgae as it satisfies almost all the quality standards defined by the American, European
and Malaysian biodiesel quality standards under study. Conclusion: Kappaphycus sp. can be
a promising source of lipid feedstock for biodiesel production. But, the low lipid content of
the species is a limiting factor to bring forth economic feasibility of biodiesel development.
We, therefore, emphasize biotechnological interventions through genetic engineering for
enhancing the lipid content and quality in the seaweed species.
Keywords: Biodiesel; Biodiesel quality standards; Fatty acid methyl esters; GC-FID;
Kappaphycus sp.; Lipid.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
129

Generation of RNA Aptamers Against Mycobacterium tuberculosis

Secretory Protein ESAT-6 - a Preliminary Study
Bakhtiar Bukari, Citartan Marimuthu and Thean Hock Tang*
Infectomics Cluster, Advanced Medical and Dental Institute (AMDI), Universiti Sains
Malaysia, Kepala Batas, Pulau Pinang, 13200, Malaysia; *corresponding author, e-mail:
tangth@amdi.usm.edu.my
Abstract: Background: ESAT-6 is a secretory protein produced by Mycobacterium
tuberculosis and is released in early stages of infection. It forms a heterodimer complex with
another protein called CFP-10 and has been implicated with Mycobacterium sp.
pathogenicity. Aptamers are chemical ligands made up of short nucleotides sequences that are
able to bind to target proteins with high affinity and specificity. They are developed using a
process called Systemic Evolution of Ligands via Exponential Enrichment (SELEX). Due to
their chemical stability and high specificity against the target, aptamers have the potential to
become very useful biological tools. Objective: The objective of this study is to develop
RNA aptamers that bind specifically to ESAT-6 protein. Methodology: Eleven SELEX
cycles were carried out using the N40-randomised RNA pool. Stringency of the binding
reaction in each SELEX cycles was increased gradually by varying the amounts of protein,
RNA pool and the competitor. The resulting RNA pool from the 11th cycle of SELEX was
subjected to filter binding assay to assess its binding against ESAT-6 protein. Results: RNA
pool was successfully derived from SELEX cycle 11. Filter Binding assay against the target
protein at 800 and 1600 nM confirmed that binding enrichment of the RNA pool has
occurred. Conclusion: Filter Binding assay suggested the presence of potential binders in the
RNA pool. Further SELEX cycles will be carried out to improve the binding enrichment of
the RNA pool and for sequence deconvolution. Sequencing will be carried out to identify the
putative aptamer.
Keywords: Aptamer; ESAT-6; Filter binding assay; M. tuberculosis; SELEX.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
130

An Expression Analysis of Salmonella Pathogenicity Island (SPI)-Derived

Non-Protein Coding RNAs in S. Typhi Biofilm formation
Kogaan Anbalagan1, Suresh V. Chinni2, Phua Kia Kien1*
1

11800, USM, Pulau Pinang, Malaysia; 2Department of Biotechnology, Faculty of Applied
Sciences, AIMST University, 08100, Bedong, Kedah, Malaysia; *corresponding author, email: kkphua7@gmail.com, Ph No: +6046534853
Abstract: Background: Chronic infection with Salmonella Typhi (S. Typhi) is associated
with long-term localization of the bacteria in the gallbladder in the form of biofilm. Recent
studies have demonstrated that genes in the Salmonella Pathogenicity Island (SPI) region of
the bacteria and many non-protein coding RNAs (npcRNAs) play a crucial role in bacterial
stress response. Therefore, expression analysis of SPI-derived npcRNAs in S. Typhi will give
an idea of their role in biofilm formation. Methods: S. Typhi biofilm was cultured in 6-well
tissue culture plates by incubating at 37C in a shaker at 350 rpm. Total RNA from 3 different
stages of S. Typhi development (planktonic, intermediate and biofilm) were extracted and
resolved on Urea-PAGE gel, and then transferred onto the nylon membrane. Northern blot
hybridization was performed using DIG-labeled specific probes to verify the expression of
the SPI-derived npcRNAs. Results: Expression of five npcRNAs, i.e. StyR-327, StyR-9,
StyR-143, StyR-161, and StyR-381, were detected. StyR-161 was equally expressed in all 3
stages of S. Typhi development, suggesting a house-keeping function for this npcRNA. StyR9 and StyR-381 were marginally down-regulated in the biofilm stage compared to the
planktonic stage. However, StyR-143 and StyR-327 was clearly up-regulated in the
intermediate and biofilm cells compared to the planktonic cells; indicating a possible role of
this npcRNA in biofilm adaptation. Conclusion: In conclusion, expression of 5 SPI-derived
npcRNAs were verified in S. Typhi cells during normal and biofilm conditions. StyR-143
was significantly up-regulated with biofilm formation, and may be related to bacterial
pathogenesis. Further studies need to be carried out to identify the mRNA target and its
regulatory mechanism.
Keywords: Biofilm; npcRNA; Pathogenesis; S. Typhi.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
131

Quantitative, Single-Step Measurement of Hemoglobin A1c in Whole Blood

for Personalized Medicine
Khor S. M.1*, Ang S. H.1, Rambeli M.1, Thevarajah T. M.2, Alias Y.1
1
Department of Chemistry, Faculty of Science, University of Malaya, 50603, Kuala Lumpur,

Malaysia; 2Department of Pathology, Faculty of Medicine, University of Malaya, 50603
Kuala Lumpur, Malaysia; *corresponding author, e-mail: naomikhor@um.edu.my, Ph No:
+603-79677022/Ext: 2520
Abstract: Background: We describe a gold nanoparticle-based sandwich immunoassay for

the dual detection and measurement of hemoglobin A1c (HbA1c) and total hemoglobin in the
whole blood (without pretreatment) in a single step for personalized medicine. Methods: The
optimized antibody-functionalized gold nanoparticles immunoreact simultaneously with
HbA1c and total hemoglobin to form a sandwich at distinctive test lines to transduce visible
signals. The applicability of this method as a personal management tool was demonstrated by
establishing a calibration curve to relate % HbA1c, a useful value for Type 2 diabetes
management, to the signal ratio of captured HbA1c to all other forms of hemoglobin.
Results: The platform showed excellent selectivity (100%) toward HbA1c at distinctive test
lines when challenged with HbA0, glycated HbA0 and HbA2. The reproducibility of the
measurement was good (6.02%) owing to the dual measurement of HbA1c and total
hemoglobin. A blood sample stability test revealed that the quantitative measurement of %
HbA1c was consistent and no false-positive results were detected. Also, this method
distinguished the blood sample with elevated HbF from the normal samples and the variants.
Conclusion: The findings of this study highlight the potential of a lateral flow immunosensor
as a simple, inexpensive, consistent, and convenient strategy for the dual measurement of
HbA1c and total Hb to provide useful % HbA1c values for better on-site diabetes care.
Keywords: Colloidal gold-based immunochromatographic assay; Hemoglobin A1c; Lateral
flow immunosensor; Single-step dual measurement; Total haemoglobin; Type 2 Diabetes
Mellitus.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
132

Development of Rapid Diagnostic Detection for Salmonella enterica

Subspecies enterica Serovar Paratyphi A using Cross Priming
Amplification
Roziana, M.H. 1,2, Tan S.C. 1, Ismail, A. 1 and Aziah, I*1
1

Health Campus, Kelantan, Malaysia; 2Universiti Teknologi MARA, Perlis, Malaysia;
*corresponding author, e-mail: aziahismail@usm.my, Ph. No: +609-7672426
Abstract: Background: Paratyphoid fever is a systemic infection caused by Salmonella

enterica subspecies enterica serovar Paratyphi A contributing one case in every four cases of
typhoid. An isothermal amplification, cross priming amplification (CPA), is amplified using
several primers by DNA polymerase with displacement activity at a constant temperature was
developed to overcome the limitation of the current PCR test. As a point of care, CPA is
developed for rapid detection of bacteria suitable for resource-limited settings with the basic
requirement of simple heating block or waterbath for the amplification. This study aims to
establish an in-house CPA combined with lateral flow assay (LFA) for the detection of S.
Paratyphi A (SPA). Methods: In-house CPA for S. Paratyphi A was developed three pairs of
primers (displacement, cross and detector) from six locations of an intergenic region between
SSPA1723a and SSPA1724 of S. Paratyphi A genome sequence. The forward and reverse
detector primers were labeled with biotin and FAM at 5end respectively. Optimisation of the
CPA-SPA components was performed using Taguchi method. The assay was further tested
with few parameters such as primer concentration, temperature and incubation time.
Analytical sensitivity of CPA was performed at DNA and bacterial level. Diagnostic
sensitivity and specificity of CPA-SPA were tested with 30 isolates of each S. Paratyphi A, S.
Typhi, other Salmonella serovars, and other bacterial strains. CPA-SPA products were
detected via 2% agarose gel electrophoresis (AGE) and compared with LFA. Results: The
optimum condition of CPA-SPA was at 63C for 60 minutes. The limit of detection of the
CPA-SPA was 10 fg of the pure genomic DNA. CPA-SPA was sensitive up to 103 CFU/ml
and 101 CFU/ml via AGE and LFA respectively. CPA-SPA was positive for all 30 S.
Paratyphi A and negative for other 90 bacterial strains. Conclusion: The present study
demonstrated high sensitivity and specificity of CPA-SPA. Rapid, sensitive and specific
detection of CPA-SPA was successfully developed. The findings suggested that the in-house
CPA-LFA has great potential for detection of S. Paratyphi A in resource-limited settings.
Keywords: Cross-priming amplification; Enteric fever; Isothermal; S. Paratyphi A.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
133

Conversion of Rice Husks to Polyhydroxyalkanoate (PHA)

Heng K.S.1, Adam F.2, Sudesh, K.1*
1
School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia;

School of Chemical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia;
*corresponding author, e-mail: ksudesh@usm.my, Ph. No: +604 6534367
Abstract: Background: Sugars obtained from the hydrolysis of rice husks have the potential
to be used as an economical feedstock for the production of polyhydroxyalkanoates (PHA), a
biodegradable polymer produced intracellularly many types of bacteria. Methods: The rice
husks were first pretreated with a combination of alkali and physical methods and then
hydrolyzed enzymatically under conditions that were optimized previously. Characterization
of the sugars in the hydrolysate was performed to determine the sugar composition. The
hydrolysate was fed to two strains, Burkholderia cepacia USM (JCM 15050) and
Cupriavidus necator NSDG-GG, an engineered strain of Cupriavidus necator H16, to
evaluate their PHA production. Results: Based on high performance anion exchange
chromatography (HPAEC) analysis, glucose and xylose were the main sugars present in the
hydrolysate, with low amounts of arabinose. B. cepacia USM utilized the hydrolysate more
efficiently compared to C. necator NSDG-GG, with a maximum cell dry weight (CDW) of
4.9 g/L and 40 wt % PHA at shake-flask scale. The CDW and PHA content of the B. cepacia
USM cultivated in a 5-L fermentor after 36 hours of fermentation were 7.80 g/L and 50%
respectively. The decrease in total phenolics at the end of fermentation suggested that B.
cepacia USM was able to metabolize phenolic compounds. Conclusion: These results
indicate that rice husks can be used as carbon sources for PHA production, thus adding value
to this agricultural by-product.
Keywords: Biosynthesis; Hydrolysis; Polyhydroxyalkanoate; Rice husks; Sugars.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
134

Salmonella typhimurium Detection Based on Electrochemical Immunoassay

using Methylene blue/MWNTs/Magnetic Particle
Ngoensawat U.a, Rijiravanich P.* b, c, Surareungchai W.a and Somasundrum M. b, c
a
School of Bioresources and Technology, King Mongkut's University of Technology

Thonburi, Bang Khun Thian, Bangkok 10150, Thailand.
b
for Genetic Engineering and Biotechnology, King Mongkut's University of Technology
Thonburi, Bang Khun Thian, Bangkok 10150, Thailand.
c
National Center Biological Engineering Graduate Program, King Mongkut's University of
Technology Thonburi, Bangmod, Bangkok 10140, Thailand;
*corresponding author, e-mail: patsamon.rij@biotec.or.th, Ph No: (+66)2-4707475
Abstract: Background: Electrochemical sensors are an attractive technology for food borne
pathogen detection, offering sensitivity, selectivity, fast response, low cost, amenability to
mass production and the possibility of miniaturization. To achieve high sensitivity and
selectivity, nanomaterials have often been integrated into detection platforms. Due to
possessing, high surface-to-volume ratios, nanomaterials have the advantage of being able to
carry high amounts of redox mediator and can often be surface-modified with biomolecules.
This has made nanomaterials highly attractive as electrochemical labels. Methods: We report
an electrochemical immunoassay using modified magnetic beads as electrochemical labels.
The labels are prepared by modifying magnetic beads with methylene blue (MB)functionalized multiwall carbon nanotube (MWNTs). The outermost layer of the beads is
coated with an antibody specific to Salmonella typhimurium. After binding, the target cells
can be easily separated from the solution by applying a magnetic field. The label-cell
conjugated is deposited on an anti-Salmonella Typhimurium-modified screen-printed
electrode for sandwich immunoassay. The signal from direct reduction of MB is recorded by
differential pulse voltammetry (DPV). Results: Each electrochemical label was found to
carry 2.35x108 molecules of MB, as determined by DPV. This is a 1000 fold increase in the
MB loadings of labels used in previous work. The peak reduction current was observed at 0.264V which is similar to MB in solution. The label signal was significantly different in the
absence of the cells. Conclusion: Magnetic beads were successfully modified with MBloaded carbon nanotubes. The resulting labels showed a high current signal for MB reduction.
The magnetic beads could be used to capture and separate target cells from solution, enabling
Salmonella typhimurium detection.
Keywords: Electrochemical immunosassay; Magnetic separation; MWNTs-methylene blue;
Salmonella typhimurium.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
135

Electrochemical Characterisation and Determination of Mycobacterium

tuberculosis by Voltammetry at Polymer Nanocomposite modified Platform
Himkusha Thakur1, Navpreet Kaur1, Dipti Sareen1, Priyanka2, Nirmal Prabhakar*1
1
Department of Biochemistry, Panjab University, Chandigarh, India.

Institute of Nano Science & Technology, Mohali, India; *corresponding author, e-mail:
nirmalprabhakar@pu.ac.in
Abstract: Background: Tuberculosis, caused by Mycobacterium tuberculosis, produces illhealth among millions of people each year and ranks as the second leading cause of death
from an infectious disease worldwide.The conventional TB detection methods are highly time
consuming, laborious and less result oriented, that can be overcome by the use of biosensors.
We have developed a novel bioelectrode for the detection of active tuberculosis using
polymer nano-composite platform. Methods: PEDOT, a polymer, exhibits relatively high
electrical conductivity and is very stable even during the electrochemical changes, while
CNT gives advantages like small size with larger surface; high sensitivity and fast response.
Biotinylatedaptamerhas been successfully immobilised onto streptavidin coated PEDOTCNT matrix. Characterisation studies like FT-IR, FE-SEM and electrochemical
characterisation (DPV) studies were done to confirm step-wise fabrication of the
aptaelectrode. Different parameters involving the aptamer concentration, binding time and
response time with the target have been optimised. The electrochemical response study of
aptaelectrode treated with a wide range of target protein was done by DPV. Results: The
electrochemical response study of aptaelectrode treated with a wide range of target protein
was done by DPV. There was increase in current response with increase of target
concentration as the 3-D complex formed improved the electron flow as comparison to sole
aptamer immobilisation. Conclusion: The detection limit of the fabricated bio-electrode has
been reported to be 0.01ng/ml. The aptaelectrode showed more than half a month stability
along with good regeneration and repeatibilty, thus establishing its potential in the field of
diagnosis in the future.
Keywords: Aptaelectrode; Biotin-Streptavidin interaction; CNT; DPV; PEDOT.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
136

Abstracts (Poster Presentations)

Cloning, Over-expression, and Purification of Hfq Protein from Klebsiella
pneumoniae
Devarubini K.1, Kishan S.1, Suresh V.C.1*
1
AIMST University, Department of Biotechnology, Faculty of Applied Sciences, Bedong,

08100, Kedah, Malaysia, *corresponding author, e-mail: cvsureshgupta@gmail.com, Ph.
No: +604-4298165.
Abstract: Background: Hfq is a RNA chaperone present in Klebsiella pneumonia and many
other bacteria. It plays a vital role in virulence of the bacteria. Hfq exhibits its function by
binding with npcRNA which will be needed for the trans-acting npcRNA, mRNA interaction.
To gain further insights on the target bacterial npcRNAs that interact with this RNA
chaperone, highly pure Hfq protein is needed. Methods: Hfq gene from Klebsiella
pneumonia was amplified and cloned into pET-28b+ vector. Ligated mixture was
transformed into TOP-10 cells. The transformed bacterial colony with recombinant plasmid
was screened by antibacterial (Kanamycin) selection and confirmed by PCR methods. The
recombinant plasmid was transformed into E.coli BL21 and induced the expression with
IPTG. The over expressed Hfq protein was purified using Ni-NTA affinity chromatography.
Results: Hfq gene had been successfully amplified, digested with appropriate restriction
enzyme and cloned in to pET-28b+ vector. Recombinant plasmid was successfully
transformed into TOP10 and BL21. A highly purified Hfq protein was obtained and
confirmed by SDS PAGE analysis. The best elution of the Hfq protein was actualized using 1
M of imidazole. Conclusion: In this study we successfully purified Hfq protein from
Klebsiella pneumonia for further RNA binding study to select the bacterial npcRNA that
binds to the protein for further identification and characterization studies.
.Keywords: Hfq recombinant protein; Klebsiella pneumonia; Ni-NTA.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
137

In vitro Anti-oxidant Assay, HPLC Profiling of Polyphenolic Compounds,

AAS and FTIR Spectrum of Malaysian Solanum torvum Swartz fruit
Sathyanarayana N.a, *, Sunitha P.b, Suresh V.C.c, Sreeramanan S.d., Marimuthu K.c, and
Xavier R.c
a
Unit of Anatomy, b Unit of Physiology, Faculty of Medicine, c Faculty of applied sciences,

Department of Biotechnology, AIMST University, Semeling, Bedong, Malaysia, d School of
Biological Sciences, UniversitiSains Malaysia, Penang, Malaysia; *corresponding author, email: drsatyanarayana.aimst@gmail.com, Ph No: +6 010-5600574
Abstract: Background: Solanum torvum Swartz, a medicinal plant of Solanaceae family and
is used in traditional systems of medicine. Since, the plant is widely used as a medicinal plant
among the Malaysian population it is important to understand the phytochemical content. The
aim of this study is to determine the phytochemical content and antioxidant potential of the
fruits of Solanum torvum. Methods: The phytochemical analysis was carried out following
standard protocol with the aqueous, ethanolic and methanolic extracts of S. torvum fruit.
Anti-oxidant potential of S. torvum fruit was evaluated by DPPH, FRAP and HPLC methods.
Elemental and functional group analysis were done by Atomic Absorption
Spectrophotometry (AAS) and Fourier Transform Infrared Spectrophotometry (FTIR)
methods respectively. Results: Quantitative assessment of total phenols and flavonoid
content, DPPH and FRAP assay was done in the ethanolic extracts of S. torvum. Qualitative
analysis of each extract showed the presence of reducing sugars, saponins, alkaloids, phenols
and flavonoids except anthraquinones. Quantitative determination of total phenols and
flavonoids showed 16.4 mg GAE/g and 2.8 mg QE/g respectively. In DPPH radical
scavenging assay, the IC50 value of the extract was found to be 1.62 mg/ml and the FRAP
value was found to be 470 mg FeSO4 E/g. The High Performance Liquid Chromatography
(HPLC) analysis revealed presence of polyphenolic compounds such as gallic acid, rutin,
quercetin and ascorbic acid. Elemental determination by AAS showed the presence of
essential elements such as Calcium (Ca), Copper (Cu), Iron (Fe), Manganese (Mn), Lead
(Pb), Zinc (Zn), Nickel (Ni), Magnesium (Mg), and Sodium (Na). FTIR results showed the
presence stretching vibrations of OH groups in phenyl, CH2 asymmetric stretch of methyl
groups, C-O stretching vibrations ring of phenyls, CH bending vibration. With this it is
concluded that S. torvum fruits are a rich source of antioxidants. Conclusion: S. torvuma wild
Malaysian medicinal plant fruit ethanolic extract possesses good amount of phenols and
flavonoids responsible for the antioxidant property.
Keywords: AAS; Antioxidant property; FTIR; HPLC; Polyphenolic compounds.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
138

Phytochemical Analysis and Antioxidant Activity of Malaysian Medicinal

Plant Abroma augustum Leaf Extract
Sunitha P.1, *, Sathyanarayana N.1, Suresh V.C.2, Sreeramanan S.3, Sam A.1 and Xavier R.2
1
Faculty of Medicine, AIMST University, Semeling, Bedong, Malaysia

Faculty of Applied Sciences, Department of Biotechnology, AIMST University, Semeling,
Bedong, Malaysia
3
School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia;
*corresponding author, e-mail: drsunitha33@gmail.com,
Ph No: +6016-4542155
Abstract: Background: Antioxidants from plant materials terminate the action of free
radicals thereby protecting the body from various diseases. Phenolic compounds from
medicinal plants possess strong antioxidant activity and may help to protect the cells against
the oxidative damage caused by free-radicals. Hence, the present study is aimed at to evaluate
the phytochemical content, antioxidant activity, functional group and elemental analysis of
the leaves of A. augustum of Malaysian origin. The investigation on the phytochemical
analysis of A. augustum is the pioneering study and the results will form the basis for
explaining the medicinal properties of this plant. Methods: Antioxidant potential and
polyphenolic compounds of A. augustum were evaluated by DPPH, FRAP assays and HPLCPDA method respectively. Elemental analysis and Functional group analysis were done by an
Atomic Absorption Spectrophotometry (AAS) and Fourier Transform Infrared
Spectrophotometry (FTIR) methods. Results: Qualitative analysis of each extract showed the
presence of reducing sugars, alkaloids, tannins, phenols and flavonoids. Quantitative analysis
of total phenolic and flavonoid content showed 15.76 mg/g GAE and 8.6 mg/g QE of A.
augustum leaf extract respectively. IC50 value of the extract was found as 790g/ml by DPPH
free radical scavenging assay. In the FRAP assay, the ethanolic leaf extract showed 367.6
mg/g FeSO4 equivalants. High performance liquid chromatography (HPLC) of the leaf extract
revealed the presence of polyphenolic compounds such as gallic acid, quercetin and ascorbic
acid. FTIR results showed the presence stretching vibrations of OH groups in phenyl, C-H
asymmetric stretch of methyl groups, C=O stretching vibrations ring of phenyl, CH bending
vibration. Presence of essential elements was detected by AAS. Conclusion: Abroma
augustum is a rich source of polyphenolic compounds (phenols and flavonoids) and
antioxidants which might be responsible for the unexplored medicinal properties of A.
augustum.
Keywords: AAS; A. augustum; Antioxidant potential; FTRI; HPLC-PDA; Polyphenolic
compounds.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
139

Reduced Reproductive Function up to Three Generations of Rats Due to

Paternal Heroin Addiction
M. Z.Farah Naquiah 1, R. J. James 1,2 *, M. I. Mohd Hafidz 2, M. Z Salleh 1,2, L.S. Lee 1, S.
Suratman 2, L. K. Teh 1,2 *
1
Integrative Pharmacogenomics Institute (iPROMISE), Level 7, FF3, Universiti Teknologi

MARA Selangor, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia.
2
Faculty of Pharmacy, Universiti Teknologi MARA Selangor, Puncak Alam Campus, 42300
Bandar Puncak Alam, Selangor. Malaysia; *corresponding author, e-mail:
tehlaykek2016@gmail.com, and ritchjj@yahoo.fr
Abstract: Background: Little is known about the long-term consequences of heroin

addiction on either the user or his future offsprings. In this study, we look into the effects of
paternal heroin exposure on the reproductive capacity and the weights of their progenies up to
three generations. Methods: Male Sprague Dawley rats (6-weeks-old) were divided into: (1)
heroin addiction induced rats (F0-H) and (2) control group treated with saline solution (F0C). Heroin or saline solution was administered intraperitoneally twice-daily for fourteen days
with increasing dosage regimen. The dosage regime started with the lowest dose of 3 mg/kg
per day of heroin followed by 1.5 mg/kg increments per day to a final dose of 13.5 mg/kg per
day for 14 days. Results: Administration of heroin in the sires resulted in a statistically
significant smaller number of litters with a 40% reduction in the size. However, this pattern
was not observed in the second and third generations. Weights of the litters were measured on
day 21 and day 90 (adult). There was no significant difference in the body weights of the rats
when compared between the control and heroin treated groups. However, on day 90, the F0H group had a lower average of weight when compared to the control group. This pattern was
also seen in the F1 and F2 generations but not in the F3 generation. Conclusion: Our results
suggest that paternal addiction to heroin caused reduction in the size of the litters and lower
body weight which is transmitted up to two generations.
Keywords: Adolescent;
Transgenerational; .
Heroin
addiction;
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Opiates;
Opioid;
Paternal
addiction;
140

Optimization of Cryopreservation Using Different Cryoprotective Agents

and Differential Temperatures on Freeze Dried Probiotics
Hassan Pyar1, 2 and K.K Peh*3
1
Faculty of Pharmacy, AIMST University, Semeling, 08100, Bedong, Kedah Darul Aman,
Malaysia; 2Hadramout University of Science and Technology, Mukalla, Hadramout, Yemen;
3
School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang,
Malaysia; *corresponding author, e-mail: kkpeh@usm.my, Ph No: +604-6533888
Abstract: Background: The use of probiotics in biotechnological applications has steadily

increased over the past decades. However, these beneficial microbes were losing viability or
activity during the preservation process. To avoid cellular damage during cryopreservation
and subsequent thawing, a wide array of cryoprotective agents has been applied. Methods:
Investigation was done on the viability and stability of probitic and the effect of different
cryoprotective agents (namely, sodium chloride, sucrose, dextran, sorbitol, monosodium
glutamate, glycerol, skim milk and skim milk with malt extract) with modified De-Man
Rogosa Sharpe (MMRS) medium were examined. Results: Commercial De-Man Rogosa
Sharpe (MRS) medium was proved to be more expensive than the modified MRS medium
with relatively low yield of probitic. Significantly high viable counts were achieved with
monosodium glutamate, skim milk and skim milk with malt extract, with optimum
concentration at 0.3% w/v. There was a reduction in cell viability at concentration above
0.5% w/v, which could be attributed to cell shrinkage associated with osmotic pressure
changes inside the cells. Probitic Lactobacillus species was found to be stable at room
temperature (28C) for eight weeks. A significant growth of probiotics was produced from
skim milk. Conclusion: modified MRS medium with skim milk is suggested for the
remarkable growth and yield of Probitic lactobacilli.
Keywords: Cryoprotective agents; De-Man Rogosa Sharpe (MRS); Probiotics.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
141

Development of a Reusable Electrochemical Immunosensor for Direct

Detection of Small Organic Molecules
Khoo M. M.1, Yatimah A.1, and Khor S. M.1*
1
Department of Chemistry, Faculty of Science, University of Malaya, 50603, Kuala Lumpur,

Malaysia; *corresponding author, e-mail: naomikhor@um.edu.my,
Ph No: +603-79677022/Ext: 2520
Abstract: Background: In order to reduce costs and waste produced, an electrochemical

immunosensor, which able to perform multiple measurements without sensor detection
ability loss, has attracted the interest of many researchers. Reusability of immunosensor
depends on the rapid reversible interaction between antibody and antigen. Dissociation of
antibodies from the immunosensor surface gives the detection signal for target analyte.
Exposure of the used immunosensor surface to antibody will give a new detective
immunosensor surface. The use of electrode polarization can help to regenerate
immunosensor surface and also eliminate the non-specific protein absorption to
immunosensor surface. In this study, the main objective was to develop a reusable and nonfouling surface for electrochemical biosensor applications. Methods: A combination of aryl
diazonium
salt
with
zwitterions
such
as
sulfanilic
acid
and
(4aminophenyl)trimethylammonium were deposited onto the electrode surface. These
zwitterions molecules are chemically stable, less subjected to oxidation and low impedance.
Besides, gold nanoparticles were employed to lower the impedance and increase the sensor
detection area. For regeneration, a constant potential of -800 mV was applied to the
immunosensor interface for 10 min to remove the surface-bound antibodies. Results: For
optimization, the electrode polarization of -800 mV at the 10-min interval time was
recognized as the optimum condition to yield the highest sensor sensitivity.The antigenantibody binding was found to be reversible with the aid of the electrode polarization, after
five replicate measurements (with an RSD of 9.14%) performed within the same day at
ambient temperature (25C). The good blocking of Fe(CN)63-/4- shown in cyclic
voltammograms indicated that the immunosensor surface was not physically damaged after
multiple times of surface regeneration. Besides, the lowest detection limit was improved from
100 ng mL-1 to 1 ng mL-1 with the aid of electrode polarization. Conclusion: This biosensor
interfacial design is suitable to be used for repeated electrochemical immunosensor use.
Keywords: Electrochemicalimmunosensor; Protein-surface interactions; Reversible affinity
interactions; Reusable biosensor; Surface regeneration.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
142

Over-expression and Purification of an RNA Chaperone, Hfq Protein of

Proteus mirabilis
Kishan S.1, Citartan M.2, Prabu S.2, Tang T.H.2, Suresh C.V.1*
1
Faculty of Applied Sciences, Department of Biotechnology, AIMST University, 08100

Bedong, Kedah, Malaysia.
2
Advanced Medical & Dental Institute, Universiti Sains Malaysia, 13200 Bertam, Kepala
Batas, Penang, Malaysia;
*corresponding author, e-mail: cvsureshgupta@gmail.com,
Ph. No: +604-4298165
Abstract: Background: Hfq, an RNA chaperone plays a vital role in virulence of various
bacteria including P. mirabilis. Hfq exhibits its function by binding with npcRNA which will
be needed for the trans-acting npcRNA, mRNA interaction. To gain further insights of the
regulating npcRNAs that interacts with this RNA chaperone, pure form of Hfq protein and a
standardized Hfq-npcRNA binding protocol need to be established. Methods: Hfq gene from
P. mirabilis was amplified and cloned in to pET-28b(+) vector and then transformed into
TOP10 cells. The bacterial with recombinant plasmid was screened by antibacterial selection
and confirmed by sequencing. The recombinant plasmid was transformed into E. coli BL21
and induced the expression with IPTG. The over expressed Hfq protein was purified using
Ni-NTA affinity chromatography. Total RNA from P. mirabilis was extracted using TRIzol
and verified by BioAnalyzer. A protocol was standardized for Hfq-npcRNA binding using
Nitrocellulose membrane affinity. Results: Hfq gene had been successfully cloned into pET28b(+) vector and transformed into TOP10 and BL21. A highly purified Hfq protein was
obtained and confirmed by SDS PAGE. The good intact of total RNA was extracted from P.
mirabilis. The first trial of the recombinant protein Hfq and total RNA binding assay was
carried out and the protein bound RNAs were precipitated and sent for Next-Generation
Sequencing. Conclusion: In this study we successfully purified Hfq protein from P.
mirabilis. Hfq bound RNAs were sent for Illumina sequencing to identify the npcRNAs and
their partners involved in Hfq mediated trans-gene regulation.
Keywords: Hfq; Nitrocellulose membrane; P. mirabilis; Recombinant protein.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
143

Peritoneal Mast Cell Stabilization and Toxicological Properties of the

Ethanolic Extract of Solanum trilobatum Linn.
Lee Yu Ren, Stephanie Wong Kah Yee, Bobby Lau Chik Chuon, Christapher Parayil
Varghese, Subramani Parasuraman*
*corresponding author, e-mail: parasuraman@aimst.edu.my,
Ph No: (+60) 103895096
Abstract: Background: Solanum trilobatum Linn., (Solanaceae) is known as thoodhuvalai

in Tamil. The leaf of S. trilobatum is commonly used as a food supplement by South Indians,
the aqueous extract from same plants has been used for the treatment of respiratory illness. In
pre-clinical studies, the extract of S. trilobatum showed hepatoprotective, antimicrobial,
larvicidal and anticancer activities. In few clinical studies, the extract of S. trilobatum showed
bronchiolitic effect. The antihistaminic effect and toxicity profile of S. trilobatum remain
unclear, hence the present study has been planned to carry out the peritoneal mast cell
stabilization activity and chronic toxic effect of ethanolic extract of S. trilobatum (EEST) in
Sprague Dawley (SD) rats. Methods: The SD rat intestinal mesentery was used to study the
peritoneal mast cell stabilization of EEST. The rat intestinal mesentery was exposed to 50,
100, 200, 300, 400 and 600 mg/ml of EEST and the peritoneal mast cell stabilization activity
was compared with that of chlorpheniramine. In chronic toxicity testing, rats were treated
once daily with 100, 200 and 400 mg/kg of EEST for 30 days. During the study, animals
behaviour and biochemical alterations were observed at regular intervals. At the end of the
study, rats were sacrificed and their organs were collected for histopathological analysis and
part of brain was preserved at-80C for dopamine assay. Result: EEST showed significant
antihistaminic activity at the dose of 300 mg/ml onwards and the effect was comparable with
that of standard. In chronic toxicity testing, EEST significantly reduced the immunization
time, locomotor activity and increased the dopamine (results were not significant) levels in
brain tissue. In histopathological analysis, EEST showed marked changes in brain, liver and
kidney. Conclusion: EEST showed significant antihistaminic activity at 300 mg/ml onwards
and had mild to moderate toxic effect at 200 mg/kg onwards.
Keywords: Antihistamine; Solanum trilobatum; Toxicology.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
144

Understanding the Host-Pathogen Interaction in Klebsiella pneumoniae

Infected Rat Model via Metabolomics Approaches
Mohd Izwan Mohamad Yusof1,2, Mohd Salleh Rofiee2, Teh Lay Kek2, Mohd Zaki Salleh*2
1
Integrative Pharmacogenomics Institute (iPROMISE), Level 7, FF3 Building, Universiti

Teknologi MARA Selangor Branch, Puncak Alam Campus, 42300 Puncak Alam, Selangor,
Malaysia.
2
Faculty of Pharmacy, Universiti Teknologi MARA Puncak Alam Campus, 42300 Puncak
Alam, Selangor, Malaysia; *corresponding author, e-mail: tehlaykek@yahoo.com, Ph No:
(+60) 332584652
Abstract: Background: Bacteremia can be defined as the presence of viable bacteria in the
bloodstream which can lead to multiple organ failures if managed incorrectly. To better
understand the interaction between pathogen-host metabolic response, we investigated the
metabolic consequences of a Klebsiella pneumoniae infection in vivo via metabolomics
approaches. Methods: K. pneumoniae was intravenously injected into rats, and serum
samples were collected at three different time points (0 hours (pre-infection), 2 hours after
infection (early infection) and 192 hours after infection (post-infection). Results: Fifteen (15)
metabolites were characterized as potential biomarkers related to K. pneumoniae infection.
The identified potential biomarkers were derived from nine (9) pathways which were found
significantly perturbed during K. pneumoniae infection. Tryptophan metabolism was the most
prominently influenced in K. pneumoniae-induced bacteremia according to the metabolic
pathway analysis (MetPA), suggesting that significant modulation of immune system activity
occurred during early infection of K. pneumoniae. In addition, we also capture several
metabolites that represent the host is in oxidative stress, inflammation, and high energy
demand state during early infection of K. pneumoniae. Conclusion: Our findings provide a
novel perspective on the metabolites signatures together with perturbed pathways in related to
bacteremia, which provided us with new insights into the pathogenesis of bacteremia, and the
discovery of targets for clinical diagnostic and treatment.
Keywords: Bacteremia; Klebsiella pneumoniae; Metabolomics.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
145

New PDE4 Inhibitors: Design, ADMET and Docking Studies on Chalcones

and Flavones for Anti-Inflammatory Activities
Idris M. H. M.1,2, Siti Norhidayu Mohd Amin1,2, Siti Norhidayah Mohd Amin1,2, Manikandan
Selvaraj1,2, Mohd Zaki Salleh1,2 and Teh Lay Kek*1,2
1
Integrative Pharmacogenomics Institute (iPROMISE), Level 7, FF3 Building, Universiti

Teknologi MARA Selangor Branch, Puncak Alam Campus, 42300 Puncak Alam, Selangor,
Malaysia.
2
Faculty of Pharmacy, Universiti Teknologi MARA Puncak Alam Campus, 42300 Puncak
Alam, Selangor, Malaysia; *corresponding author, e-mail: tehlaykek@yahoo.com, Ph No:
(+60) 332584652
Abstract: Background: Phosphodiesterase type 4 (PDE4) regulates cyclic adenosine

monophosphate (cAMP) which acts as intracellular secondary messenger by hydrolysis.
Selective inhibition of PDE4 therefore elevates cAMP which then downregulate
inflammation. Chalcones and flavones with anti-inflammatory properties which inhibit PDE4
are potential lead compounds as anti-inflammatory and analgesic agents. Methods:
Computational studies were undertaken to test the inhibitory scaffold of 18 synthesized
chalcones and flavones on PDE4. Structures of chalcones and flavones were drawn using
Marvin Sketch 16.2.8. Protein crystal structure with rolipram (PDB ID: 1OYN) was retrieved
from Protein Database Bank (PDB), US. Docking study was performed using Glide 6.9.
ADMET properties of the compounds were calculated using QikProp and ACD/I-Lab.
Results: The docking result of chalcones showed that the binding energies were in the range
of -4.258 kcal/mol to -10.209 kcal/mol. For flavones, the range of binding energies were 5.282 kcal/mol to -7.552 kcal/mol. However, five (5) out of the fifteen (15) chalcones and
one (1) of the three (3) flavones showed good binding pose as compared to rolipram. For
druglikeness properties, ten (10) chalcones and three (3) flavones fulfilled the Lipinskis
Rule of Five criteria while four (4) chalcones and one (1) flavone were predicted to cause
genotoxicity. In addition, eleven (11) chalcones and three (3) flavones were predicted to have
good ADMET properties. Conclusion: Based on the binding energies, chalcones and
flavones are potentially new PDE4 inhibitors.
Keywords: Anti-inflammatory; Cyclic adenosine monophosphate; In silico.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
146

Effect of Telfaira occidentalis in Mice Fed Aflatoxin Contaminated Feed

Ndanusa Abdullahi Hassan1*, T.A Gbodi2, Rohini Karunakaran1, Uma Sankar A1, Khin Mar
Aye1, Ahmad Alhaji2, Umar Muazu2
1
Unit of Biochemistry, Faculty of Medicine, AIMST University, Malaysia.

Department of Biochemistry, Ibrahim Badamasi Babangida University, Lapia, Niger state,
Nigeria; *corresponding author, e-mail: ndanusabb@gmail.com, Ph No: 0149321340
Abstract: Background: Aflatoxin is potent hepatotoxic and hepatocarcinogenic agents. This

hepatotoxicity is thought to be mediated by its ability to generate reactive oxygen species and
cause peroxidative damage. This study investigated possible effect of pumpkin (Telfaira
occidentalis) in ameliorating the toxic effect of aflatoxin using animal model. Methods:
Twenty albino mice were procured, grouped into four of five groups each and allowed to
acclimatize for one week. Aflatoxigenic Aspergillus flavus, cultured groundnut cake was used
for oral aflatoxin exposure. Group 1 served as the control and fed commercial feed. Group 2
received 2.5g of aflatoxin contaminated diet and commercial feed. Group 3 received
2.5g+0.1g of T.occidentalis leaves powder+ commercial feed. Group 4 received 2.5g+0.2g of
T.occidentalis leaves powder+ commercial feed. Group 5 received only 0.1g of T.occidentalis
leaves powder+ commercial feed. The diet was administered daily for the period of 3weeks.
Blood glucose test was carried out at the end of each week using acute check active
glucometer. At the end of the experiment blood samples were collected from the mice
through ocular puncture into clean containers. The samples were analyzed for liver function
test (Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline
phosphatase (ALP), Total protein, Albumin, Bilirubin.) and renal function test (RFTs) (Urea,
Sodium, Potassium, Chloride, creatinine). Results & Conclusion: From the results, there was
significant (p<0.05) decrease in blood glucose levels of all the groups of animals after week 2
and week 3 as compared to the control group 1. There was significant difference in renal
function test values of urea, sodium, potassium and creatinine in all the groups and there was
no significant difference between group 2, 3, 4 and 5 of chloride as compared to the control
group 1. Also there was significant difference between AST, ALT, TP, ALB, and BL. The
significant changes in the above mentioned parameters were likely to be due to toxic effect of
aflatoxin contaminated feed. From the findings of these studies, T. occidentalis may have a
potential to reduce the toxic effect of aflatoxin in diet.
Keywords: Aflatoxin; Antioxidant; Aspergillus flavus; Telfairia occidentalis.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
147

Accuracy of Rapid Point-of-Care Diagnostic Tests for Acquired Immune

Deficiency Syndrome - A Systematic Review and Meta-analysis
Paramasivam R.1,*, Veeramachineni A.K.1, Janarthanan P.1, Sathasivam T.2, and Langford
S.J.1
1
School of Science, Monash University Malaysia, Jalan Lagoon Selatan, 46150 Bandar
Sunway, Selangor Darul Ehsan, Malaysia.
2
School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, 46150 Bandar
Sunway, Selangor Darul Ehsan, Malaysia; *corresponding author, e-mail:
rpar21@student.monash.edu.my, Ph No: (+60)1111887169
Abstract: Background: Rapid point-of-care tests provide a feasible diagnostic strategy for
HIV detection in low resource areas. However, rapid assays are relatively fallacious than
conventional ELISA and Western blot technique. We performed a systematic review to
assess the diagnostic accuracy of multiple point-of-care rapid assay platforms for HIV
detection according to standard methods and summarized test performance using metaanalysis. Methods: A computer-aided search of MEDLINE (1950-March 2016), EBSCO
(1966-June 2016), OVID database (1966 to January 2016), and EMBASE (1974- January
2016) was performed for relevant publications. Reports meeting inclusion criteria of rapid
assays performed with serum, oral and urine samples for antigen and/or antibody detection of
HIV was assessed for methodological quality by using the QUADAS 2. Meta-DiSc, a
Windows-based, user-friendly, freely available software was used to perform and validate
diagnostic meta-analysis. Results: Out of 2724 citations which were identified and screened
from four databases, 86 rapid assays which met the inclusion criteria were included in the
study. Four themes were identifies 1) serological assays yield a higher pooled sensitivity and
specificity than urine or oral saliva-based assays, 2) antibody + antigen detecting combination
assays are relatively better than antigen or antibody detection assay, 3) Multi-antigen assays
yield a better pooled sensitivity and specificity than single-antigen or whole cell antigen
assays and 4) HIV 1/2 detecting combination assays are relatively better than HIV 1 and HIV
2 detection assays. Conclusion: Most reported studies are conducted on small sample number
which misleads the diagnostic accuracy of the assay, this is overcome by understanding the
pooled sensitivity and specificity using meta-analysis of the reported studies. It is clear that
serological assays that can detect both HIV 1/2 antibody and antigen will yield higher pooled
sensitivity and specificity.
Keywords: Acquired immune deficiency syndrome; Meta-analysis; Meta-disc; Rapid assay.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
148

Biocontrol of Macergen Infestation on Plants using Bacteriophage Cocktail

Sasireigga J.*, Ravichandran M., and Kurunathan S.
Bedong, Kedah, Malaysia; *corresponding author, e-mail: sasi7278@gmail.com, Ph No:
+60194451285.
Background : Macergens is a term used to describe bacteria capable of releasing pectic

enzymes that degrades the plant cell wall which leads to maceration, a symptom associated to
soft rot disease. Among these macergens, Dickeya chrysanthemi has been reported to cause
stem rot disease in several important crops such as potato, tomato, lettuces and papaya. As
bacteriophages are well known potent biocontrol agents, we were interested in isolating and
examining the efficiency of novel bacteriophages in controlling the progression of soft-rot
disease in plants infected by D. chrysanthemi. This has been our interest as there has been
limited studies on isolation of bacteriophage and its application against D. chrysanthemi
infected plants. Methods : Bacteriophages were isolated from soil and water sample by
primary enrichment method. Host range of bacteriophages was assessed using spot tests.
Biocontrol test was performed on three weeks old young seedlings of chill, papaya and
tomato. The young seedlings were inoculated by syringe infiltration with D. chrysanthemi
bacterial cell suspensions after wounding the stems with sterilized scalpers. Bacteriophage
cocktail were applied on the young seedlings by spraying method after infected with D.
chrysanthemi. Results: Five lytic bacteriophages were isolated against D. chrysanthemi. All
the five bacteriophages showed high specificity against D. chrysanthemi. Interestingly, stem
rot disease caused by D. chrysanthemi on these plants were greatly reduced by cocktail of
bacteriophages within 24 h. It was also observed that the growth of plants treated with
bacteriophage cocktail after D. chrysanthemi infection were similar with untreated sample
and it had no stem rot symtoms were recorded. Conclusion: This study had successfully
isolated bacteriophages that have the potential to be used as a biocontrol agent to prevent
stem rot disease caused by D. chrysanthemi.
Key words: Bacteriophages; Biocontrol; Dickeya chrysanthemi; Macergen.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
149

Oral Bacterial Diversity Study in Malay Ethnic Group in Malaysia

Kah Man Woh1, Kameswari K.2, Sivakumar P.2, Lay Kek Teh3, Moh Zaki Salleh3, Subhash J
Bhore*1
1

Semeling Road, Semeling 08100, Kedah, Malaysia; 2Faculty of Dentistry, AIMST University,
Bedong Semeling Road, Semeling 08100, Kedah, Malaysia; 3Integrative Pharmacogenomics
Institute (iPROMISE), Universiti Teknologi MARA, Puncak Alam Campus, 42300 Puncak
Alam, Selangor DE, Malaysia; *corresponding author, e-mail: subhash@aimst.edu.my,
subhashbhore@gmail.com, Ph No: (+60) 4 429 8176
Abstract: Background: Human body contain several indigenous microorganisms that vary
at different anatomical sites. Human oral cavity is one of the most diverse sites of
microorganisms. It is estimated that approximately 500 to 700 different bacterial species
might be existing in human oral cavity. Malaysia is a multi-racial country with different
lifestyles, cultures and eating habits. In Malaysia, Malay is a major ethnic group and limited
information is available about this communitys oral bacterial diversity. Therefore, this
research project was undertaken. The objective of this research project was to elucidate the
oral bacterial diversity among healthy subjects of Malay community. Methods: Based on
consent, eleven (11) healthy subjects were selected randomly from Malay community. From
oral cavity of selected subjects, saliva and sub-gingival plaques samples were collected.
Separately, saliva and sub-gingival plaques samples were pooled together and bacterial DNA
samples were prepared using kit. Metagenomics approach was used to elucidate DNA
sequence based identities of the bacteria using Illumina, a next generation sequencing (NGS)
platform. Results: The primary analysis of results indicates that 441 types of bacteria were
present in the saliva samples of Malay subjects. The analysis of the data generated from subgingival plaques samples suggest that 325 types of bacteria were embedded in the subgingival plaque samples. Data analysis suggests that 166 bacterial species were common in
saliva and sub-gingival plaque samples. Conclusion: Based on primary analysis of the results
data, we conclude that about 600 different types of bacteria are harboured in the oral cavity of
the healthy subjects from the Malay community of Malaysia. Our research findings clearly
elucidate the oral bacterial diversity and these finding could serve as foundation for the
further study in understanding the connection between oral bacterial diversity and the oral
health or overall health of the individuals.
Keywords: 16S rRNA; Bacteria; Human oral cavity; Illumina; Malay ethnic group;
Ribosomal DNA; Saliva; Sub-gingival plaque.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
150

Isolation and Characterization of Seven Lytic Bacteriophages As

Candidates for Phage Therapy
Sanirbandha C., Vickneswaran N.M., Sivachandran P., Ravichandran M., Lee, S.Y.*
Department of Biotechnology, Faculty of Applied Sciences, AIMST, University, 08100
Bedong, Kedah, Malaysia; *corresponding author, e-mail: su_yin@aimst.edu.my, Ph. No:
+604-4298177
Abstract: Background: Bacteriophages are bacteria-specic viruses that can infect and
destroy their host bacteria. Phage therapy has been used to combat bacterial infections. In
order to understand the mechanism in which bacteriophage uses its lytic enzymes to kill its
host, we have isolated and characterized 7 novel bacteriophages from various environmental
samples. Methods: Water and soil samples from different environmental sources were used
to isolate bacteriophages. The samples were processed through membrane filtration and
eventually spotted on a range of bacterial strains to identify the possible host. After
enrichment with the bacterial host for 72 hours, the bacteriophage was recovered by
membrane filtration. Morphological characterization of each bacteriophage was performed
using transmission electron microscopy (TEM) to determine the morphology and the type of
nuclear material was also determined by S1 nuclease and DNase digestion. Results: Seven
different bacteriophages were isolated from the environmental samples. The phages were
found to be specific against Salmonella paratyphi A (SPA-S), Salmonella paratyphi B (SPBS and SPB-V), Salmonella paratyphi C (SPC-S and SPC-V), Salmonella typhi (ST-S) and
Citrobacter freundii (CF-S). Nuclease digestion revealed that SPA-S, SPC-S, ST-S, CF-S,
SPB-V, SPC-V were double-stranded DNA phages, while SPB-S was a single-stranded DNA
phage. TEM analysis showed that the six double-stranded DNA phages had T4 or phagelike structure with non-enveloped contractile tail, while the single-stranded DNA phage
(SPB-S) was seen to be non-enveloped rod-shaped in structure. Conclusion: The results of
this study paves the way for further studies into whole genome sequencing of the
bacteriophages and to understand the function of specific genes, such as lysin that can be
used for phage therapy in the future.
Keywords: Bacteriophage; Enrichment; Environmental samples, Isolation; TEM.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
151

Transcriptome Analysis for the Identification of Novel ncRNAs in

Acinetobacter baumannii
Saw H. S., Sumitha S., Kishan Raj S., Xavier R., Suresh V.C.*
Semeling Road, Semeling 08100, Kedah, Malaysia;
*corresponding author, e-mail: cvsureshgupta@gmail.com, Ph. No: +604-4298165
Abstract: Background: Multidrug-resistant Acinetobacter baumannii is recognized to be

among the most difficult antimicrobial-resistant Gram-negative bacilli to control and treat.
Study of ncRNA of A. baumannii would shed light on the unexplored regulation in
understanding the pathogenicity of this organism. In general, recent reports indicated that
ncRNA plays important role in regulation of metabolic pathways, gene expression and
pathogenicity of several bacteria. Methods: In our study, we used A. baumannii
transcriptome data from NCBI to identify the novel ncRNA candidates. In parallel, we also
performed genome wise search for ncRNA genes using computational prediction software
nocoRNAc. The transcriptome data of A. baumannii was analyzed through the genome
viewer Artemis to screen the un-annotated transcripts, which were further analyzed for the
absence of ORF and having no hit in Rfam and Genbank database. The RPKM value and
read count of the transcripts were also calculated by creating ncRNA candidates gff file.
Results: Total 637 ncRNA transcripts are predicted by nocoRNAc. Transcriptome data
disclosed 50 possible ncRNA candidates. Among these, 5 could be possible novel protein
coding genes as they are possessing possible novel ORFs. Finally 13 were identified as the
potential ncRNA candidates, which are further validating their expression by Northern blot
analysis. Conclusion: In this study we identified 13 novel ncRNAs in A. baumannii and
validation of their expression is under progress. This could serve as foundation for further
understanding of the gene regulation in this bacteria.
Keywords: Acinetobacter baumannii; Bioinformatics; Non-coding RNA; Transcriptome.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
152

Computational Modelling of the Newly Synthesized Chalcone Derivatives

in Inhibiting 5-lipoxygenase
Siti Norhidayah M.A., Teh L.K., Manikandan S. and Mohd Zaki S.*
Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA Puncak
Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia; *corresponding author, email: zakisalleh@puncakalam.uitm.edu.my, Ph No: (+603) 32584652.
Abstract: Background: 5-lipoxygenase (5-LOX) is an enzyme that is involved in

inflammation. Thus, inhibiting this enzyme will reduce undesired inflammatory reaction.
Currently, the commercially available 5-LOX inhibitor is Zileuton, but it is less widely
prescribed due to its side effects. Therefore, this study aims to design new alternative 5-LOX
inhibitor using molecular docking approach. Methods: 5-LOX crystal structure was retrieved
from Protein Database Bank. The structures of the compounds which are derivatives of
chalcones synthesised in-house were drawn using Marvin Sketch. The active site of 5-LOX
enzyme was predicted using SiteMap. GOLD and AutoDock softwares were used to
investigate the parameters of the complex of synthetic chalcones with 5-LOX enzyme. The
docking results were compared with the standard reference ligand (Zileuton). Results: 10
(ten) out of 15 (fifteen) synthetic chalcones had higher fitness score and lower binding energy
compared to the standard reference ligand when docked with 5-LOX. The fitness scores
shown by GOLD ranged from 54.19 to 66.21. On the other hand, the binding energies of the
compounds as shown by AutoDock were within the range of -8.98 to -5.76 kcal/mol.
Conclusion: Ten newly synthesised derivatives of the chalcones have potential as 5-LOX
inhibitor. The in vitro and in vivo studies elucidating the mechanism of the compounds are
currently being carried out.
Keywords: Bioinformatics; In vitro; Lipoxygenase chalcone; Molecular docking.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
153

Computational Design of Flavone and Chalcone Derivatives as

Cyclooxygenase-2 (COX-2) Inhibitor
Siti Norhidayu M.A., Teh L.K., Manikandan S. and Salleh, M.Z.*
Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA Puncak
Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia; *corresponding author, email: zakisalleh@puncakalam.uitm.edu.my, Ph No: (+603) 32584652
Abstract: Background: Cyclooxygenase-2 (COX-2) is an enzyme responsible for the

conversion of prostaglandin H2 to prostanoids; which are the crucial mediators of
inflammation. Inhibition of the COX-2 enzyme is reduced or inhibited the proinflammatory
enzyme activity. We aim to evaluate the inhibitory efficiency of chalcone and flavone
derivatives for COX-2 and to identify compounds that are selective to COX-2. Methods: In
the present work, we evaluated the interaction of compounds with COX-1 (PDB ID: 1Q4G)
and COX-2 (PDB ID: 3LN1) using GLIDE and GOLD modelling software. The flavone and
chalcone derivatives were synthesized, and the structures of the compounds were drawn
using Schrodinger. Structure of the new compounds was verfied for their similarity with other
existing compounds in ChemSpider database. Before proceeding to molecular docking,
Qikprop were used to screen the compounds for their ADME properties, and compounds
were excluded if it violates more than one Lipinski's rule of five. Results: Most of the
compounds have shown pi interactions with TYR385, TYR355, TRP387, and ARG120 of
COX-2. 8-iodo-5,7-dimethoxy-2-phenyl-4H-chromen-4-one (F3) was found to have
interaction with COX-2 only. Conclusion: We conclude that F3 could be a potent antiinflammatory compound based on the molecular interaction studies.
Keywords: Chalcone; Cyclooxygenase; Flavone; Molecular docking.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
154

Identification of Novel npcRNA Candidates in Klebsiella pneumoniae

Sridevi V., Sumitha S., Kishan S. and Suresh CV.*
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling,
Bedong 08100, Kedah, Malaysia; *corresponding author, e-mail:
cvsureshgupta@gmail.com, Ph No: +604 42988165
Abstract: Background: Small non protein-coding RNAs (npcRNAs) have emerged as the
important regulators of many cellular pathways in bacteria. These npcRNAs regulate the
virulence of bacteria by interfering with the target mRNAs involved in bacterial
pathophysiology. The Klebsiella pneumoniae, a gram negative bacterium, is associated with
various infectious diseases in human, including pneumonia and urinary tract infections. Due
to the emergence of antibiotic resistance strains, there is an increasing need to identify novel
npcRNAs involved in virulence and antibiotic resistance of Klebsiella pneumoniae.
Methods: We used various computational tools to identify novel npcRNA candidates from
the transcriptome of Klebsiella pneumoniae HS11286. The intergenic (un-annotated)
transcripts were selected by viewing transcriptome bam file on Artemis. The most possible
npcRNA candidates were identified by further screening for the absence of ORF and no hit in
Rfam database. Total RNA of bacteria during different growth phases and stress conditions
were extracted using Trizol reagent and transferred to the membrane for Northern blot
hybridization. Results: A total of 238 intergenic transcripts were selected from the
transcriptome of Klebsiella pneumoniae. By using ORF finder, 137 of these are possessing
possible ORF and could be potential novel protein coding genes. Interestingly, 14 out of these
remaining 101 transcripts were found in Rfam database as known npcRNA in other bacteria.
So, finally we have identified 87 most potential npcRNA candidates in Klebsiella
pneumoniae. Total RNA extracted was highly intact and was confirmed by Bioanalyzer.
Conclusion: Totally 87 potential npcRNAs were identified and confirmation of the
expression of these npcRNAs during different stress conditions is in progress. This novel
npcRNA candidates can fill the gaps in further understanding of the regulation of virulence of
the organism.
Keywords: Klebsiella; Non-coding RNA; npcRNA; Trascriptome.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
155

Arduino Microcontroller Based Heart Rate Monitor using Fingertip

Sensors
Srilahari Namani1,*, Manickam Ramasamy1, Sunitha Namani2, Satyanarayana Namani2
1
2
Faculty of Engineering and Computer Technology, AIMST University, Malaysia.

Faculty of Medicine, AIMST University, Malaysia; *corresponding author, e-mail:
srilahari4u@gmail.com, Ph No: +60164375166
Abstract: Background: The main aim of this project is to develop a portable device which
helps to record heart rate of a person. The heart rate also referred as pulse rate, has been
recognized as the most important vital parameter which helps the individual as well as doctor
to spot out developing health problems. Using Heart Rate Monitor (HRM) is a more accurate
way to monitor heart rate than manually taking your pulse at carotid and radial pulse. A Heart
Rate Monitor (HRM) detects the electronic signal of heart and automatically computes the
heart rate in BPM. Method: It is a portable heart rate device developed using Arduino
microcontroller and infrared sensors to detect the heartbeat. It is the non-invasive method of
determining heart rate. Transmittance and Reflectance are two basic types of
photoplethysmography (PPG). Reflectance (PPG) is used here.An Infrared Light Emitting
Diode is used as a transmitter and a Phototransistor is used as receiver. The Infrared (IR)
Sensors use principle of reflectance plethysmography (PPG) to sense the pulse signal from
finger tip. The light source and the light detector are both placed on the same side of a body
part. The light is emitted into the tissue and the reflected light is measured by the detector. As
the light doesnt have to penetrate the body, the reflectance PPG can be applied to any parts
of human body.The sensor output is read by the arduino board, computes the Beats per
Minute (BPM) and display the instantaneous heart rate on LCD display module. Results:
Heart beat waveform was observed in Oscilloscope. It was able to record heart rate of
different people and display on LCD module. Conclusion: Arduino Based heart monitor is
able to detect Heart rate of a person. It is able to measure heart rate of the person and display
BPM on LCD display module.
Keywords: Arduino microcontroller; BP; Heart Rate Monitor (HRM); Health; Infrared
sensors.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
156

Transcriptome Analysis of Proteus mirabilis during Oxidative Stress

Adaptation
Sumitha S.1, Kishan S.1, Yukgehnaish K.1, Laurence J.C.2, Xavier R.1, Suresh C.V.*1
1
AIMST University, Department of Biotechnology, Faculty of Applied Sciences, 08100

Bedong, Kedah, Malaysia; 2Malaysian Genomics Resource Centre Berhad, Mid Valley City,
59200 Kuala Lumpur, Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com,
Ph. No: +604-4298165
Abstract: Background: Bacteria are well known for their fast adaptation to the stress
condition. Bacteria undergo some changes in the gene expression which enable us to
understand their adaptation to the stress condition. A transcriptome study is conducted to
compare the differential gene expression of ncRNA and mRNAs between normal and
oxidative stress condition of P. mirabilis. This study elucidates novel ncRNAs and also
enables us to understand the adaptation of P. mirabilis during stress. Methods: P. mirabilis
cultured in Luria-Bertani broth and the total RNA was extracted during exponential and
oxidative stress. These total RNA were sequenced via Illumina HiSeq 2000 platform. The
fastQ format transcriptome sequences were analysed using bioinformatics software tools such
as Trimmomatic and Bowtie2. Un-annotated intergenic regions were screened for the
possible novel ncRNA candidates using Artemis. Differential expression of mRNAs and
ncRNAs was analysed using HTSeq and DESeq software. Results: A total of 207 possible
ncRNA candidates were identified. By verifying the annotation in other bacteria and Rfam
database, 52 were selected as potential ncRNAs. Interestingly, 26 ncRNAs are up-regulated
while other 26 ncRNAs are down-regulated during oxidative stress condition. Out of 3460
genes, 1693 and 1688 showed significantly up and down regulations respectively. Most of the
phage protein and dimethyl sulfoxide reductase genes are up-regulated. The genes coding for
respiratory nitrate reductase, tetrathionate reductase, flagellar related proteins, tryptophan
synthase and alkyl hydroperoxide reductase were shown to be down-regulated during
oxidative stress. Conclusion: This transcriptome analysis data reveals both protein coding
and non- coding genes are playing a major role in bacterial oxidative stress adaptation. A
total of 26 novel and potential ncRNAs have been identified in P. mirabilis.
Keywords: Differential expression; ncRNA; Oxidative stress; P. mirabilis; Transcriptome.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
157

Safety and antiobesity effect of Garcinia atroviridis

Suriati Mohd Nasir1, Teh Lay Kek1,2, Mohd Zaki Salleh*1,2
1
Integrative Pharmacogenomic Institute (iPROMISE), Level 7, FF3 Building, Universiti

Teknologi MARA (UiTM) Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor
Darul Ehsan, Malaysia. 2Faculty of Pharmacy Universiti Teknologi MARA (UiTM) Puncak
Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia;
*corresponding author, e-mail: zakisalleh@puncakalam.uitm.edu.my, Ph No: (+603) 3258
4652
Abstract: Background: Obesity is a global health problem affecting both males and
females. Many attempt to reduce body weight by different approaches, including the
consumption of natural product with antiobesity effect. Garcinia atroviridis (GA) was
claimed to have antiobesity properties due to the presence of hydroxycitric acid (HCA).The
aim of this study is to elucidate safety andantiobesity effect of methanolic extract of Garcinia
atroviridis fruit. Methods: Acute and sub-acute toxicity study was conducted according to
OECD 407 and 423 guidelines, respectively. Forty-eight(48) female sprague-dawley rats
were divided into six groups (n=8). Obese rat model was developed using high fat diet (60%
fat, Research diet, USA). Then the treatment was given orally until significant reduction in
body weight gain was observed. Results: No toxicity effect was observed in the
animalfollowing the treatment. Histological study revealed no lesions or pathological changes
in the organs of either sex of the GA treated rats compared to control groups. Obese rats
treated with GA showed significant reduction in body weight gain. Conclusion: We conclude
that GA is a potential antiobesity agent.
Keywords: Antiobesity; Diet; Fat; Health; Oil; Rats.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
158

Development of Ochratoxin A Detection Based on Electrochemical Sensor

by Using Au-ball Labels
Suttiporn. P.*1, Patsamon. R2 and Werasak. S3
1
Food Technology, School of Agro-Industry, Mae Fah Luang University,333 Moo 1 Thasud
Muang, Chiang Rai, Thailand; 2National Center for Genetic Engineering and Biotechnology
(BIOTEC), 113 Thailand Science Park Phahonyothib Road Klong 1 Klong Luang,
Pathumthani, Thailand; 3School of Bioresources and Technology, King Mongkuts University
of Technology Thonburi, 83 Moo 8 Thakham Bangkhuntein, Bangkok, Thailand;
*corresponding author, e-mail: suttiporn.pin@mfu.ac.th, Ph No: (+66)923808281
Abstract: Background: There are increasing international attention to the problem of

ochratoxin A (OTA) contamination in coffee. This toxin represents a risk for human and
animal health when ingested through contaminated food.Therefore, developing a highly
sensitive and rapid method for OTA detection is necessary. Methods: In this study, an
electrochemical peptidesensor for OTA detection was developed using numerous number of
Au particles coated on the surface of silica particle (Au-ball). Moreover, this assay was
performed in 384 well plate, so the multiple detections was done. Results: The synthesized
silica particle was spherical in shape and size was 27517 nm. After coated with Au layer,
size of Au-ball was 28014 nn. Au particles loaded can be taken up resulting in approx. 1 x
107 Au3+ molecules per silica particle. Moreover, the optimization conditions were studied.
The limit of detection of this assay showed as low as 2 ppb. Conclusion: This platform
showed rapid, sensitive and specific to ochratoxin A detection. In spite of these advantages,
this Au-ball based peptidesensors is suitable to use in the detection of ochratoxin A
contaminated in coffee or seed industry.
Keywords: Anodic stripping voltammetry; Biosensor; Ochratoxin A; Peptide.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
159

Effect of Different Diets on the Growth and Survival of Silver Arowana

(Osteoglossum bichirrosum)
Tan W.S, Sivachandran P., Kurunathan S., Marimuthu K.*
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, 08100 Bedong,
Kedah, Malaysia; *corresponding author, e-mail: aquamuthu2k@gmail.com, Ph. No:
+60164723672
Abstract: Background: Silver arowana, (Osteoglossum bichirrosum) is a bony-tongue fish

native to South America and is one among the highly demanded ornamental fish species in
Asian countries. The present study investigated the effect of different diets on the growth and
survival of silver arowana. Methods: The experimental set up consists of 12 glass aquarium
tanks and each tank was bifurcated into three compartments and stocked with one fish for
each compartment. The initial length and weight were 9.0 0.26 mm and 4.05 0.30 g,
respectively. The fish were fed with four different types of diets namely, fish pellet,
mealworms, frozen bloodworms and mosquito fish for six months. Total weight and total
length of the fish and growth and survival was monitored each month. The weight gain and
specific growth rate (SGR) were calculated. Results: The study results found that, the highest
weight gain (56.335 9.003 g) and SGR (1.471) was observed in fish fed with fish pellets
when compared to all the diets tested. Significantly lower weight gain (16.062 1.808) and
growth performance was observed in fish fed with bloodworms. Conclusion: The present
study is the first kind of its type and evaluated the growth performance of silver arowana fed
with four diets under laboratory conditions. Results obtained suggest that artificial pelleted
feed is the best for larval rearing of silver arowana.
Keywords: Feed; Growth; Osteoglossum bichirrosum; Silver arowana.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
160

Effect of Aqueous and Methanol Extracts of Polygonum minus Leaves on

Drug-Induced Hepatotoxicity in Rats
Thanapakiam G, Lim S.Y, Grace K.Y.X, Loo S.W, Amutha V. V, Pavitra L, Neoh H. F,
Ahmad H.R, *Christapher P.V
Faculty of Pharmacy, AIMST University, Semeling 08100, Malaysia;
*corresponding author, e-mail: christapher@aimst.edu.my,
Ph No: +60 44298000 (extn: 1029)
Abstract: Background: Polygonum minus, a commonly available plant in Southeast Asia.

This plant is traditionally used for ailments such as pain relief, as a tonic after child birth and
to remove dandruff. Presence of high content of flavonoids and phenolic compounds is
responsible for its excellent antioxidant activity. The reported pharmacological studies
include anti-inflammatory, analgesic, antiulcer and cognitive improvement effects. Our study
aimed to evaluate the hepatoprotective activity of aqueous and methanol extract of P. minus
leaves in paracetamol-induced hepatotoxicity in rat models. Method: Dried leaves of P.
minus were powdered and macerated for 5 days using distilled water and methanol,
separately, filtered and evaporated to obtain dry crude extracts. Hepatic cell damage was
induced in Sprague Dawley rats by the administration of paracetamol (750 mg/kg;
paracetamol at every 72 h for 10 days) for all groups except the normal control (1 ml of CMC
was administered). All the treated groups were administered with either standard or extract
according to the treatment protocol. Parameters such as body weight, biochemical and
histopathology studies were studied. Results: Animals body weights did not show
significant variation in any treated group, compared with normal control, but was
significantly decreased in paracetamol control group. The biochemical analysis showed that
the higher dose of both extracts treated groups exhibited significant variations from the
paracetamol control group. The histopathology study also confirmed the hepatoprotective
effects of both extracts. Conclusion: Aqueous and methanolic P. minus extracts possess
significant hepatoprotective activity. More detailed studies are required to establish its
hepatoprotective efficiency.
Keywords: Hepatoprotective effect; Liver damage; Paracetamol; Polygonum minus.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
161

Identification of Novel Non-protein Coding RNAs (ncRNAs) in

Staphylococcus haemolyticus Biofilm
Thurga Devi N., Saw H.S., Kishan S., Sumitha S., Xavier R., Suresh V. C.*
Bedong, Kedah, Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com, Ph.
No: +604-4298165
Abstract: Background: Staphylococcus haemolyticus is an opportunistic nosocomial

bacterial pathogen often cause wide range of infections as simple as acne to serious infections
like endocarditis, peritonitis and UTI. Its ability to form biofilms especially on medical
devices is a major virulence factor associated with S. haemolyticus. Non-coding RNAs, the
newly emerged class of RNAs, having increasing evidence on their role in the regulation of
majority of the bacterial pathways including virulence and biofilm formation. This study
elucidates novel ncRNAs and also enables us to understand the adaptation of S. haemolyticus
to the different environment. Methods: S. haemolyticus was cultured in specialized media to
form biofilm. Total RNA was extracted and sequenced using Illumina platform. The
transcriptome sequences were aligned with reference genome of S. haemolyticus using
Bowtie2. Using genome viewer, un-annotated transcripts were selected and performed
sequence search similarity in other organisms using Blastn and Rfam database respectively.
The total RNA extracted from S. haemolyticus grown under different stress conditions,
resolved on denaturing gel and trans-blot to nylon membrane for Northern hybridization.
Results: A total of 147 possible ncRNA candidates identified and after filtering through
blastn, ORF search and Rfam, 68 were selected as most potential novel ncRNAs. 13 ncRNAs
with the highest RPKM value has been chosen for Northern hybridization. Interestingly 64 of
these 68 ncRNAs are specific to S. haemolyticus. Conclusion: This is the pioneering study
reveals 68 novel ncRNAs in S. haemolyticus whose expression is under the progress of
validating by Northern blot hybridization. Further research need to be done to deeply
understand the possible function of ncRNA of S. haemolyticus.
Keywords: Biofilm; ncRNA; Staphylococcus haemolyticus; Transcriptome.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
162

Application of a Novel Lytic Bacteriophage Strain as Biocontrol Agent for

Water Sanitization
Vickneswaran N.M., Sanirbandha C., Sivachandran P., Ravichandran M., Lee, S.Y.*
Bedong, Kedah, Malaysia; *corresponding author, e-mail: su_yin@aimst.edu.my, Ph. No:
+604-4298177
Abstract: Background: Bacteriophages are viruses that are parasitic on bacteria, and they
have been considered as one of the agents for treating bacterial infections. Bacteriophages
have a special adaptation to lyse the specific host cell by using the lysin enzyme. The aim of
this study is to investigate the use of bacteriophages as biocontrol agent for water
sanitization. Methods: Bacteriophages were isolated from various environmental samples by
filtration using 0.22m and 0.45m membrane filters. Spot test on different bacterial lawn
cultures was used to detect presence of life phages and determine host specificity.
Morphological characterization was done by using transmission electron microscopy (TEM).
The biocontrol study was done by inoculating the bacteriophage in pond water and LB
culture (as control), followed by incubation for 5 days. Each day, the phage titer (PFU/ml)
and the bacteria titer (CFU/ml) were enumerated to determine the effectiveness of the
bacteriophage as biocontrol agent. Results: Three novel strains of lytic bacteriophages were
isolated, which was specific towards Salmonella paratyphi a, Salmonella enteritidis and
Salmonella typhimurium. TEM analysis revealed that the unique structure of protein coat of
this strain as icosahedral head with longitudinal tail that is from the myoviridae family. The
biocontrol studies with the 3 phages showed that the bacterial titer reduced after 20 hours
post-inoculation, while the phage titer continued to increase until 100 hours post-inoculation
in both pond water and LB control media. The inverse relationship of the bacterial and phage
titres showed that the phages were able to control the bacterial growth and subsequently
multiply. Conclusion: The study showed that the isolated 3 strains of bacteriophage have the
potential to be used as effective biocontrol agent for sanitization of water and infection
control.
Keywords: Bacteriophage; Biocontrol agent; Lysin; Plaque assay.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
163

Phytochemical Analysis and Pharmacological Screening (Dopamine level)

of the Ethanolic Extract of Solanum trilobatum Linn.
Stephanie Wong Kah Yee, Bobby Lau Chik Chuon, Lee Yu Ren, Christapher Parayil
Varghese, Subramani Parasuraman*
*corresponding author, e-mail: parasuraman@aimst.edu.my, Ph No: (+60) 103895096
Abstract: Background: Solanum trilobatum Linn., (Solanaceae) is known as thoodhuvalai

in Tamil. The leaves of S. trilobatum are most widely used as food supplement in southern
part of Indian subcontinent and also this plant was traditionally used for the treatment of
respiratory illness. In pre-clinical studies, the extract of S. trilobatum showed favorable
antimicrobial, hepatoprotective and anticancer activities. The complete phytochemical profile
and dopaminergic activity of the S. trilobatum remain unclear, hence the present study was
planned to carry out the Gas Chromatography Mass Spectrometry (GC-MS) analysis and
dopaminergic activity of ethanolic extract of S. trilobatum (EEST). Methods: The
phytochemical analysis was carried out using chemical and instrumental (GC-MS) analytical
methods. The dopaminergic activity of EEST was determined in Sprague Dawley (SD) rats.
The rats were divided into two groups each of five animals. Rats were treated with vehicle
and EEST at 400 mg/kg, once daily for 30 days. During the study, body weight variations
and behavioral alterations were monitored. At the end of the study, rats were sacrificed, brain
was collected and weighed. The brain homogenate was used for the estimation of dopamine
levels using UV-visible spectrophotometer. Results: The phytochemical analysis showed the
presence of carbohydrates, saponins, flavonoids, alkaloids, tannins and phenolic compounds,
and GC-MS analysis showed the presence of 44 fragmented compounds which included
epoxylinalol, himachalol, illudol, epibuphanamine, baimuxinal and edulan IV. On chronic
administration, EEST increased the dopamine (*P<0.05) levels in brain tissue and not having
any significant effect on immunization time, locomotor activity when compared with the
control. Conclusion: EEST has significantly increased the dopamine levels in rat brain and
doesnt alter the behaviour of the animals.
Keywords: Dopamine; GC-MS analysis; Solanum trilobatum.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
164

Computational Analysis of Common Bean (Phaseolus vulgaris L.) Sadenosyl-L-methionine dependent methyltransferase gene cDNA Isolated
from Bean-pod-tissue cDNA Library
Cheah V.S.1, Amelia K.2, and Bhore S.J.*1, 2
1
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling

08100, Kedah, Malaysia; 2Department of Molecular Biology, Melaka Institute of
Biotechnology, Lot 7, Melaka International Trade Centre City, 75450 Ayer Keroh, Melaka;
*corresponding author, e-mail: subhash@aimst.edu.my / subhashbhore@gmail.com, Ph
No: +604 429 8176.
Abstract: Background: Common bean (Phaseolus vulgaris L.) is one of the legumes
cultivated in many countries. Common beans are important in diet as a source of proteins
especially for the poor people; because, it is inexpensive as compare to other sources of
proteins. In order to study the expressed genes, transcriptomics study was initiated and about
6000 ESTs were generated. While processing data, we identified a complimentary DNA
(cDNA) clone of S-adenosyl-L-Methionine-dependent Methyltransferase (PvSAM MTase)
gene and to understand more about it this study was undertaken. The objective of this study
was to annotate PvSAM MTase cDNA and deduced amino acid sequence using
computational tools. Methods: Both strands of PvSAM MTase cDNA clone were sequenced
using M13 forward and M13 reverse primer to reveal the nucleotide sequence. Online
bioinformatics tools were used for analysis and annotation of both cDNA and deduced
protein sequences. Sequence comparison was carried out using Blastp whereas split tree
program was used to construct a phylogenetic tree. The secondary and tertiary structure was
predicted using PHYRE automatic fold recognition server. Results: The cDNA and deduced
protein sequence analysis showed that PvSAM MTase gene cDNA is 1312 bp in length and
its open reading frame (ORF) encodes for 344 amino acids residues. The phylogenetic
analysis suggest that PvSAM MTase protein is closely related to its counterpart from Vigna
radiata L. The PvSAM MTase protein structure was successfully predicted and will be
discussed. Conclusion: The cDNA was annotated and primary analysis of the PvSAM
MTase deduced protein sequence suggested that it contains catalytic sites essential for its
function. Further study is required to validate the predicted structure of PvSAM MTase.
Keywords: cDNA; Common bean; Phaseolus vulgaris L.; Protein structure prediction; SAM
MTase.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
165

Biochemical Changes in African catfish, Clarias gariepinus Exposed to

Buprofezin
Gobinath R., Marimuthu K., Xavier R., Suresh C.V.*
No: +604-4298165
Abstract: Background: Buprofezin, is an insecticide widely used in Malaysia for paddy
cultivation. The study was carried out to investigate the biochemical changes in a freshwater
African catsh, Clarias gariepinus exposed to different concentrations of buprofezin under
laboratory conditions. The biochemical markers represent the most sensitive and relatively
early events of pollutant damage. Thus, it is imperative to study the impacts of pollutants in
aquatic organisms and delineate the mechanisms of pollutant action, and possible ways to
mitigate the adverse effects. Methods: The 96 h LC50 value of buprofezin to C. gariepinus
was estimated using probit analysis method. The experimental fishes were exposed to sublethal concentrations of the LC50 values of buprofezin at 0.15 mg/L, 0.30 mg/L and 0.60 mg/L
for a period of 8 days. At the end of the 2nd, 4th and 8th day of the exposure periods, the fish
blood sample was withdrawn and the concentrations of metabolites (glucose and total
protein) and enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST],
and alkaline phosphatase [ALP]) in all experimental fishes were determined. Results: The
fish exposed to sub-lethal concentration of buprofezin showed several abnormal behaviours
which including restlessness, loss of equilibrium, increased opercular activities, strong spasm,
paralysis and sudden erratic movements. Biochemical analysis showed that, on the 2nd, 4th
and 8th day of exposure, the amount of aspartate transaminase (AST), alkaline phosphatase
(ALP), alanine transaminase (ALT) and total protein levels were significantly declined. The
increased amount of total glucose was observed in all the higher concentrations of buprofezin
exposed fishes compared to control group. Conclusion: The present results elucidate that, the
insecticide, buprofezin exposure had induced biochemical alterations in fishes. These
biochemical parameters offer a rapid and sensitive means of monitoring towards the impact
of buprofezin in fish model organisms.
Keywords: African catfish; Biochemical parameters; Buprofezin; Clarias gariepinus; Sub
letahl toxicity.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
166

Haematological Changes in African catfish, Clarias gariepinus exposed to

Buprofezin
Gobinath R., Marimuthu K., Xavier R., Suresh C.V.*
No: +604-4298165
Abstract: Background: The study was carried out to investigate the haematological changes
in a freshwater African catsh, Clarias gariepinus exposed to buprofezin under laboratory
conditions. Buprofezin, is an insecticide widely used in Malaysia for paddy cultivation.
Effects of different pesticides on hematological parameters of several fish species have been
reported and most of the studies described on the effects of pesticides on the biochemical and
physiological changes, but very little attention has been paid to the hematological modulation
induce by buprofezin. Methods: The 96 h LC50 value of buprofezin to C. gariepinus was
estimated using probit analysis method. The experimental fishes were exposed to sub-lethal
concentrations of the LC50 buprofezin at 0.15 mg/L, 0.30 mg/L and 0.60 mg/L for a period of
8 days. At the end of the 2nd, 4th and 8th day of the exposure periods, fish blood sample was
withdrawn and haematological parameters were determined. The cells with two nuclei were
considered as binuclei (BN). The blood parameters white blood cell (WBC), neutrophil
(NUE), lymphocyte (LYM), monocyte (MONO), eosinophil (EOS), basophil (BASO), Mean
Corpuscular Haemoglobin (MCH), Mean Corpuscular Haemoglobin Concentration (MCHC),
red blood cell distribution width (RDW) and platelets (PLT) were analyzed by "Cell-Dyn
Haemotology Analyzer". Results: Haematological changes have been observed in buprofezin
treated fish in which, the amount of white blood cell, neutrophil, lymphocyte, monocyte,
eosinophil, basophil, mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin
concentration (MCHC), red cell distribution width (RDW) and platelet were decreased from
control to higher concentration of buprofezin exposed for the periods of 8 days. Amount of
lymphocyte was increased from control to higher concentration of this treatment for the
period of 8 days. These alterations have been attributed to direct responses of structural
damage to RBC membranes resulting in haemolysis and impairment in haemoglobin
synthesis induced by buprofezin exposure. Conclusion: The present study results showed
that, the sub-lethal effect of buprofezin on adult catfish C. gariepinus, by measuring a set of
haematological parameters. This study therefore gives an insight and baseline information
about the toxicity of buprofezin in fish model organisms, African catfish, Clarias gariepinus.
Keywords: African catfish; Buprofezin; Clarias gariepinus; Haematological parameters.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
167

Identification of Novel Non-coding RNA (ncRNA) from Tannerella

forsythia
Kavithannjali K.1, Saw H.S. 1, Sumitha S. 1, Jawahar D.2, Suresh C.V1.*
1

Bedong, Kedah, Malaysia; 2AIMST University, Faculty of Dentistry, 08100 Bedong, Kedah,
Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com, Ph. No: +6044298165
Abstract: Background: Tannerella forsythia is an anaerobic, Gram-negative bacteria of the

Cytophaga Bacteroidetes family. It has been strongly implicated in the onset of periodontitis.
T. forsythia is associated more frequently in higher levels with various forms of the diseases,
including gingivitis, chronic and aggressive periodontitis, than normal. Thus, it is important
to identify the virulence functions of the organism. One of the way to understand the
virulence of T. forsythia through identification of ncRNAs as they play a significant role in
regulation of fundamental bacterial adaptive processes including bacterial virulence.
Methods: nocoRNAc software was used to identify the ncRNA genes in T. forsythia. Artemis
viewer was used to select the predicted ncRNA genes at intergenic region of T. forsythia. The
obtained candidates were screened with blastN and Rfam. Results: NocoRNAc predicted 429
ncRNA candidates in T. forsythia. Among these, 82 are derived from intergenic regions of T.
forsythia. Further sequence similarity search against the annotated Genbank and Rfam
databases showed 343 are annotated as protein coding genes and 80 as known ncRNA genes
respectively. Finally 80 are identified as most possible ncRNA genes in T. forsythia.
Interestingly, 53 of these are highly specific to T. forsythia. Conclusion: The study predicts
80 ncRNAs in T. forsythia which need to be further confirmed for their expression. The
function and regulatory roles of these ncRNAs will be a great insight in understanding the
virulence of T. forsythia.
Keywords: DNA; ncRNA; nocoRNAc; Periodontitis; Tannerella forsythia.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
168

Microbial Load, Antimicrobial Sensitivity and Plasmid Profiles of Vibrio

cholerae in Fruit Juice
Md. Mehdi Hasan2, 5, Sharmin Islam*1., Rayhan Ahmed 2, 5 ., Md. Rowfur Rahman 2., Md.
Shahidul Islam 3 and Md. Masudur Rahman Khalil 1,4.
1
Center for Communicable Diseases, International Center for Diarrhoeal Disease Research,
Bangladesh (icddr,b), Dhaka-1212, Bangladesh. 2Department of Microbiology, Gono
Bishwabidyalay (University), Dhaka-1344, Bangladesh. 3Department of Microbiology, Prime
Asia University, Dhaka-1213, Bangladesh. 4Department of Microbiology and Biotechnology,
Jagannath University, Dhaka-1100, Bangladesh. 5Department of Microbiology, faculty of
medicine, AIMST University, Bedong-08100, Kedah, Malaysia; *corresponding author, email: mukta.sharmin@gmail.com, Ph No: +8801710365279
Abstract: Background: Vibrio cholerae is the causative agents of cholera, an acute

dehydrating diarrhea. The research report was designed for isolation and identification of V.
cholerae from juices. A total of forty samples of different fruit juices such as orange, papaya,
grape, malta, sugarcane , lemon, aloe vera and isupgul juice were collected from different
areas of Dhaka city. Methods: The samples were subjected to inoculate into TCBS agar
media upon dilution for the isolation of V.cholerae, followed by biochemical identification of
the bacteria. Different biochemical tests were performed such as oxidase, TSI, MR-VP,
Citrate, Motility etc. And finally for plasmid isolation agarose gel-electrophoresis has been done.
Results: The total viable count was varying from 7.73 x104-7.62 x108 cfu/ml where the
highest count observed in sugarcane juice and lowest count in orange juice. V. cholerae was
present in 20% of samples where the 75% of them were detected from sugarcane juice which
was also contaminated with high bacterial and fungal load. The occurrence of drug resistance
was also determined in the 8 isolated V. cholerae. With all the investigation it is bearing on
mind that Vibrio cholerae cause severe diarrhoea and occurance of Vibrio cholerae in juice
may cause a serious health hazards. Conclusion: About 40% isolates were resistant to
tetracycline and neomycin and 80% isolates were resistant to amikacin. Plasmid profiling of
eight isolates also performed, where most of the strain harbor two plasmids. All the isolates
were resistant to polymixin B, and sensitive to gentamycin and ciprofloxacin.
Keywords: Bangladesh; Coliform; E. coli; Fruit juice; Plasmid; Vibrio cholerae.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
169

Isolation of Arsenite Resistant Bacteria from Ground Water and Soil of

Dhaka, Bangladesh
Rayhan Ahmed2,3, Ezazul Haque 2,3, Md. Jewel Rana2 and Taslin Jahan Mou1*
1
Dept. of Microbiology, Faculty of Biological Science, Jahangirnagar University, Savar,

Dhaka-1342, Bangladesh. 2Dept. of Microbiology, Faculty of Health and Medical Science,
Gono University, Savar, Dhaka-1340, Bangladesh, 3Unit of Microbiology, Faculty of
Medicine, AIMST University, Semeling, Bedong-08100, Kedah, Malaysia; *corresponding
author, e-mail: moumicro@juniv.edu, Ph No: +88 01717562770
Abstract: Background: Worldwide more than 100 million people have been chronically
exposed to arsenic by either drinking high level arsenic containing water or through soil.
Arsenic resistant bacteria can tolerate high level of arsenic by the mechanism of energydependent efflux of either arsenate or arsenite from the cell mediated via the ars operon. The
objective of the study was isolation and characterization of arsenite resistant bacteria from
arsenic prone area in Bangladesh. Methods: Total of 6 samples (three each for groundwater
and soil) were collected in May, 2014 from different places of Dohar, Dhaka, Bangladesh.
100 mL of each groundwater sample and 1 g of each of the soil samples were diluted with
distilled water and 10-2 dilution of water and 10-3 dilution of soil sample inoculated to the 300
mL of minimal salt medium (MSM) supplemented with sodium arsenite. Arsenic tolerance of
the bacteria was determined at 2,3,5,8 & 10 mM of sodium arsenite concentration
respectively on minimal salt media. Bacterial strains were presumptively identified according
to Bergeys manual of systemic bacteriology. Results: A total of 60 bacterial isolates
were found among which E.coli, Bacillus and Pseudomonas were presumptively prevalent
according to the biochemical tests. 63% and 55% of the isolates were resistant against 8mM
and 10mM respectively. Conclusion: From the study it can be concluded that arsenite
resistant bacteria are prevalent in the water and soil sample of Dohar, Dhaka which are
tolerant to high level of arsenic. Further investigation is required for molecular analysis and
screening of the arsenite oxidizing bacteria.
Keywords: Arsenite resistance; Bangladesh; Soil; Water.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
170

Compared to Serum Media, Serum-Free Medium Enhanced the

Generation of Mesenchymal Stem Cells Derived from Full-Term Human
Amniotic Fluid
Ghoraishizadeh P.1, Rohayu I.M.R.1, Ramasamy R.2, Singh G.3, Tan B.C.4, Rejali Z.5, Rosli
R.1,6 Nordin N.1,6 and Thilakavathy K1,6*
1
Medical Genetics Unit, Department of Biomedical Sciences, Faculty of Medicine and Health
Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 2Department
of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400
UPM Serdang, Selangor, Malaysia.
3
Stempeutics Research Sdn Bhd, Technology Park Malaysia, 57000 Bukit Jalil, Kuala
Lumpur, Malaysia; 4Britannia Women and Children Specialist Centre, Selangor, Malaysia;
5
Department of Obstetrics and Gynaecology, Faculty of Medicine and Health Sciences,
Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 6Genetics and
Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences,
Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; *corresponding
author, e-mail: thilathy@upm.edu.my, Ph No: +603-89472652
Abstract: Background: Mesenchymal stem cells (MSCs) are multipotent stem cells that are
abundantly found in human amniotic fluid and possess therapeutic potential. DMEM
supplemented with fetal bovine serum is the standard culture medium used to generate the
human amniotic fluid-derived MSCs (hAF-MSCs). However, culturing of these cells in
serum and xeno contained medium has challenged their usage in clinical applications.
Hence, we aimed to use serum media and MesencultTM-XF xeno- and serum-free medium
(XSFM) to generate, characterize and compare the properties of full-term hAF-MSCs.
Methods: Amniotic fluid cells obtained from caesarean sections were cultured and
propagated in XSFM, DMEM low glucose supplemented with 15% fetal bovine serum
(DMEM-FBS), and 15% human serum (DMEM-HS). Immunophenotyping, differentiation
and CFU-F assays were carried out to characterize the hAF-MSCs. Results: Spindle-shaped
fibroblast-like cells were observed at passage 0 in all culture media used. These cells
proliferated beyond passage 7 in XSFM medium and up to passage 3 in DMEM-FBS,
however, failed to proliferate beyond passage 0 in DMEM-HS. Immunophenotyping analysis
revealed that the cells generated in XSFM and DMEM-FBS expressed MSCs markers. hAFMSCs generated in XSFM were able to differentiate into adipocytes, osteoblasts and
chondrocytes, however in DMEM-FBS, they did not differentiate into chondrocytes.
Population doubling time and CFU-F of hAF-MSCs generated in XSFM was 14 times shorter
and 11 folds higher, respectively, compared to DMEM-FBS. Conclusions: XSFM medium
was found to be better in culturing and generating the hAF-MSCs isolated from full-term
pregnancy compared to DMEM-FBS and DMEM-HS, suggesting its potential therapeutic
usage in clinical applications.
Keywords: Fetal bovine serum; Full-term pregnancy; Human amniotic fluid; Human serum;
Mesenchymal stem cell; Xeno-and serum-free.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
171

Hybrid Assembly of Salmonella enterica subsp. enterica ser. Typhi Isolates

PM016/13 and B/SF/13/03/195 from a Typhoid Outbreak in Pasir Mas,
Kelantan in 2013
Salwani Muhamad Harish1, Nazalan Najimuddin2, Ismail Aziah1*
1
Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
2
School of Biological Science, Main Campus, Universiti Sains Malaysia, 11800 USM, Pulau
Pinang, Malaysia; *corresponding author, e-mail: aziahismail@gmail.com
Abstract: Background: The ability to sequence the entire genome of individual bacteria
helps to improve understanding of the organization and evolution of the organism at the
molecular level. The generation of complete genome sequences provides a blueprint that
facilitates the genetic characteristics of pathogens and their hosts. In this study, two isolates
of S. enterica subsp. enterica ser. Typhi PM016/13 from untreated well water and also
B/SF/13/03/195 from food handler were sequenced using next generation sequencing
platforms provided by Pacific Biosciences and Illumina. Methods: The two isolates were
assembled using the method of hybrid assembly. De novo assemblies of PacBio reads from
both isolates were generated using PacBios SMRT Portal (v2.3.0) and hierarchical genome
assembly process. Illumina reads were first screened by FastQC to identify low quality reads
and adapters. The low quality reads and adapters were trimmed off the reads using NGS QC
Toolkit before mapped onto PacBio assembled contigs using SSPACE Standard. The contigs
obtained from the mapping were aligned according to S. enterica subsp. enterica ser. Typhi
CT18 genome (GenBank accession number: NC_003198) using CONTIGuator2 to determine
their correct order and orientation. The gaps from the scaffolds obtained were filled by
Gapfiller. Results: The hybrid assembly resulted in one contig for both isolates meaning a
complete genome of both isolates were obtained from this method. Isolates PM016/13 was
assembled with 4,803,901 bp in size while isolates B/SF/13/03/195 was assembled with
4,801,617 bp in size. The GC content of both isolates was 52.00%. Conclusion: Hybrid
assembly of the isolates had successfully assembled the reads into complete genome. The
combination of short read Illumina and long read PacBio can give better accuracy to the
assembly since the use of single molecule third generation sequencing data only give low
accuracy, which causes inherent errors in the sequenced DNA. Using solely second
generation sequencing data on the other hand lead to incomplete assembly of important part
of a genome. Combination of both sequencing data can overcome these limitations.
Keywords: Genomics; Hybrid assembly; Illumine; Next generation sequencing; Pacific
biosciences; S. enterica subsp. enterica ser.; Typhi; Typhoid.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
172

Identification and Characterization of Novel Non-protein Coding RNAs in

Salmonella serovar Typhi Biofilm
Satisvar S.1, Kogaan .A.2, Phua K.K. 2, Suresh C.V.1*
1

Bedong, Kedah, Malaysia.
2
11800, USM, Pulau Pinang, Malaysia; *corresponding author, e-mail:
cvsureshgupta@gmail.com, Ph. No: +604-4298165
Abstract: Background: Salmonella enterica serovar Typhi (S. Typhi) is the sole cause of
typhoid fever, which is common among travellers to third world countries. The battle against
typhoid fever is intensified due to the ability of S. Typhi to form biofilm in the gallbladder.
The biofilm provides a rudimentary protection against commonly used antibiotics. Further
understanding of the role of non-coding RNA (ncRNA) in its formation will aid physicians
and scientists alike to counter biofilm formation. Methods: Total RNA was extracted from
various growth phases of S. Typhi including biofilm formation. The transcriptome sequences
of S. Typhi were aligned with the reference genome S. Typhi Ty2 using Bowtie and analyzed
using genome viewer. Online ORF finder was used to detect the presence of possible ORFs.
The candidates were then cross-checked with known ncRNAs in Rfam database. The total
RNA was resolved on denaturing gel and transferred to a nylon membrane using semi-dry
transfer protocol. Results: A total of 754 possible ncRNA transcripts were obtained from the
intergenic regions of S. Typhi. These were screened for their annotation as protein coding
genes or ncRNA genes in other organisms using Blastn/BlastP and Rfam search, respectively.
Nine potentially novel ncRNAs were found in S. Typhi biofilm. Interestingly, one of these
novel ncRNA gene was located adjacent to topoisomerase gene and having the conservation
of STAXI motif which may interact with the anti-restriction enzyme proteins. Conclusion: In
summary, we have identified 9 novel ncRNAs from S. Typhi biofilm. Current work is in
progress to analyze the variations and specificity of their expression during biofilm formation
using Northern-blot. This study could help better understand the mechanism of biofilm
formation in S. Typhi which is worldwide becoming more resistant to antibiotics.
Keywords: Biofilm; Non-coding RNA; S. Typhi; Transcriptomics.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
173

Identification of Novel Non-coding RNA (ncRNA) from Bacillus

thuringiensis
Shereenrani S., Saw H.S., Sumitha S., Maheswaran S., Xavier R., Suresh C.V.*
No: +604-4298165
Abstract: Background: Bacillus thuringiensis (Bt) is a Gram-positive, rod-shaped, sporeforming bacterium. It grows at body temperature and produces a different shaped crystal
proteins. It used to fend off insects, predators, and other pathogens. Bt toxin is speciesspecific and different shape and activity depends on unknown regulatory pathways. The
objective of this study is to identify the possible ncRNA candidates in Bt which can fill the
gaps of understand the gene regulation. Methods: Bt transcriptome data was downloaded
from DNA Data Bank of Japan (DDBJ) database. FastQC analysis was performed to view the
quality of the transcriptome. Trimmomatic is done to remove the adapter sequences. The
sequences were then aligned to Bt genome using Bowtie2. the un-annotated transcripts were
selected using genome viewer Artemis. These possible ncRNAs were filtered further using
blastN, ORF Finder and Rfam to exclude the annotated genes. ncRNA genes also were predicted
computationally using nocoRNAc software. Results: A total of 418 candidates were identified
as un-annotated transcripts. Finally 12 candidates were selected as potential ncRNA
candidates after filtering through ORF Finder, blastN and Rfam. Interestingly, all these 12
candidates are genus specific to the Bacillus sp. In parallel, 1454 ncRNA candidates were
predicted by nocoRNAc. Seven of these 12 candidates were overlapping with nocoRNAc
prediction list. Conclusion: The preliminary study reveals 12 novel ncRNAs from Bt
transcriptome. However, need to confirm their expression again by real-time PCR.
Keywords: Bacillus thuringiensis; ncRNA; nocoRNAc; Transcriptome.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
174

Morphological and Differential Protein Expression Analysis of Placentas

from Term and Spontaneous Preterm Labor with Intact Membrane
Tan N.J.1, Daim L.D.J.2, Jamil A.A.M.3, Mohtarrudin N.4 and Thilakavathy K.1,5*
1
Medical Genetics Unit, Department of Biomedical Sciences, Faculty of Medicine and Health
Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 2Sime Darby
Technology Centre Sdn. Bhd, 1st Floor, Block B, UPM-MTDC Technology Centre III, Lebuh
Silikon, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia; 3Department of
Obstetrics and Gynaecology, Faculty of Medicine and Health Sciences, Universiti Putra
Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 4Department of Pathology, Faculty of
Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor,
Malaysia; 5Genetics and Regenerative Medicine Research Centre, Faculty of Medicine and
Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor,
Malaysia;*corresponding author, e-mail: thilathy@upm.edu.my, Ph No: +60389472652
Abstract: Background: Spontaneous preterm labor with intact membrane (sPTL-IM)

accounts for approximately 45% of the total preterm delivery worldwide. Many major
pregnancy complications had been association with placenta abnormalities. Hence,
evaluating the histology and protein profiles of the placenta will provide us better
understanding on the cause of sPTL-IM, which will help in improving the current diagnosis,
prognosis, treatment and decision making. Therefore, this study aimed to determine the term
and sPTL-IM placentas histopathology, and differentially expressed proteins in fetal site of
the placenta cotyledon. Methods: Term and sPTL-IM placentas were obtained from Hospital
Serdang, Malaysia. The whole thickness of placenta disc, umbilical cord and membrane were
formalin fixed, paraffin-embedded, stained with hematoxylin and eosin (H&E) stain, and
slides were viewed under the light microscope. Subsequently, to study the differential protein
expression, fetal site of the placentas tissues were pulverized and proteins were extracted
using DNase/Lithium chloride-dense sucrose homogenization coupled dichloromethanemethanol precipitation. Extracted proteins were separated on 2D-gel electrophoresis and
visualized using silver stain. Results: The H&E appearance of the whole thickness of
placental disc including decidua and chorion, distal and proximal site of the umbilical cords
and the membrane tissues showed no difference for both term and preterm delivery placentas.
Interestingly, 7 protein spots found in 2D-gel were found differentially expressed between
normal and sPTL-IM placentas at the fetal site. Conclusion: Although there is no difference
morphologically between term and sPTL-IM placentas, but some proteins were differentially
expressed, suggesting molecular investigation might benefit in future clinical advancement.
Keywords: 2D-gel electrophoresis; Histology; Protein profile; Placenta; Spontaneous preterm
labor with intact membrane.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
175

Expression of Salmonella Typhi Ty21 TolC Protein in Different Host Cells

Teh Boon Auna, Amy Amilda Anthonyb, Ismail Aziahb, Asma Ismaila, Eugene Boon Beng
Onga, and Phua Kia Kien*a
a
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Penang,
Malaysia. b Institute for Research in Molecular Medicine, Health Campus, Universiti Sains
Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia; *corresponding author, e-mail:
kkphua@usm.my
Abstract: Background: Salmonella enterica serovar Typhi 50 kDa outer-membrane protein

was reported to be antigenic. Here we investigated the effects of recombinant (r) protein
expression in different host cells on the antigenicity of S. Typhi TolC protein, a component of
the OMP. Modification and over-expression in E. coli could render the antigenicity of rTolC
useless. Epitopes on rTolC appear to be conformational as native TolC reacted with typhoid
patient serum while denatured TolC had much lower reactivity. Methods: Bacteria strains
used as host cells in this experiment were S. Typhi Ty21a, TolC deletion mutan, E. coli
DH5a, and E. coli Lemo21. Recombinant TolC was cloned into pBAD Myc-His A plasmid
and the protein was subsequently overexpressed in S. Typhi Ty21a and E. coli. Recombinant
TolC was then purified and analyzed by MALDI-TOF/TOF. ELISA was used to measure the
antibody reactivity with rTolC antigens expressed in the different host cells. Results: The
ELISA results demonstrated that antibodies were reactive with rTolC expressed in S. Typhi
with a significant difference in the mean OD value between typhoid sera (OD405 = 3.15, n=2)
and normal sera (OD405 = 1.19, n=2). However, for rTolC expressed in E. coli, no significant
difference in the mean OD was observed between normal sera (OD405 = 0.30, n=2) and
typhoid sera (OD405 = 0.32, n=2), whereas for rTolC purified under denaturing condition a
slight difference in the mean OD values was found between pooled typhoid sera (OD405 =
0.23 ) and pooled normal sera (OD405 = 0.11, n=2) while for rTolC expressed in E. coli host
cells, a significant difference in OD was observed between pooled typhoid sera (OD405 =
0.25) and pooled normal sera as controls (OD405 = 0.15). Thus, the ELISA results showed
that the rTolC was highly reactive with typhoid sera. Conclusion: This study we showed that
recombinant TolC from S. Typhi TolC deletion mutant had the potential to be used as the
antigen for development of a rapid ELISA diagnostic test for typhoid fever. Also found was
that rTolC purified from S. Typhi host cells were more antigenic compared with rTolC
purified from E. coli host cells. Future studies include determining the analytical sensitivity
and specificity of the antigens on various diagnostic platforms for diagnosis of typhoid fever.
Keywords: Antigen; ELISA; Salmonella; TolC; Typhoid.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
176

Stemness of Spontaneously Transformed Murine Bone Marrow

Mesenchymal Stem Cells is Maintained Upon Prolonged In Vitro
Expansion
Lye K.L.1, Vidyadaran S.2,3, Nordin N.1,2 and Thilakavathy K.1,2*
1
Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti

Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 2 Genetics and Regenerative
Medicine Research Centre, Faculty of Medicine and Health Sciences, Universiti Putra
Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 3Department of Pathology, Faculty of
Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor,
Malaysia; *corresponding author, e-mail: thilathy@upm.edu.my, Ph No: +603-89472652
Abstract: Background: Mesenchymal stem cells (MSCs) are a unique population of stem
cells that possess immunomodulatory properties, making them one of the ideal candidates for
regenerative medicine. MSCs are also known to suppress or activate cancer formation. In
this study, we aimed to investigate the spontaneous transformation of murine bone marrow
MSCs (mBM-MSCs) and evaluate their stemness characteristic. Methods: mBM-MSCs were
isolated from the femur of C57BL/6 mice and cultured in DMEM high glucose supplemented
with 15% fetal bovine serum and 1% antibiotic/antimycotic. Immunophenotyping and
differentiation assays were carried out to characterize the stemness of the isolated MSCs
population. Chromosome analysis and selected oncogenes expressions analysis were
performed to confirm the occurrence of spontaneous transformation in these cells. Results:
Homogeneous population of mBM-MSCs with spindle shaped fibroblast-like morphology
was successfully generated from passage 5 onwards. Variations in chromosome numbers
were observed at passage 20 and beyond, indicating spontaneous transformation. This was
further supported by the over-expression of c-Myc, MDM2, and Fos oncogenes as compared
to the earlier passages. However, these transformed cells were still expressing positive MSCs
markers and able to differentiate into adipocytes and osteoblasts, denoting the stemness.
Conclusion: This study clearly indicates that mBM-MSCs undergo spontaneous
transformation at molecular level but still maintain their stemness in prolonged culture. This
study also suggests that early passage MSCs to be used in research studies, and molecular
characterization should be made obligatory prior to their application in regenerative medicine
and therapy.
Keywords: Bone marrow; Chromosomes; Mesenchymal stem cells; Oncogenes; Spontaneous
transformation.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
177

16S Ribosomal DNA Sequence Based Identification of Bacterial

Endophytes Isolated from Seeds of Starfruit (Averrhoa carambola L.)
Narmataa M. and Bhore S. J.*
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, BedongSemeling Road, 08100 Bedong, Kedah, Malaysia; *corresponding author, e-mail:
subhashbhore@gmail.com / subhash@aimst.edu.my
Abstract: Background: Starfruit plant (Averrhoa carambola L.) is cultivated in tropics for
its fruits (Starfruit). Fruits are sold, imported and exported extensively in South East Asia
region and beyond. However, this plant is poorly studied at molecular level as well as for its
endophytes. Endophytes are the microbes (usually, bacteria and fungi) that stay inside plant
body without causing any visible symptoms of disease. Endophytes can be used to promote
plant growth and development and have a great potential in agriculture sector and other
sectors of biotechnology. The objective of this study was to isolate and identify bacterial
endophytes associated with seeds of Starfruits. Methods: Starfruits (Clone B10) were
purchased in local market of Sungai Petani, Kedah, Malaysia. Surface sterilisation of fruits
was done by following a standard method and extracted seeds were macerated and plated on
sterile Luria-Bertani Agar. Plates were incubation at 37C for period for 36 hours and
bacterial endophytes were isolated. 16S ribosomal DNA fragments were amplified using
genomic DNA from pure cultures of respective isolated bacterial endophytes. Isolates were
identified based on sequence of amplified 16S ribosomal DNA. Results: A total of five
endophytic bacterial isolates (EBIs) were obtained from the seeds of the Starfruit. The
sequences of amplified 16S ribosomal DNA were analysed using nucleotide database of
National Centre for Biotechnology Information (NCBI). Analysis revealed the identity of the
isolated EBIs as Bacillus anthracis, Bacillus cereus, and Bacillus toyonensis. The nucleotide
sequence data of 16S ribosomal RNA gene (partial sequences) fragments was submitted to
nucleotide sequences database of NCBI. The annotated 16S rRNA gene fragment nucleotide
sequences of all five isolates has been submitted to the nucleotide database at
GenBank/DDBJ/EMBL under accession numbers: KT861455 - KT861459. Conclusions: In
conclusion, Starfruit (Clone B10) seeds do possess bacterial endophytes. This study reveals
the presence of Bacillus anthracis, Bacillus cereus, and Bacillus toyonensis in Starfruit seeds.
Further study is required to find out the benefits of bacterial endophytes to the Starfruit plant.
Keywords: 16S Rrna; Averrhoa carambola L.; Endophytes; Seeds; Starfruit.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
178

An Alternative Method of Screening the M2/ANXA5 Haplotype for

Repeated Pregnancy Loss
1
Kai-Cheen Ang*, 2Sushilnathan Kathirgamanathan, 3Yan-Yeow Lee, 4Anna Liza binti

Roslani, 3Krishna Kumar, 5Bavanandan Naidu, 2Ridzuan bin Abdullah, 6Nadja Bogdanova,
1
Narazah Mohd Yusoff, 7Wan Zaidah Abdullah, 6Arseni Markoff, 1Thean-Hock Tang*
1
Advanced Medical and Dental Institute, University Sains Malaysia, Bertam, Penang,
Malaysia. 2Department of Obstetrics and Gynaecology, Hospital Sultan Abdul Halim, Sungai
Petani, Malaysia. 3Department of Obstetrics and Gynaecology, Hospital Tuanku Jaafar,
Seremban, Malaysia. 4Department of Obstetrics and Gynaecology, Hospital Tengku Ampuan
Afzan, Kuantan, Malaysia. 5Department of Obstetrics and Gynaecology, Hospital Sultanah
Bahiyah, Alor Setar, Malaysia. 6Institute of Human Genetics, University of Muenster,
Muenster, Germany. 7Department of Haematology, School of Medical Sciences, Universiti
Sains Malaysia, Kubang Kerian, Malaysia; *corresponding author, e-mail:
jocelyn85ang@yahoo.com, Ph No: (+60) 174059103
Abstract: Background: Repeated pregnancy loss (RPL) has an adverse psychosocial impact
in women and their families, resulting in depression or anxiety for the next pregnancy.
Recently, M2/ANXA5 haplotype, a variant in the proximal core promoter region of the
annexin A5 (ANXA5) gene, was suggested as a risk factor in thrombophilia-associated RPL
impeding embryonic anticoagulation. M2/ANXA5 haplotype is suggested as the third
hereditary predisposition gene for thrombophilia-associated RPL. We developed and
validated of an Allelic specific PCR (AsPCR) for the detection of M2/ANXA5 haplotype. The
AsPCR is simple, sensitive, less time consuming and cheap assay. Methods: Allele specific
PCR for M2/ANXA5 haplotype is designed based on the concept of nested PCR by using a
common primers and allele specific primers sets. All the primers, DNA, MgCl2 and Taq were
adjusted to an optimum amount until the specific bands were produced. The annealing
temperature was optimized using a gradient PCR to determine the optimum annealing
temperature at which all the primers react to anneal with the template DNA at their optimum
conditions. A total of 105 blood samples were collected for genotyping with AS PCR and
also validated with DNA sequencing. Results: The AsPCR was successfully discriminated
the M2 and normal haplotypes. Sequencing results confirmed and validated the AsPCR
result with 100% reliability. Conclusions: AsPCR was developed for the detection
M2/ANXA5 haplotype. This assay provides an easier, faster and more cost effective method
for screening the M2/ANXA5 haplotype.
Keywords: Allelic specific PCR; M2/ANXA5 haplotype; Repeated pregnancy loss.
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Computational Analysis of Class IV Chitinase Gene cDNA Isolated from

American Oil Palm (Elaeis oleifera) Fruit Mesocarp Tissue cDNA Library
Vengadan S.1, Amelia K.2 and Subhash J. Bhore*1, 2
1
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, BedongSemeling Road, Bedong, 08100, Kedah Darul Aman, Malaysia; 2Molecular Biology Division,
Melaka Institute of Biotechnology, Lot 7, Melaka International Trade Centre City, 75450
Ayer Keroh, Melaka, Malaysia; *corresponding author, e-mail: subhashbhore@gmail.com /
subhash@aimst.edu.my, Ph No: +604-429 8176
Abstract: Background: Elaeis is a genus of palms which contains two Oil-palm species.
The African Oil-palm, Elaeis guineensis Jacq (variety Tenera) is native to west and southwest
Africa and cultivated widely in tropics because of its high yield of oil. The American Oilpalm, Elaeis oleifera is native to tropical central and South America and it is used locally for
oil production. However, it is not cultivated on a commercial scale to produce palm oil
because of its low yield. In order to study the genes expressed in fruits of the American Oilpalm, about 3200 expressed sequence tags (ESTs) were generated and while processing the
ESTs, class IV Chitinase gene cDNA was identified. The objective of this study was to
analyze and annotate Elaeis oleifera class IV Chitinase (Cht) full-length cDNA and its
deduced protein sequence. Methods: The cDNA sequence and its deduced protein sequence
was analyzed and annotated using online bioinformatics tools including JustBio. The multiple
sequence alignment (MSA) and the phylogenetic analysis of Elaeis oleifera class IV chitinase
protein was carried out by using CLUSTALW and treeviewX. Secondary structures and
tertiary (3D) structure of class IV Chitinase protein was predicted by using Phyre 2 server.
Results: In total, 3258 ESTs (cDNA) clones have been isolated from mesocarp cDNA library
by using random method of gene isolation. Class IV chitinase protein cDNA gene was found
when the generated ESTs were processed and analysed. Result analysis suggest that class IV
chitinase protein cDNA sequence is 1049 bp in length. The open reading frame analysis
suggest that it encodes for a protein which contains 268 amino acids. Phylogenetic analysis
indicated that Elaeis oleifera class IV chitinase protein is closely related to its counterpart
from African Oil-palm. The 3D structure prediction work is in progress and will be reported
in due time. Conclusion: The Elaeis oleifera class IV chitinase protein cDNA clone sequence
and deduced protein sequence is successfully annotated. Further research is required to
understand the role of class IV chitinase in the Oil-palm biology. These primary research
findings will be presented in the conference.
Keywords: American oil-palm; BLAST; Class IV chitinase; Elaeis oleifera; Oil.
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In Silico Analysis of Omega-6 Fatty Acid Desaturase Gene cDNA Isolated

from Common Bean (Phaseolus vulgaris L.) Pod Tissue cDNA Library
Teh S.Y.1, Amelia K.2 and Subhash J. Bhore*1, 2
1
Abstract: Background: Common beans (Phaseolus vulgaris L.) are widely consumed
worldwide because of its high protein content. However, to meet the demand of growing
population, its yield needs to be increased. In line with the agenda of Phaseomics (an
international consortium), work of expressed sequence tags (ESTs) generation from bean
pods was initiated. Altogether, 5972 ESTs have been isolated which we have deposited in
GenBank. Omega-6 fatty acid desaturase (O6FAD) encoding gene cDNA was one of the
clones among generated ESTs. The O6FAD is an important enzyme in fatty acid biosynthesis
pathway; hence, to understand more about it this study was undertaken. The objective of this
study was to elucidate P. vulgaris L. O6FAD (PvO6FAD) gene cDNA sequence and to
predict the three-dimensional (3D) structure of deduced protein. Methods: Positive and
negative strands of the PvO6FAD cDNA clone were sequenced using M13 forward and M13
reverse primers to elucidate the nucleotide sequence. Deduced PvO6FAD cDNA and protein
sequence was analyzed for their basic features using online bioinformatics tools. Sequence
comparison was carried out using bl2seq program, and tree-view program was used to
construct a phylogenetic tree. The secondary structures and 3D structure of PvO6FAD
protein were predicted by using the PHYRE automatic fold recognition server. Results: The
sequencing results analysis showed that PvO6FAD cDNA is 1470 bp in length and it's open
reading frame (ORF) encodes for a protein that contains 383 amino acids. Deduced protein
sequence analysis showed the presence of essential domains of the enzyme. Protein structure
prediction is in progress and will be reported in due time. Conclusions: The 1470 bp
long PvO6FAD cDNA encodes for 383 amino acid long protein that contains conserved
domains required for biological functions of O6FAD. Further research is needed to predict
and annotate the PvO6FAD protein's 3D structure as well as to validate it. These primary
research findings will be presented in the conference.
Keywords: cDNA; Omega-6 fatty acids; Omega-6 fatty acid desaturase; Phaseolus vulgaris;
Proteins.
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Computational Analysis of Common Bean (Phaseolus vulgaris L.)

Palmitoyltransferase Gene cDNA Isolated from Bean Pod Tissue cDNA
Library
Archana.E.1, Amelia K.2 and Subhash J. Bhore*1, 2
1
Abstract: Background: The common bean (Phaseolus vulgaris L.) is a main grain legume
consumed worldwide for its edible seeds and pods. Common beans are a rich and affordable
source of proteins especially for people from low income category. To meet the demand of
growing population, we need to increase its yield. As a part of Phaseomics (an international
consortium) consortium, work of expressed sequence tags (ESTs) generation from bean pods
was initiated. Altogether, 5972 ESTs have been isolated and during analysis of data we
identified a cDNA clone of palmitoyltransferase (PvPT) gene and to understand more about it
this study was conducted. The objective of this study was to annotate PvPT cDNA and
deduce amino acid sequence using computational tools. Methods: Online bioinformatics
tools were used for analysis and annotation of both cDNA and deduced protein sequences.
Blastp was used to compare the sequence and the phylogenetic tree was constructed using
Treeview after sequence alignment is done in ClustalW. The secondary structure and tertiary
(3D) structure was predicted using PHYRE automatic fold recognition server. Results: Based
on complimentary DNA and deduced protein sequence analysis, PvPT gene cDNA is 1420 bp
in length and its open reading frame (ORF) encodes for 343 amino acids residues. The
predicted tertiary (3D) structure of PvPT protein shows that it is analogous to protein S-acyl
transferase (PAT). Conclusions: We have successfully annotated the cDNA and deduced
protein sequence of PvPT. Although 3D structure is predicted for the protein, further research
is needed to annotate the predicted structure by docking study and predicted structure needs
to be validated.
Keywords: cDNA; Common beans; ESTs; mRNA; Phaseolus vulgaris L., Protein structure.
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Annotation of Elaeis oleifera CAP cDNA and its Deduced Amino Acid
Sequence using Computational Tools
Jihan M.R.1, Amelia K.2 and Subhash J. Bhore*1, 2
1
subhash@aimst.edu.my, Ph No: +604-429 8176.
Abstract: Background: American Oil-palm (Elaeis oleifera) is an important oil producing

crop; but, it yields relatively less palm oil as compared to African Oil-palm, Elaeis guineensis
Jacq. Hence, Oil-palm plantation companies and farmers prefer African Oil-palm over
American Oil-palm for commercial plantation. In order to study the gene expression in
developing fruit tissue, transcriptomics study was initiated and about 3250 ESTs were
generated. While processing data, we identified a cDNA clone of cytosolic ascorbate
peroxidase (CAP) gene and to understand more about this gene this study was undertaken.
The main objective of this study was to annotate E. oleifera CAP cDNA and its deduced
amino acid sequence using computational tools. Methods: Both strands of CAP cDNA clone
were sequenced using M13 forward and reverse primer to elucidate the nucleotide sequence.
Online bioinformatics tools were used for analysis and annotation of both cDNA and deduced
protein sequence. Sequence comparison was carried out using Blastp whereas split tree
program was used to construct a phylogenetic tree. The secondary structure and tertiary (3D)
structure of protein was predicted using PHYRE automatic fold recognition server. Results:
Complimentary DNA and deduced protein sequence analysis showed that E. oleifera CAP
gene cDNA is 1101 bp in length and its open reading frame (ORF) encodes for 249 amino
acid residues. The predicted 3D structure of CAP protein shows that it is analogous to
ascorbate peroxidase. Conclusions: E. oleifera CAP cDNA is 1101 bp in length and its ORF
encodes for 249 amino acid residues. The primary analysis of cDNA and deduced protein
sequence suggest that it contains catalytic sites essential for the enzymes function. Further
study is required to annotate the predicted 3D structure of the protein and to validate the
predicted structure.
Keywords: American palm oil; Ascorbate peroxidase; cDNA; Cytosolic ascorbate peroxidase;
DNA; mRNA.
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Comparison of Methods for Pure Quality RNA Isolation from

Polysaccharide Rich Averrhoa carambola L. Fruits (Starfruits)
Narmataa M. and Bhore S. J.*
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, BedongSemeling Road, 08100 Bedong, Kedah, Malaysia; *corresponding author, e-mail:
subhashbhore@gmail.com / subhash@aimst.edu.my, Ph No: +604-429 8176
Abstract: Background: Starfruit plants (family: Oxalidaceae) are cultivated commercially

for its fruits in several countries, especially in tropical belt. This plant species belongs to
Averrhoa genus and A. carambola and A. blimbi species are cultivated widely. Starfruit is
fleshy, juicy, slightly tart, acidic and sweet in taste. Traditionally, Starfruit is used in
Ayurvedic and Traditional Chinese Medicines (TCM) preparations to relieve ailments such as
chronic headache, fever, cough, gastro-enteritis, ringworm infections, and skin
inflammations. Fruits are good source of antioxidants. However, this fruit contains high
oxalate which gives adverse effect to high uremic patients because of their limited ability to
excrete waste substances from bloodstream. The main objective of our research project is to
study the transcriptomics of the Starfruit. However, we are reporting the comparative analysis
of RNA extraction methods to obtain high yield and pure quality of RNA from starfruits.
Methods: Starfruits (Clone B10) were purchased in local market of Sungai Petani, Kedah,
Malaysia. The fruits were washed thoroughly to remove dirt from the fruit surface and cut
into small pieces; seeds and ridges were removed and Starfruit tissue samples were instantly
freezed using liquid nitrogen and then stored at 80 C. The samples were used for RNA
extraction using three different methods. The qualitative and quantitative analysis of isolated
total RNA samples was carried out by using agarose gel-electrophoresis and
spectrophotometry, respectively. Results: Based on the qualitative and quantitative analysis
of isolated total RNA samples, Phenol Chloroform based method of total RNA extraction
appears more effective in comparison to TriZol Reagent and Chloroform with LiCl
Precipitation based methods. The Nanodrop based quantitative analysis of isolated total
RNA samples clearly suggest that Phenol Chloroform based method of total RNA
extraction gives maximum yield of pure quality total RNA. Conclusions: For the extraction
of the pure quality total RNA from polysaccharide rich Starfruit samples, the Phenol
chloroform based method is the most efficient method.
Keywords: Averrhoa carambola; Polysaccharides; RNA quality; Starfruit; Total RNA
extraction.
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Keynote and plenary speakers

who delivered their talk in 3rdRC4Bs-2016
Appendices
Appendix I: Brief Biography of Keynote and Plenary Speakers who
delivered their talk in 3rdRC4Bs-2016.
Biography of YBhg. Dato' Professor Dr. Asma Binti Ismail (Keynote Speaker
1)
Asma Ismail is currently the first woman appointed as the

Director General of Higher Education, Ministry of Higher
Education, Malaysia. She graduated from the University of
Nevada, Reno, USA with distinction in biology, received her
MA in Microbiology from Indiana University, Bloomington,
USA and obtained her PhD in the field of Cellular and
Molecular Biology at the University of Nevada, Reno, USA
in 1986.
Her areas of expertise include Medical
Microbiology, Clinical Microbiology and Medical
Biotechnology.
Prof. Asma started her career as a lecturer in the Department
of Medical Microbiology and Parasitology, School of
Medical Sciences, Universiti Sains Malaysia (USM) in 1986.
She was a visiting scientist at University of Tokyo in 1989 and a visiting fellow at the
Medical College, St Bartholomews Hospital in London in 1992. She was promoted to
Associate Professor in 1993 and served as Deputy Dean of Administration in 1994. She was
promoted to Professor in 2000 and became Deputy Dean of Research in the same year. In
2001, she became the Director for the Centre for Medical Innovations and Technology
Development, USM. In 2003, she was appointed as the Founding Director, Institute for
Research in Molecular Medicine (INFORMM), the first multi-disciplinary cluster based
research institute for Universiti Sains Malaysia. In May, 2008 she became the first woman in
USM to hold the post of Deputy Vice-Chancellor (Research and innovation). In December
2012, she became the first woman to hold the Vice-Chancellors post at Universiti Sains
Islam Malaysia and the fifth woman to be appointed as Vice-Chancellor of a public
university.
Prof. Asmas research interest is in the scientific discovery of biomarkers and the
development of rapid diagnostics for infectious diseases especially for typhoid and
paratyphoid fever. Prof Asma is especially interested in the development of technology
platforms for diagnostics in low resource settings. Her efforts in this area was given due
recognition when she was conferred the Honorary Doctor of Science from University of
Glasgow, Scotland, United Kingdom in June 2013. Indiana University, Bloomington, USA
has also honored her work in development of rapid diagnostics for typhoid by conferring her
the Indiana Universitys Thomas Hart Benton Mural Medallion for her contributions in
medical diagnostics which was awarded on 23rd May of 2015.
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Prof Asma is among the countrys research leaders. She is actively involved in research
having attained more than RM19 million (USD 5.6 million) worth of grants within the last 5
years, presented 340 papers and received more than 184 invitations as a plenary or keynote
speaker at national and international levels to share her scientific findings and experience in
the commercialization of R&D products. Prof Asmas R&D achievements and research
impact at national and international levels have led to more than 132 scientific publications,
30 patents filed world wide of which 13 have been granted, and received more than 162
awards and recognitions at national and international levels. The prestigious awards received
include the National Young Scientist Award in 1991, Malaysian Toray Science and
Technology Award for outstanding contribution in science in 2002, National Inventor Award
in 2003, National Innovation Award, 2006 and the National Academic Award for Product
and Commercialization in 2007. She was conferred Top Research Scientist Malaysia in 2012
by Academy of Sciences Malaysia.
For her significant contribution in science, she was made a Fellow of the Academy of
Sciences Malaysia in 2003 and served as its council member from 2007-2011. In 2010, she
was made a member of the Third World Academy of Sciences (TWAS). In 2012, she
became the first woman elected as the Vice-President for Academy of Sciences Malaysia for
a period of 4 years. Based on her expertise, she served as the WHO temporary Advisor for
vaccine and diarrheal diseases since 2002.
At the National level, she is the founding chairperson for the establishment and
implementation of the National Academic Award (Anugerah Akademik Negara), a job that
she maintained for seven years to ensure the successful set up, branding and positioning of
the award. She was also entrusted to chair the Implementation of Innovative Human Capital
for the Ministry of Higher Education that developed action plans for to develop innovative
talent from cradle to career. She also chairs the Cluster Development Committee for the
Nobel Laureate grants in Physiology or Medicine under the Academy of Sciences Malaysia
and the Fundamental Research Grant Scheme (medicine) for the Ministry of Higher
Education. She is a committee member for the Establishment of Research Universities in
Malaysia and is also a member of the Assessment Committee for Research Universities by
the Ministry of Higher Education that led to the landmark establishment of Research
universities in Malaysia. She also serves as the chair for the development and direction of
R&D towards implementation of the national Strategic Planning for Higher Education. She
also helped determine the direction in Biotechnology by being a member of the Cluster
Working group on Healthcare biotechnology, Member of the Penang Biotech, Pharma and
Neutraceutical Advisory panel for Invest Penang and member for the National Strategic
Planning and Advisory for Biotechnology, Ministry of Higher Education.
Prof Asma played a key role as she co-helmed the development of the landmark Malaysian
Education Blueprint (Higher Education) 2013-2025. She is one of the authors as well as
Deputy to the blueprint project taskforce. Prof Asma is responsible for making strategic
decisions on policy directions, coordinating the blueprint development process, manage key
resources of input, engaging a broad range of stakeholders and is one of the two people who
has to sign-off the document. She is currently overseeing the implementation of the Blueprint
including the soon to be launched University Transformation Programme.
As a researcher, Prof Asma is passionate about the idea of developing indigenous health
technologies and innovations that can generate wealth for the country and improve the quality
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of life of society at large. She is committed to the development of rapid and affordable
diagnostics for infectious diseases as a means for ensuring availability and accessibility to
quality healthcare especially for the people from the underdeveloped countries. To drive this
passion, she specializes in the area of proteomics and its application in the rapid diagnosis of
infectious diseases, especially typhoid fever. Her studies on specific biomarkers led to the
discovery of an antigenically specific 50kDa of Salmonella Typhi. Prof Asma is one of the
scientists credited with the translation of the scientific discovery into four rapid diagnostic
kits for typhoid that have been successfully commercialized globally to more than 18
countries since 1994. Commercialization of TYPHIDOT, a rapid diagnostic test to diagnose
for acute typhoid, has generated sales, publications, improved the quality of healthcare
especially among the poor, generated more than 500 jobs worldwide and supported growth of
the local industries in Malaysia. In partnership with Malaysian Technology Development
Corporation, she has helped to create a startup biotech company pioneering in Biodiagnostics for the country. TYPHIDOT became the flagship product of the company which
generated at least RM 16 million in gross sales and helped to diagnose more than 2 million
cases.
Biography of Professor Dr. Eiichi Tamiya (Keynote Speaker 2)
Eiichi
Tamiya is Professor, Department of Applied physics,

Graduate School of Engineering, Osaka University. He received
Ph.D. in Science and Engineering, Graduated School, Tokyo
Institute of Technology in 1985. He has been Assistant
Professor at Tokyo Institute of Technology from1985 to 1987,
Lecturer at Tokyo Institute of Technology from 1987 to 1988
and Associate Professor at Tokyo University from 1988 to 1993
and Professor at Japan Advanced Institute of Science and
Technology from 1993 to 2007. He has been Professor at Osaka
University from 1993 until now.
His research topics have been (1) Biochips and Biosensors, (2)
Nanotechnology based bioscience and bioengineering, (3) Biomass energy conversion
systems, (4) POC (point-of-care) biosensors for medical diagnosis, food safety and
environmental protection, (5) Cell based chips for tissue and stem cell engineering.
Biography of Professor Ravichandran Manickam (Plenary Speaker 1)
Professor
M. Ravichandran obtained his M.Sc.-Medical

Microbiology from Christian Medical College, Vellore in 1991
and gained his PhD degree from Anna University, Chennai, India
in 1997. Currently his research interests are cholera vaccine,
molecular diagnostics and phage therapy. He has constructed
several cholera vaccine candidates for O139 Vibrio cholerae and
developed several simple cold chain free molecular diagnostic
kits. He has 59 publications in international journals (Cumulative
impact factor 119), supervised 36 postgraduate students, filed 8
patents and commercialized 3 products on diagnostics. He is an
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associate member of Academy of Sciences Malaysia (ASM) and Expert panel member for R,
D & C grants- Sciencefund, Technofund, Community Innovation Fund (CIF) and Enterprise
Innovation Fund (EIF), and Innovation (MOSTI); He was the Cluster Working Group (CWG)
Committee member on Human Capital Development, Malaysian Biotechnology Corporation
and the Committee member on TOP RESEARCH SCIENTISTS MALAYSIA(TRSM) of
Academy of Sciences Malaysia (ASM), Biotechnology Road map of the Kedah state was
formulated under his supervision. He is currently the Chief Executive and Vice Chancellor of
AIMST University, Kedah, Malaysia.
Biography of Dr. Bent Petersen (Plenary Speaker 2)
Bent
Petersen is an Associate Professor at the
Department of Systems Biology, Technical University of

Denmark. He has 8 years of experience within
Bioinformatics,
Computational
biology,
DNA
Sequencing, NGS data analysis, Metagenomics, Protein
structure prediction and Genome assembly of a variety of
organisms, such as bacteria, insects, mammals, parasites,
complex organisms and metagenomics samples.
Currently, he is working within the area of Metagenomics
with special focus on Next Generation Sequencing
technologies. He has made a significant contribution in exciting large variety of exciting
projects - spanning from ancient horses to modern breeds, extinct animals, malaria parasites,
and metagenomics samples from exotic places. The results have been published in highimpact journals such as Science, Nature, Cell and PNAS. In 2014, he started his own
company together with his colleagues, Bison-SeqTech, which offers fast and highly
professional bioinformatics services that are tailored to the customers needs. While
providing easily affordable access to a top 100 High-Performance Computer Systems BisonSeqTech is not hindered by traditional cluster and cloud limitations, such as insufficient
memory, processing capabilities, and disk space. He is also a passionate blogger and science
enthusiast. Read more at www.bpetersen.dk and www.bison-seqtech.dk
Biography of Professor Dr. Prakash P. Kumar (Plenary Speaker 3)
Professor
Prakash Kumars early education was from

India, and PhD (1988) from the University of Calgary,
Canada. He joined the National University of Singapore
(NUS) in 1989, where he currently serves as a full
professor at the Department of Biological Sciences. He is a
prominent scientist in the fields of plant molecular
developmental biology and biotechnology. He has
supervised 25 PhD students at NUS. He is Editor of four
international journals, namely, BMC Plant Biology,
Frontiers in Plant Science, Plant Cell Reports and Plant
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Biotechnology Reports. Several of his 90 scientific papers are published in prestigious
journals. In addition to a mangrove salinity tolerance work, he leads a major rice research
program funded by the National Research Foundation, Singapore, in collaboration with TLL
Singapore and IRRI Philippines. He has held several department and University-level senior
administrative positions at the NUS and serves on National and International committees,
including, Genetic Modification Advisory Committee, Singapore. He is the Elected
Secretary/Treasurer of Plant Biotechnology Section (2014 to 2016), Society for In Vitro
Biology, USA.
Biography of Professor Dr. Phua K.K. (Plenary Speaker 4)
Professor
Phua K.K. pursued his undergraduate and
postgraduate studies at the University of Liverpool, United

Kingdom, where he obtained his PhD in Immunology from the
Medical School in 1986. Upon his return to Malaysia, he
joined the School of Medical Sciences, Universiti Sains
Malaysia (USM) and was appointed lecturer at the Department
of Chemical Pathology in 1986, Senior lecturer at the
Department of Immunology in 1990, and Associate Professor
of Immunology in 1994. In 2004, Dr. Phua joint the Institute
for Reseach in Molecular Medicine (INFORMM), and in 2008
he was appointed Acting Deputy Director for Research and
Commercialization. In 2010 he was appoint Deputy Director for Quality and Sustainability.
In 2012, he was transferred to Chancellory USM to resume the post of Director for Industry
and Community Network. In 2013, he was appointed Research Cluster Head. Today, he is
the Director for the Division of Institutional Development, USM.
Dr. Phua has held several academic administrative posts at the School of Medical Sciences,
USM, including Program Coordinator for Diploma in Medical Laboratory Technology
(MLT) course, Block Coordinator for undergraduate Medical Doctor (MD) and Postgraduate
Masters in Medicine (M.Med) courses. Being a pioneer in Computer-Aided Instruction
(CAI), and Chairman of the first committee for implementation of CAI in the medical
curriculum, he has written many CAI programs for MLT, MD and M.Med courses for the
Medical School undergraduate and postgraduate programs. He has published many articles
on the development of CAI programs for teaching, and have shared his insights in designing
and integrating Interactive multimedia (IM) for teaching and administrative purposes in local
and international conferences, for which he was awarded the prestigous Best Technology
Using Educator ISTE (1994), APPLE Distinguished Educator award (2008) award by the
Vice President, Education Division, Apple Inc. In scholarly pursuit of excellence in teaching,
Dr. Phua had secured international grants from Apple Inc. and IBM Inc. and published his
research findings in peer-reviewed journals.
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Dr. Phua has been actively involved in research and have secured many grants for research in
Immunology, Biotechnology, ICT, Microbiology and recently Community-engagement work
in support of USMs APEX agenda for sustainability. These have yielded many publications
and learning resource materials, such as Handbooks, Student Study aids, PBL and CAI
materials, that are relevant to Malaysias health problems. Eg., his research in viral hepatitis
not only brought in grants from IDRC but also helped developed the National Immunization
Policy EPI for Hepatitis B immunization and mandatory Hepatitis C blood screening in all
blood banks in Malaysia.
He has also received many awards including the Young Scientist of the Nation Award
(Medical Sector) by the Malaysian Government in 1992, Best Invention Product 1993
(Research Exhibition, SIRIM, MOSTE), PTJ Excellence Service Awards (1994, 2003, 2006),
USMs Quality Award 2000 for development of the Postgraduate Information System, Best
Oral Fee Paper Presentation awards for Basic Sciences (2005) and Medical Education (1993)
at the National Conference in Medical Sciences, many Gold awards for Innovative and
Creative Circles 2007 (ICC, National Productivity Corp. Malaysia), AKSA 2002 Public
Services Sector Quality Innovation Award MAMPU, Gold medal from the Malaysian
Invention and Design Society (MINDS) 23rd International Invention, Innovation &
Technology Exhibition (ITEX) 2008, Bronze award (Salon International Des Inventions
Geneve, 2009), Gold medal ITEX 2012, MPC ICC Anugerah Inovasi Terbaik, Malaysian
Productivity Corporation (MPC) (Public University category), 2012. In addition Dr. Phua has
received several USM Sanggar Sanjung awards for achievements in Quality, Publications and
Research products.
As Head of the Enteric Diseases Research Cluster at USM, Prof Phua has worked with Prof
Asma the Founding Director of INFORMM, to promote Typhoid Fever research to the next
level with the help of 10 researchers and 25 postgraduate students. Amongsts the successes
of the Research Cluster include 58 publications in Citation Indexed Journals, Cumulative
impact factor of 70, 3 patent filings, and 22 awards for diagnostic products. The group has
helped to significantly reduce the incidence of Typhoid fever in the state of Kelantan.
Todate, Prof Phua has trained over 15 M.Sc. & Ph.D. students and many M.Med students,
and published over 100 papers and hold 2 IPRs.
Amongst his quality pursuits include serving as Chairman, Secretary, and Management
Representative for ISO 9001:2000, ISO 9001:2008 and ISO 17205 implementation in USM,
and have achieved the necessary accreditations from IRCA, Moody Inc. and GCP to support
these efforts.
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Biography of Dr.T.Theivasanthi (Plenary Speaker 5)
Dr.
(Ms) T.Theivasanthi is a Physicist (with nanomaterials /

nanotechnology interests) working as Assistant Professor of
Physics in International Research Centre of Kalasalingam
University, Krishnankoil. She has completed Ph.D. (Synthesis
and Characterisations of Metal Nanopowders) during 2014. She
has 12 years teaching experience, published many research
articles / books and h-index 8. She is a life member of The
Indian Science Congress Association and Magnetics Society of
India. She has achieved some awards/ honours/ recognitions
including - Madurai Women Achievers Award - 2013,
Motivational Award - 2015 and 2015 Womens Day Award of
St.Annes College.
She serves, as Editorial Board Member in Nanoscience and Nanotechnology (ISSN:21632588). Her publications have been cited 211 times which shows the high impact / value. Her
nanotechnology based innovations Worlds first plants materials based superparamagnetic
particles named Santhi Particles (has been honoured as World Record in LIMCA Book
of Records-2015 and also received award from Kalasalingam University
http://goo.gl/1F5Ru2 -Entry in GUINNESS World Record Book is under process), "The
Worlds lowest priced graphene" & "Worlds Smallest Particles of Vegetables" (have been
recognized as World Records by Limca Book of Records 2016) - http://goo.gl/QbbdFo ,
http://goo.gl/jGATFk
superparamagnetic
nanoparticles
of
graphene,
lead
(http://goo.gl/ftS0hr), semiconducting Pb Nanoparticles, agricultural nanofertilisers and
nanoparticles for treatment of diabetes, psoriasis and EBOLA, DENGUE, HIV & H1N1 virus
infection. News about her research Santhi Particles in The Economic Times & The
Telegraph. She has been invited to deliver lectures (more than 35) in many International /
National scientific conferences and foreign countries (China & Malaysia). She was an invited
speaker, for the 7th Bangalore INDIA NANO organized by the Govt. of Karnataka.
She delivered an EXPERT TALK in 2nd National Seminar on Nanotechnology: New
Materials & Applications at ITM Universe, Vadodara, Gujarat (www.prlog.org/12442698),
National conference (NCIEST-2015) in Ramco Institute of Technology, Rajapalayam,
Advances in Computational Material Science (ACMS 2015) in Central University of
Tamilnadu (http://goo.gl/SxlmmD ).
She was an Invited Speaker for PLENARY Session and Co-Chairman for Oral Presentation
in 2nd International Conference on Nanotechnology (ICNT - 2015) & Indo-USA Joint
Symposium Organized by Indian Institute of Chemical Engineers (Haldia Regional Centre) at
Haldia Institute of Technology, Haldia (Kolkatta).
She has been invited to deliver talk in Central University of Tamilnadu, Thiruvarur, FDP at
Anna University, Trichy and National Institute of Technology, Rourkela. She is delivering
Motiva tional Lectures for women, students and staff of institutes / Multi-national corporate
companies. Prime Minister Office / Dept.of Biotechnology (Govt. of India) shows interests to
develop her inventions related to nano-biotechnology for the benefits of public.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
191

Biography of Dr. Subash C.B. Gopinath (Plenary Speaker 6)
Dr. Subash Gopinath has earned his doctorate degree

from the University of Madras. Then he was working in
the Institute of Academia Sinica, Taiwan (1999-2003).
Following this he received Japan Society for the
Promotion of Science-Fellow award to advance his
research in National Institute of Advanced Industrial
Science and Technology, Tsukuba, Japan and continued
as Senior Researcher (2003-2014). Recently, he moved
to the Institute of Nano Electronic Engineering,
Universiti Malaysia Perlis (UniMAP). Dr. Gopinaths
main expertise includes biosensors, aptamer technology and
diagnosis of infectious diseases. In the past 15 years of scientific career, he produced 9
patents including an immune-aptamer that has been successfully commercialized
internationally by Wako Chemicals, Japan. His research works (total of 120) were published
in refereed scientific journals and he has 9 book chapters. His research is frequently referred
(by fellow scientific communities) with total citations of 2000 and H-index 24. The outcome
of his research and academic recognition includes the appointment as Editorial and Advisory
member of scientific journals, including Plos One and BioMed Research International. He
was awarded the Excellent Academic Award by Optical Society of Singapore, and
Distinguished Visitor, Brain Gain Malaysia by MOSTI.
Biography of Professor Dr. K. Sudesh (Plenary Speaker 7)
Professor Sudeshs main research interest is in the design and

synthesis of biodegradable polyhydroxyalkanoates (PHAs)
using microbial systems. He started research in this area in
1992 and obtained his Masters in Biotechnology from
University Malaya. Then, he continued his research for his
PhD, which was sponsored by Japanese Government
(Monbusho). The research was conducted at RIKEN Institute,
Japan under the supervision of Prof Y. Doi.
He obtained his PhD in 1999 and then continued as a Special
Postdoctoral Researcher at RIKEN. He returned to Malaysia
under the Brain Gain program and joined the School of
Biological Sciences, Universiti Sains Malaysia as a lecturer in
Nov., 2001 and became a full professor in 2011.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
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He has significantly contributed to the research and development of biodegradable plastics in
Malaysia from palm oil products. In addition to the numerous scientific publications in both
local and international journals, he has 5 granted patents, two of which has been successfully
licensed.
Biography of Professor Aziz Amine (Plenary Speaker 8)
Professor
Aziz Amine, has been Head of Department of

Chemical Engineering and Environment of the Hassan II
University of Casablanca during the period 1999-2003.
Professor Amines research over the last 25 years has focused
on sensors and biosensors and there use in Analytical
Chemistry. He is author of more than 110 papers and has served
as coordinator of several national and international research
projects. He is a reviewer for several scientific international
journals.
He is one of the Editors of the International Journal Biosensors
and Bioelectronics published by Elsevier with Impact Factor
6.5.
Chairman of the International Workshop Biosensors for Food Safety and Environmental
Monitoring organized every two years in Morocco (www.biocap.ma).
He was invited speaker in several congresses. He has more than 3400 citations in Scopus and
has an h-index of 32.
Biography of Professor Dr. Quamrul Hasan (Plenary Speaker 9)
Professor Dr.
Quamrul Hasan has a Ph.D. in Biotechnology
from Kyoto University, Japan. Prior to joining at Universiti

Utara Malaysia (UUM) as a full professor in August 2014 he
was managing his own firm-Bioinnovare Co., Ltd., an
international business development consulting company, based
in Kobe, Japan which he founded in 2009. Prior to that he was a
Professor at Japan Advanced Institute of Science and
Technology (JAIST), a national postgraduate university, in
Ishikawa, Japan (1997-2005). He also worked for Procter &
Gamble Company, as an R&D Scientist and Manager (19942001).
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
193

Prof. Dr. Quamrul Hasan established a non-profit organization Japan Halal Research
Institute (JAHARI) in Hyogo, Japan in 2014 and he became the founder Chairman of this
organization. A Japanese national, he had been living in Kobe, Japan with his family since
1994.
Some of the key achievements of Prof. Dr. Quamrul Hasan are:
1. Professional biotechnologist with more than twenty five years of experiences in
research and management (in Japan and USA)
2. Extensively experienced in both the Western and Japanese (multi-cultural) business
settings.
3. Invented, co-developed and successfully launched a health-care consumer product,
from original idea and laboratory test to prototyping and field-testing (FebrezeAllergen Reducer has been globally marketed by Procter & Gamble since 2004)
4. Recipient of Procter & Gamble Innovation Award
5. Published more than 50 patents and articles.
At UUM, currently Prof. Quamrul Hasan is also the Director of an international research
center collaborating with the universities and companies in Japan, which were initiated by
him.
Biography of Professor Dr. Mohd Zaki Salleh (Plenary Speaker 10)
Professor
Dato Dr. Mohd Zaki Salleh started his academic

career at the Department of Medical Microbiology and
Parasitology, School of Medical Sciences, Universiti Sains
Malaysia in 1995 at the age of 40 years old after obtaining PhD
from Universiti Sains Malaysia under the supervision of Prof.
Dato Dr. Asma Ismail and Prof. Dr. Zainul F Zainuddin. Prior
to that, he was a marketing executive at an American Firm, a
Bank Officer at Chung Khiaw Bank Ltd (Singapore
Incorporated) and a Credit Officer at Mayban Finance Bhd.
In 2004, he left Institute for Research In Molecular Medicine (INFORMM), Universiti Sains
Malaysia to join UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan
Malaysia. In 2005, he joined Malaysian BioDiagnostics Research Sdn Bhd (MBDr) as the
Technical and Business Development Director. His passion in academic and research
prompted him to join the Faculty of Pharmacy, Universiti Teknologi MARA (UiTM) in 2007.
In 2008, together with Prof Teh Lay Kek, they started the Pharmacogenomics Centre
(PROMISE). In September 2013, the centre was upgraded to an institute and was named as
Integrative Pharmacogenomics Institute (iPROMISE) and he was appointed as the founding
director of the institute. Their research areas include pharmacogenomics, genomics,
proteomics, metabolomics and epigenetics. They had managed to develop and innovate a few
PCR based genotyping kits to identify patients of different genetic backgrounds for CYPs,
MDR1 and VKORC1 to help make therapy more personalized for patients. Some of these
kits have been made available to hospitals. They are also in the forefront in Malaysia for
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
194

whole genome sequencing and metabolomics studies. They had performed whole genome
sequencing of a few Malay Human Genomes, some subtribes of the Orang Asli of Malaysia,
some pathogens and more genomes are in the pipeline. In metabolomics, they look for
potential biomarkers in cancers and responses to drugs using LCMS-QTOF. From the
research in natural products, a few compounds were isolated and are being optimized using in
silico models to produce drugs for anti-inflammatory disorders.
They hope to generate high quality research knowledge and information in genome sequences
and useful metabolomics and proteomics markers for better patients management in
Malaysia. They also provide services and consultations in whole genome sequencing and
metabolomics related studies to the local scientists. iPROMISE had established some
collaborations in research which include the local universities, Malaysian Ministry of Health,
Malaysian Bureau of Pharmacy, Agilent Technologies, CSIR-IGIB India and the National
University of Singapore.
In 2014, Prof. Zaki was appointed as an Adjunct Professor to the Institute of Genomics and
Integrative Biology, Council of Scientific and Industrial Research (CSIR), India. Together
with a few other researchers, the Malaysian Metabolomics Society (MyMs) was established
in 2011 and Prof Zaki was appointed as the founding president.
Biography of Dr. Werasak S. (Plenary Speaker 11)
Dr. Werasak Surareungchai received his bachelor degree in

Chemistry and Biology from Silpakorn University, Thailand
and his Ph.D. from Cranfield University, UK in 1997. He is
now an associate professor at KMUTT. He chairs the Sensor
research lab and the Nano research cluster at KMUTT.
He is the founder of a spinoff company, Quasense which is
commercializing custom-made screen printed electrodes and
electrochemical devices. His research interests are biosensors,
electroanalytical chemistry and bionanotechnology.
Biography of Professor J.-F. F. Weber (Plenary Speaker 12)

Natural product chemist: Pharmacy degree (Reims, France,
1981), Master in Pharmacochemistry of Natural Products
(Paris, France, 1984), PhD in Phytochemistry under the
supervision of Prof. Bruneton (Angers, France, 1988).
1991: First academic appointment at U. of Bordeaux, Faculty
of Pharmacy, Laboratory of Pharmacognosy; main research
theme: polyphenols and tannins from grape vine and other
plants.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
195

Associate Prof. at the National University of Malaysia UKM, Pharmacy Department

(1997); main research theme: oligostilbenes (polyphenols) from Malaysian timber trees
Professor at the MARA University of Technology UiTM, Faculty of Pharmacy (2003)

o Conceived and set up Atta-ur-Rahman Institute for Natural Product Discovery
o Head of the Microbial Metabolite Lab ( 20 members)
o Main research themes:
oligostilbenes (polyphenols) from Malaysian timber trees
drug discovery from bioactive fungal secondary metabolites
40 research papers in peer-reviewed journals, 10 PhD graduates, 2 post-docs. Currently
supervisor or co-supervisor of 10 MSc and PhD students by research only.
Biography of Dr. Latiful Bari (Plenary Speaker 13)
Dr.
Latiful Bari completed his Ph.D. in Veterinary public

health in 2001 from Osaka Prefecture University, Japan and
M.Sc in Microbiology in 1994 from the University of Dhaka.
He did his post-doctoral work under JSPS fellowship at
National Food Research Institute, Tsukuba, Japan and
thereafter continued his research work on food safety and
hygiene and the improvement of food safety status in different
countries. His research work produces more than 100 peer
reviewed publications in Journals, books-chapters, booklets
etc. Many of his articles are very popular in the field of food
protection.
Dr. Bari, joined at the University of Dhaka in June 2010 and working on the same field and
trying to improve the hygiene and food safety status in Bangladesh.
Dr. Baris current research including intervention strategies for controlling foodborne
pathogens and develop strategies for performing the technology in commercial scale:
Microbiological safety of minimally processed foods; fresh produce, meat and meat products
etc. Develop predictive modeling for agricultural products and used in microbial risk
assessments. Emerging technologies in food preservation, Processes to reduce pathogen
contamination in foods, Food irradiation, chemicals and biochemicals used in food
preservation and application of combined factors technology in the shelf life and safety of
minimally processed foods including fruits and vegetables.
His recent research develop lot of bio based products for water treatment, soil fertility
improvement using microbiological model, natural de-coloring agents and so on. Besides his
research, In addition to his research, he has social connection worldwide to the scientific
community, and he is serving as general secretary for Asian Food Safety and Security
Association (AFSA).
Dr. Bari provides laboratory training to the scientists, students, working people, and govt.
employee, on microbial detection and intervention procedure in food.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
196

Biography of Prof. M. Anwar Hossain (Plenary Speaker 14)
Professor M. Anwar Hossain awarded PhD in Pharmaceutical

Analytical Chemistry 1991 from University of Tokyo, Japan,
and B.Sc (Hons) & M.Sc in Biochemistry and Molecular
Biology in 1981 & 1983 from the University of Dhaka. He did
his post-doctoral work in industry Suntory Crop., Osaka, Japan
and in academia UMDNJ, Robert-Wood Johnson Medical
University, NJ, USA. Dr. Hossain joined as faculty member in
the department of Biochemistry and Molecular Biology in 1984
and currently serving as a Professor since 1997 in Department
of Microbiology, University of Dhaka.
He was Chairman of Microbiology Department from 20092012 and 1997. Dr. Hossain served as Visiting Professor,
Department of Cultural Fisheries, Faculty of Agriculture, Kochi University, Japan and RheinWaal University, Germany. Dr. Hossain served or has been serving in the highest
authoritative bodies like Senate, Syndicate, Regent bodies, Academic Council and Board of
Advance studies in more than six national or private universities in Bangladesh. He has been
serving as National Expert member in National Task Force on National Biotechnology for
Bangladesh since 2009 to date the committee which is chaired by the Prime Minister,
Government of the Peoples Republic of Bangladesh. Dr. Hossain had served as Board
Governors Member Bangladesh Council for Scientific and Industrial Research, (BCSIR),
(S&IT Minister Nominee) & Council Member, BCSIR, 2009-2015.
His research work produced more than 120 peer reviewed publications in Journals, bookschapters, proceedings and abstract in international conferences. Dr. Hossain awarded
international bodies fellowships Common Wealth, Monbusho, USESCO, FOBMB, Fida
Foundation etc. Dr. Hossain awarded best researcher award in 2011 by University Grants
Commissions of Bangladesh. Currently, Dr. Hossain and his team have been working in
Foot-and Mouth Disease Virus and vaccine development; Antibiotics resistant mechanism
and pollutions; Poultry Salmonella and route of transmission of pathogens and resistant
bacteria by migratory birds; Arsenic microbiomes and bioremediations; and bioinformatics.
In addition to his research, he has social connection worldwide to the scientific community,
and he is serving as Vice-President and also founder of Asian Food Safety and Security
Association (AFSA).
He had served as secretary general and president of Bangladesh Society of Microbiologists;
and also organized two international conferences one in 2010 and other in 2015. He is/was
also member of large numbers of national and international professional organization like
American Society of Microbiology, NY academy of Science, USA and The Society for
Neuroscience, USA.
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
197
Scientific programme of the 3rdRC4Bs-2016
Appendix II: A copy of scientific programme of the 3rdRC4Bs-2016.
Day 1: 20/04/2016 (Wednesday)

Preconference Workshop
Programme
Time
08.00-09.00
Registration
Workshop-1: Drug-Protein Interaction and Personalized Medicine.
Speaker: Ms. Monisha Hajra, ScientiaBio, Bangalore, India
09.00-17.00
Time
Workshop-2: Basic Statistics using Microsoft Excel and SPSS &

Basic Concept of Effective Scientific Communications.
Speakers: Snr. Assoc. Prof. Dr. K. Marimuthu and Snr. Assoc. Prof.
Dr. P. Balakumar, AIMST University, Malaysia
Registration of Participants (Main Conference)
Programme
15.00-19.00
Registration
15.15-19.00
Check-in (Pre-booked participants only)
09.00 - 10.45
Venue
Medical Building,
AIMST University
Guest House /
Apartment /
Hostels of AIMST
University
Day 2: 21/04/2016 (Thursday)

Programme
Time
07.30 - 08.45
Venue
Medical/ Dental
Building
Ground Floor
Computer Lab,
Medical Building,
AIMST University
Third Floor
Computer Lab,
Medical Building,
AIMST University
Registration
Venue
Great Hall
Opening Ceremony
Welcome Address
Snr. Assoc. Prof. Dr. Subhash J. Bhore
Chairman, 3rd Regional Conference on Biosensors, Biodiagnostics,
Biochips and Biotechnology 2016
Felicitation Speech
Snr. Prof. Dr. M. Ravichandran
Vice Chancellor and Chief Executive, AIMST University, Malaysia
Introduction of Keynote Speaker
Prof. Dr. Mohd. Baidi bin Bahari
Deputy Vice Chancellor (Research and Innovation), AIMST University,
Malaysia
Officiating and Keynote Address
YBhg. Dato' Prof. Dr. Asma Binti Ismail
Director General, Department of Higher Education, Ministry of Higher
Education, Malaysia
Keynote Address Talk Title: Moving the Regional Biotechnology and
Bioeconomy Forward
10.45-11.00
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Tea break
Great Hall
Foyer
198

Session: 1
Chair: Prof. Dr. K. Sudesh, University Sains Malaysia, Malaysia
Co-chair: Prof. Dr. Mohd. Baidi Bahari, AIMST University, Malaysia
Current Progress in Cholera Diagnostics
11.00-11.30
P1
Snr. Prof. Dr. M. Ravichandran, AIMST University, Malaysia
Great Hall
Supercomputing in Biotechnology: making sense of big data

11.30-12.00
P2
12.30-12.40
Associate Prof. Bent Petersen, Technical University of Denmark,

Denmark
Relevance of Biotechnological Applications for Global Food
Security and Sustainability
P3
Prof. Prakash P. Kumar, National University of Singapore,
Singapore
Summary and conclusion of the session
12.45-14.00
Lunch
Cafeteria,
1st Floor
13.15-14.00
Poster evaluation
Medical
Building
12.00-12.30
Great Hall
Session 2
Session 2A
Chair: Prof. M Anwar Hossain, University of Dhaka, Bangladesh
Co-chair: Dr. Lee S. Y., FAS, AIMST University, Malaysia
Molecular Approaches to Fundamental Studies on Biomarkers and
Development of Sustainable Rapid Nano-biodiagnostics to Enteric
Diseases for Low Resources Settings
14.00-14.30
P4
Prof. Dr. Phua K. K, Universiti Sains Malaysia (USM), Malaysia
14.30 -14.45
OP1
Paper-based visual detection of Salmonella bacteria using

Isothermal DNA amplification and magnetic beads aggregation
Dr. Ahmed M.U., Universiti Brunei Darussalam, Brunei
14.45 -15.00
OP2
15.00 -15.15
OP3
Development of a Reverse Hybridization Assay (RHA) for

Simultaneous Identification of Salmonella Serotypes Causing
Enteric Fever
Carlos S., Universiti Sains Malaysia (USM), Malaysia
Decrypting the Evolutionary Path of Antimicrobial Resistance of
Acinetobacter baumannii via Next-Gen Sequencing
Mohamad Izwan Ismail, Universiti Teknologi MARA (UiTM)
Puncak Alam, Malaysia
15.15- 15.25
Summary and conclusion
15.25-16.00
Tea Break
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Lecture
Theatre- 1,
Medical
building
Medical
building
199

Session 2B
Chair: Assoc. Prof. Dr. S. Kathiresan, AIMST University, Malaysia
Co-chair: Snr. Assoc. Prof. Dr. V. Ravichandran, AIMST University, Malaysia
Bio-Applications of Innovative Nano-materials
14.00-14.30
P5
Dr. T. Theivasanthi, Kalasalingam University, India
Isoluminol-functionalized gold nanoparticles and graphene oxide
nanoribbons composite for development of enzyme-based
14.30 -14.45
OP4 electrochemiluminescence biosensors
Nur Syakimah Ismail, Universiti Malaysia Perlis, Malaysia
14.45 -15.00
OP5
15.00 -15.15
OP6
Disposable Screen-Printed Electrodes Modified With Nanoparticles

for Sucrose Sensor
Detpisuttitham W., King Mongkut's University of Technology,
Thailand
Cloning and expression of the urease operon from Helicobacter
pylori J99
Lecture
Theatre- 2,
Medical
building
Mohamad CWSR, Universiti Malaysia Perlis, Malaysia.

15.15- 15.25
15.25-16.00
Tea Break
Medical
building
Session 2C
Chair: Assoc. Prof. Dr. Bent Petersen, Technical University of Denmark, Denmark
Co-chair: Dr. Ramesh Kumaresan, AIMST University, Malaysia
14.00-14.30
14.30 -14.45
14.45 -15.00
15.00 -15.15
15.15- 15.25
15.25-16.00
P6
OP7
Aptasensors: Bench to Bedside and Beyond

Dr.
Gopinath
S.C.B,
Universiti
(UniMAP),Malaysia
Malaysia
Perlis
Analysis of chalcone-flavanone isomerase (CHI) gene cDNA

isolated from American oil-palm (Elaeis oleifera) mesocarp tissue
cDNA library
Yu Chuen Leow, AIMST University, Malaysia
OP8
OP9
Lecture
Theatre- 3,
Medical
building
Non-Protein coding RNA genes as novel diagnostic markers to

detect pathogenic bacteria
Suresh C.V., AIMST University, Malaysia
Herbal based stabilisers of native and misfolded state of nuclear
co-repressor (N-CoR)
Matiullah Khan, AIMST University, Malaysia
Lecture
Theatre- 3,
Medical
building
Medical
building
Tea Break
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
200

Session 3
Session 3A
Chair: Prof. Dr. Tang Thean Hock, University Sains Malaysia
Co-chair: Dr. Yu Chye Wah, AIMST University, Malaysia
16.00-16.30
P7
16.30 -16.45
OP10
16.45 -17.00
OP11
17.00 -17.15
OP12
Recent progress in the production of biodegradable plastics from

palm oil in Malaysia
Sudesh K., Universiti Sains Malaysia, Malaysia
Hepatoprotective effect of methanol extract of Polygonum minus
leaves in carbon tetrachloride-induced liver damage in rats
Christapher V, AIMST University, Malaysia
Molluscicidal Effect of Poly herbal Extracts on Golden Apple
Snail, Pomacea maculata
Guruswamy Prabhakaran, AIMST University, Malaysia.
Production of Butter Flavour Concentrate from Butter fat with
Lactic Acid Bacteria by Solid Substrate Fermentation
Thambirajah J.J, Department of Biotechnology, AIMST
University, Malaysia.
17.15- 17.30
17.30-18.00
Poster evaluation
Lecture
Theatre-1,
Medical
building
Medical
Building
Session 3B
Chair: Assoc. Prof. Dr. Chan Yean Yean, University Sains Malaysia
Co-chair: Dr. Sawri Rajan, AIMST University, Malaysia
16.00-16.30
P8
Recent advances in biosensors based on enzyme inhibition

Prof. Aziz Amine, Hassan II University of Casablanca, Morocco
Design and characterization in time of an on-off DNA biosensor
16.30 -16.45
16.45 -17.00
17.00 -17.15
17.15- 17.30
17.30-18.00
OP13
OP14
OP15
Guajardo-Yvenes C. F, King Mongkut's University of Technology

Thonbury, Bangkok, Thailand.
Optimization of PCR for rapid detection of CTX-M gene in ESBL
producing Klebsiella pneumoniae clinical isolates
Rasheeda B, Lahore College for Women University Lahore,
Pakistan
Lecture
Theatre-2,
Medical
building
Coenzyme Q10 dietary supplementation during antitubercular

therapy prevents renal damage in rats
Evan Prince Sabina, School of Biosciences and Technology, VIT
University, Vellore, India
Poster evaluation
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Medical
Building
201

Session 3C
Chair: Dr. Gopinath S.C.B, Universiti Malaysia Perlis (UniMAP),Malaysia
Co-chair: Dr. Subhash J. Bhore, AIMST University, Malaysia
16.00-16.30
P9
16.30 -16.45
OP16
Nano- and Bio-technological Advancement to assist in the

Prof. Quamrul Hasan, Universiti Utara Malaysia (UUM),
Malaysia & Chairman, Japan Halal Research Institute for
Products and Services (JAHARI), Japan
Lecture
Theatre- 3,
Medical
building
Umami Tasting Detection Based Electrochemical Sensor
17.15- 17.30
Chaiboon T, King Mongkut's University of Technology Thonburi,

Bang KhunThian, Bangkok 10150, Thailand.
Detection of Salmonella enterica serovar Typhi Form Water
Samples and Its Association with Geographical Clustering of
OP17
Enteric Fever
Ismail Aziah, Universiti Sains Malaysia, Malaysia
The fabrication of membrane-based pneumatic microvalves in
microfluidic system
OP18
Ngamchana S,
King Mongkuts University of Technology
Thonburi (KMUTT), Bangkok 10150 Thailand.
17.30-18.00
Poster evaluation
19.00-22.30
Cultural Programme and Conference Dinner
16.45 -17.00
17.00 -17.15
Lecture
Theatre- 3,
Medical
building
Medical
Building
Great Hall
Day 3: 22/04/2016
09.00-09.10
09.10-09.55
09.55-10.10
Introduction of Key note Speaker by Snr. Prof. M. Ravichandran, VC &

CEO, AIMST University, Malaysia
Keynote Address
Tamiya E., Osaka University, Japan
Nanotechnology oriented biosensors and biomedical application
Tamiya E., Professor, Department of Applied Physics, Graduate School of
Engineering, Osaka University, Japan
Tea Break
Lecture
Theatre- 1,
Medical
building
Session 4
Session 4A
Chair: Snr. Assoc. Prof. Dr. P. Balakumar, AIMST University, Malaysia
Co-chair: Dr. Annie Jeyachristy, AIMST University, Malaysia
10.10-10.40
10.40 -10.55
P10
OP19
Genomics of the endangered Orang Asli: disease susceptibility

and sustainability
Dato Prof. Dr. Mohd Zaki Salleh., UiTM, Malaysia
Role of Outer member proteins (OMP) and lipopolysaccharides
(LPS) in antibody response against Pasteurella multocida type B2 in bovines
Imran A, University of Veterinary and Animal Sciences
(UVAS),Pakistan
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Lecture
Theatre- 1,
Medical
building
Lecture
Theatre- 1,
Medical
building
202
11.40- 11.55
12.00-14.30
Exploration of Novel Endophytic Bacterial Isolates for Their

Antioxidant and Pro-oxidant Properties
Monowar T, AIMST University, Kedah, Malaysia
Sensitivity analysis of graphene based surface plasmon resonance
biosensor
OP21
Toloue H., University Teknologi Malaysia, Kuala Lumpur,
Malaysia
Ameliorative Effect of Curcumin on Olanzapine Induced Obesity
OP22 in Sprague Dawley Rats
Parasuraman. S, AIMST University, Malaysia
Lunch
13.10-14.30
Poster evaluation
10.55 -11.10
11.10 -11.25
11.25 -11.40
OP20
Cafeteria
Medical
building
Session 4B
Chair: Prof. Md. Latiful Bari, University of Dhaka
Co-chair: Dr. Suresh C.V., AIMST University, Malaysia
11.40- 11.55
12.00-14.30
Highly Sensitive Detection of DNA Hybridization and

Immunoassay Based on Nanomaterials
P11
Werasak Surareungchai, King Mongkuts University of
Technology Thonburi, Thailand
Study of Nanoparticle-Modified Screen-Printed Electrodes for
detection of Sudan I contamination in chili
OP23
Phanthong C,
King Mongkuts University of Technology
Thonburi (KMUTT), Thailand.
Bioengineering of Tacca integrifolia (Bat flower): effects of
hormones on in vitro rooting and production of Taccalonolides
OP24
Fatimah A.L,
UniversitiTeknologi MARA, Shah Alam,
Selangor,Malaysia
Isolation, characterization and potential application of
OP25 bacteriophages for phage therapy
Bhandare, S.G, Universiti Malaysia Kelantan, Kelantan, Malaysia
Reconfigurable Filter Bank for Accurate Spectral Decomposition
OP26 of EEG Signals
Girish Kumar C., AIMST University, Malaysia
Lunch
13.10-14.30
Poster evaluation
10.10-10.40
10.40 -10.55
10.55 -11.10
11.10 -11.25
11.25 -11.40
Lecture
Theatre-2,
Medical
building
Lecture
Theatre-2,
Medical
building
Cafeteria
Medical
building
Session 4C
Chair: Prof. Dr. Pandurangan, AIMST University, Malaysia

Co-chair: Dr. Leela A. J, AIMST University, Malaysia
10.10-10.40
P12
P12 - Fungal Secondary Metabolites - A Pharmaceutical Chemist

Perspective
Prof. Dr. J.-F. F. Weber, Universiti Teknologi MARA, Kampus
Puncak Alam, Malaysia
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
Lecture
Theatre-3,
Medical
building
203
10.40 -10.55
OP27
10.55 -11.10
OP28
11.10 -11.25
OP29
11.25-11.40
OP30
11.40-11.55
OP31
Eco-friendly Biosynthesis of Atrocarpusaltilis Mediated Silver

Nanoparticles - Characterization and Evaluation of its
Antimicrobial and Antioxidant Potential
Dr. Ravichandran V, AIMST University, Malaysia
Mutiplex Isothermal Amplification for Detection of Melioidosis
Jilien M. W. T, School of Medical Science, Universiti Sains
Malaysia, 16150 KubangKerian, Kelantan.
Effective Pulmonary Therapeutic Delivery via Surface Acoustic
Waves Nebulization and Phononic Crystal Structures
Mohd H. Ismail, School of Microelectronic Engineering,
Universiti Malaysia Perlis, Malaysia.
Development of a Novel Duplex PCR Assay for Specific
Detection of Salmonella enterica subspecies enterica serovar
Typhi Based on Single-Gene Target
Goay Y. X., INFORMM, Health Campus, Universiti Sains
Malaysia, Kelantan, Malaysia.
Lecture
Theatre-3,
Medical
building
Assessment of Biodiesel Properties From the FAME Composition

of a Malaysian Rhodophyte (Kappaphycus sp.)
Md. Sayedur Rahman, Aimst University
11.55- 12.10
12.10-14.30

Lunch
13.10-14.30
Poster evaluation
Cafeteria
Medical
building
Session 5
Session 5A
Chair: Snr. Assoc. Prof. Dr. K. Marimuthu, AIMST University, Malaysia

Co-chair: Dr. Kazi A Selim, AIMST University, Malaysia
14.30-15.00
P13
15.00 -15.30
P14
15.30 -15.45
OP32
15.45 -16.00
OP33
16.00- 16.15
Safe Water as the Key to Food Safety & Global Health

Associate Prof. Md. Latiful Bari, University of Dhaka, Bangladesh
Foot-and-Mouth Disease: Current Scenario in Asia and
Bangladesh
Prof. M Anwar Hossain, University of Dhaka, Bangladesh
Generation of RNA aptamers against Mycobacterium tuberculosis
secretory protein ESAT-6 - a preliminary study
Lecture
Theatre-1,
Medical
building
Bakhtiar B, AMDI, Universiti Sains Malaysia, Kepala Batas,

Malaysia.
An Expression Analysis of Salmonella Pathogenicity Island (SPI)Derived Non-Protein Coding RNAs in S. Typhi Biofilm formation
Anbalagan, INFORMM, Universiti Sains Malaysia, Malaysia.
Lecture
Theatre-1,
Medical
building
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
204

Session 5B
Chair: Snr. Prof. Dr. M. Ravichandran, AIMST University, Malaysia
Co-chair: Assoc. Prof. Dr. Matiullah Khan, AIMST University, Malaysia
14.30-14.45
OP34
14.45 -15.00
OP35
15.00 -15.15
15.15 -15.30
15.30- 15.45
15.45- 16.00
Quantitative, Single-Step Measurement of Hemoglobin A1c in

Whole Blood for Personalized Medicine
Khor S. M., University of Malaya (UM), Malaysia
Development of rapid diagnostic detection for Salmonella enterica
subspecies entericaserovar Paratyphi A using cross priming
amplification
Roziana, M.H, INFORMM, Universiti Sains Malaysia, , Kelantan,
Malaysia
Conversion of rice husks to polyhydroxyalkanoate (PHA)
OP36
Heng K.S, Universiti Sains Malaysia, Penang, Malaysia
Lecture
Theatre-3,
Medical
building
Salmonella typhimurium Detection based on Electrochemical

Immunoassay using Methylene blue/MWNTs/Magnetic Particle
OP37
Ngoensawat, U, King Mongkut's University of Technology,
Bangkok 10150, Thailand.
Electrochemical Characterisation and Determination of
Mycobacterium Tuberculosis by Voltammetry at Polymer
OP38
Nanocomposite modified Platform
Himkusha Thakur, Panjab University, Chandigarh, India
Valedictory function
15.45-16.15
16.15- 17.00
Welcome
Prizes and awards
Vote of thanks
Lecture
Theatre- 1,
Medical
building
Tea Break
Note: K, Keynote address; P, plenary talk; OP, oral presentation
------- End of the Conference ----
ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1
205
WITH BEST COMPLIMENTS
206
207
208
About Editor
Subhash Bhore, PhD:
Subhash completed his
BSc (Botany) and MSc
(Botany)
degree
education at University of
Pune, India. Immediately
after completing his MSc
(May 1996), he got an
opportunity to work at
Biochemical Engineering
Department and Plant Tissue Culture Pilot
Plant of the National Chemical Laboratory,
Pune, India. In June 2000, he received a
Doctoral Fellowship (GRA) to pursue a PhD
Degree in Molecular Genetics at the National
University of Malaysia (UKM). In 2004, he was
appointed as Senior Research Officer at
Melaka Institute of Biotechnology (MIB), a
research wing of Melaka Biotechnology
Corporation,
Malaysia.
Based
on
his
performance, in April 2005, he was promoted
as Principal Investigator & Head of R&D
Department at MIB, Malaysia. In 2008, he was
invited by the AIMST University as a Visiting
Faculty for their Department of Biotechnology
and now serving as a Senior Associate
Professor. In 2009, he was nominated for the
AASIO (Association of Agricultural Scientists of
Indian Origin) Young Scientist Award. He has
published more than 45 peer-reviewed articles,
4 books, and submitted more than 11,900 DNA
sequences in Gene Bank, and got more than
10 awards/fellowships. As of May 2016, he has
supervised more than 67 students including
postgraduates, undergraduates and industrial
trainees. He is actively involved in research as
well as teaching and advising of postgraduate
and undergraduate students. You may contact
him using email, subhash@aimst.edu.my or
subhashbhore@gmail.com

Published by AIMST University

Research Highlights in 4Bs: Biosensors, Biodiagnostics, Biochips and Biotechnology

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Research Highlights in 4Bs: Biosensors, Biodiagnostics, Biochips and Biotechnology

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Research Highlights in 4Bs

Biosensors, Biodiagnostics, Biochips and Biotechnology

Proceedings of the 3rd Regional Conference on Biosensors,