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**Research Highlights in 4Bs**

**Biosensors, Biodiagnostics, Biochips and Biotechnology**

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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

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**Research Highlights in 4Bs**

**Biosensors, Biodiagnostics, Biochips and Biotechnology**

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

Biosensors, Biodiagnostics, Biochips and Biotechnology

2016 (3rdRC4Bs-2016) and for this Book was provided

by:

Conference was jointly organized by:

Conference was supported by:

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**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 this conference a successful

event.

**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**

**Publicity & Exhibitions Committee**

Dr. Annie Jeyachristy

Dr. Sivachandran P.

Dr. Heera Rajandas

Mr. Siventhiran B.

Mr. M. K. Faiz

**Treasurer**

Mr. Dhanaraj R.

Mr. Arvinth Murugiah

Mr. P. K. Karuna

Mr. Vijayan Krishnan

****

**Reception Committee**

**Logistics Committee**

Ms. Sridevi Visvanathan

Ms. Musalinah Buzri

Ms. Elil Suthamathi

Mr. V. Krishnan

Dr. Tahmina Monowar

Ms. Yoganandhini

Ms. Azdlina

Mr. Christapher V.

**First Aid and Safety Committee**

Mr. Mahes D.S.

Dr. Sawri Rajan

Mr. G. Prabhakaran

Dr. Leela A. J.

Dr. D. Jawahar

Mr. Maheswaran S.

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ iv

**Foreword**

**I** t 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 3rdRC4Bs-

2016. 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_ v

## Preface

****

**T** he 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_

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 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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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

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ viii

Exploration of Novel Endophytic Bacterial Isolates for Their Antioxidant and Pro-

oxidant 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 Non-

Protein 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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_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 pneumo_ niae 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

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ x

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 Drug-

Induced 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-L-

methionine 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

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 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

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ xii

Men love to wonder, and that is the seed of science.

\--- Ralph Waldo Emerson

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ xiii

**Research Highlights in 4Bs**

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_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 activator–like 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**

based on a plenary talk delivered at a

conference. Based on scientific evidence and

The use of genetically modified (GM) crops

first-hand experience with GM plant

has been made into a controversial topic that

production, the intended take-home message

elicits quite polarized views. This article is

of this talk was that we should not hesitate to

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 1

_Res. Highl. 4Bs (2016)_

_Global Food Security and Sustainability_ Kumar use

modern

technologies

for

crop

b) Expression of introduced (foreign) gene

improvement. It is acknowledged that

to confer a new/modified trait to the crop

having adequate tests to verify the general

plant, e.g., carotenoid production in the

safety of the GM crops is important, but

endosperm tissue of the Golden Rice grains.

once proven safe (or not significantly

different from the conventional crop

_**The main examples of GM plants**_

variety), the GM crop should be used as a

The main examples of GM plants that have

valuable alternative. With judicious use of

been commercially cultivated for the last 20

technology and crop management practices,

or so years include herbicide resistant and

we can achieve global food security.

insect resistant plants. The major crops with

such GM varieties include corn, soybean,

**GM FOOD**

canola and cotton. In 2013, GM crops were

****

grown in about 175 million hectares of land

_**What is Genetic Modification (GM) and is**_

by 18 million farmers in 27 countries

_**it vastly different from conventional plant**_

(James, 2013). The global market value of

_**breeding in its principle?**_

GM crops for 2013 was estimated to be

Crop modification has been ongoing for

about US$15.6 billion.

thousands of years. The use of ever-

changing technologies has been a constant

Some of the other traits being introduced to

theme in this as with all other modern

crop plants include drought tolerance,

human endeavors. We recognize that use of

disease tolerance (both fungal and viral),

any novel technology involves risks and

resistance towards root nematodes, modified

benefits, it is up to us to evaluate them and

nutritional parameters such as altered lipid

decide to adopt them if the benefits vastly

profiles in oil crops and fortified grains.

outweigh risks. Also, human ingenuity is

Although the Golden Rice has been fully

such that technology can be constantly

developed and safety tested – it has gone

refined to minimize the initial risks to

through the so called deregulation process

negligible levels as was done with the

mandatory for GM crops – it has not yet

minimization of radiation emission in the

received

the

necessary

Government

present day cell phone or mobile phone

approvals. The Golden Rice has been touted

technology. The yield enhancement as a

as a staple grain that will lead to vast

result of the Green Revolution has reached

improvements in the nutritional status (with

its limit now. Although it is known that crop

respect to vitamin A and iron) of millions of

plants have not reached their biological

poor

people

in

Asia

and

Africa.

upper limits for yield increase, any further

Nevertheless, it has not been released to the

increases in crop yield will have to rely on

growers. Scientists at the International Rice

novel tools. One of the relatively recent

Research Institute, Philippines (IRRI) have

novel technologies is genetic modification or

developed Golden Rice varieties suitable for

genetic

engineering

(also

known

as

several rice growing regions with the help of

biotechnology,

recombinant

DNA

generous

funding

from

philanthropic

technology, etc.).

organizations such as the Bill and Melinda

Gates

Foundation.

Therefore,

when

Two main approaches in GM plant

approved, the Golden Rice seeds can be

production are: a) Modifying existing genes

distributed to growers without profiteering.

in plants - e.g., fruit ripening by modifying

The IRRI has established a highly positive

ethylene biosynthesis or ethylene action, and

and remarkable reputation for successfully

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_Global Food Security and Sustainability_ Kumar releasing the flood tolerant SUB1 rice that

common in cheese making now, with

was developed by non-GM methods (marker

estimated 70% to 90% of cheeses in the

assisted breeding). However, it must be

USA manufactured using such microbial

highlighted that this could not have been

GM rennet now. Also, the US Food and

achieved without data from GM tools. Prior

Drug Administration (FDA) has approved

studies using molecular biological tools

over 30 other recombinant enzymes for use

helped to identify the flood tolerant variant

in food production (including α-amylase,

of the _Sub1a_ gene in rice. Experimental GM

used to make glucose or fructose syrups).

rice plants demonstrated that when this

resistant form of the gene was introduced to

Another noteworthy development in the

flood sensitive rice varieties they could be

recent years is the FDA approval of the first

turned into flood tolerant forms. Therefore,

transgenic animal for food use, namely, GM

without GM tools scientists could not have

salmon. After an exhaustive and rigorous

so easily developed the marker assisted

review that lasted nearly twenty years, the

SUB1 rice varieties in such a short time

FDA has approved the use of fast growing

span. These varieties dubbed 'Scuba Rice'

GM salmon (AquAdvantage) in November

are being grown for the past several years by

2015.

FDA

determined

that

the

thousands of happy farmers in millions of

AquAdvantage salmon is as safe to be

hectares of land, for example, in the flood

consumed by humans as the commercial

prone areas of India and Bangladesh.

non-GM Atlantic salmon. This was a long-

awaited positive decision welcomed by

**OTHER GM FOOD**

biotech scientists around the world.

Besides the examples cited above, there are

**AGRICULTURAL ECONOMY**

several other minor successes in GM plants

_****_

including the virus resistant GM papaya in

_**How important are GM crops to the**_

Hawaii, the delayed ripening tomatoes, as

_**agricultural economy**_?

well as some ornamentals such as long-vase-

An economic assessment of the impact of

life carnation flowers and the Blue Rose

commercial agricultural biotechnology on

marketed

by

Suntory

Biotechnology

global agriculture was done (Brookes and

company.

Barfoot, 2009). The study highlighted the

significant impacts on the production of

The use of GM products such as

soybeans, corn, cotton and canola crops.

recombinant proteins, mainly enzymes in

According to this study, GM technology led

food industry is noteworthy development.

to net economic benefits at the farm level of

GM or 'Recombinant proteins' from bacteria

about US$10.1 billion in 2007. The other

and fungi are used in cheese making. Cheese

benefits associated with the use of GM had a

production requires the use of a protein

positive impact to the tune of 25% of the

called chymosin (also known as rennin or

total direct farm income benefit in the USA.

rennet). Chymosin was previously obtained

Biotech crops contributed to increasing

only from calf stomachs, which had several

global production levels of the four main

implications including health concerns,

crops examined. For example, the use of

conflict with nutritional preferences due to

GM soybeans corresponded to adding 68

religious restrictions or personal choices of

million tons and GM corn added 62 million

various groups of people. The use of

tons to global production levels in 2007.

chymosin from GM microbes has become

Their use was equivalent to 172 million kg

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_Global Food Security and Sustainability_ Kumar reduction

in

pesticide

use

(which

Further complications are already evident

corresponds to 14% reduction in the

from the global climate change. The

environmental footprint associated with

predicted impacts of climate change include,

pesticide use). In addition, other factors

reduced wheat production in South Asia by

from

growing

these

biotech

crops

2030, and rice production in Southeast Asia,

significantly

reduced

greenhouse

gas

particularly

in

the

Greater

Mekong

emissions from agriculture equivalent to

Subregion (ADB, 2009). These will almost

removing 5 million cars from the roads

certainly lead to increased food prices. The

(Brookes and Barfoot, 2009).

ADB estimates that rice, wheat, and soybean

prices could increase by 10%–50%, while

**THE NEED TO INCREASE FOOD**

corn price may double by 2050. This should

**PRODUCTION**

be recalled in connection with the global

food and energy price surge in 2007–2008,

Why not just stick to old ways of producing

which exposed the vulnerabilities of

food? Why resort to new tools? According

households and of the international food and

to a UN study, the world population is

nutrition insecurity. Increase in extreme

expected to grow to ~9.7 billion by 2050.

weather events (floods, droughts, typhoons)

According to that study, Africa and Asia

will

have

serious

consequences

for

would be adding 1.14 billion more people to

agriculture, food, and forestry production in

their population. By 2050, Africa (2478

the coming decades. Therefore, it is

million) and Asia (5267 million) collectively

imperative that crop yield has to be raised to

will have a population size larger than the

ensure food security.

current world population.

**FOOD PRODUCTION AND WATER**

Average Asian rice consumption is about

100 kg per person per year. The number of

The growing worldwide shortage of water is

undernourished people in the world is close

extremely worrisome. The global demand

to 1 billion and nearly two-thirds of the

for water is estimated to double by 2050.

world's hungry people reside in Asia and the

However, about 35% of the world

Pacific (FAO, 2009).

population is already facing water shortages.

Asia is facing acute water shortage, with

**POPULATION**

**AND**

**CLIMATE**

available

freshwater

being

unevenly

**CHANGE V/S FOOD SECURITY**

distributed. Under these circumstances, it is

obvious that to grow more food with less

It is well recognized now that crop yield has

water, crop productivity needs to be

to be raised to ensure food security in the

improved. However, the traditional breeding

coming decades. Global food production

tools are often inadequate to achieve such

will need to increase by 40% by 2030 to

spectacular increases in yield.

keep pace with global demand (FAO, 2009),

because the world population is increasing.

Water and rice - how are the two related? To

The maxim 'Less food, more mouths to

grow the current world production of

feed' is made relevant when we realize the

approximately 500 million tons of rice it is

fact that the land area available for crop

estimated that about 1,750 km3 fresh water

cultivation is continually decreasing as the

is needed per year [1 km3 = 1000 billion

population keeps increasing.

liters (=1012 liters)]. If we take overall

agriculture, it accounts for about 80% of

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_Global Food Security and Sustainability_ Kumar world

water

use

in crop yield has to be through plant

(www.adb.org/publications/asian-water-

breeding and biotechnology.

development-outlook-2013).

Currently,

~40% of this water is lost due to inefficient

In view of the above, the answer to the

agricultural practices (crops and farm

question of whether we should use novel

animals). The water required for production

technologies (e.g., GM technology) for crop

of some of the key agricultural produce

improvement

should

be

obviously

illustrate the vastness of water needed in

affirmative! I cite the example of breast

agriculture. Liters of water necessary per kg

feeding v/s infant formula in support of this

of produce (indicated in parentheses): for

view. We all know breast feeding is the best

rice (2,500), other grains in a favorable

nutritional option for infants. But, global

climate (2,000), grains in arid climate

market for baby food is huge (over US$10

(5,000) and beef (15,000). Therefore, the

billion/year) with 50% to 70% of it as the

total world water use is 7,450 to 8,160

value of infant formula! North America and

km3/year, and the water used for food

Europe constitute 33% of this market, while

production is estimated to be 6,390 to 7,200

Asia forms 53% of the infant food market.

km3/year, with domestic needs accounting

Over 120 companies manufacture and sell

for 180 to 344 km3/year (IRRI, and various

infant formula! What has happened here is

other sources).

the need for convenience (in most cases,

necessity in some) has led to the adoption of

Some of the major challenges for rice and

an alternative to breast feeding as an

grain production include abiotic and biotic

acceptable alternative for our infants. In a

stresses. The major abiotic stresses are (1)

similar rational manner, we need to develop

cold (a climatic factor that affects ~66% of

modern technologies for crop improvement

land worldwide), (2) drought, (3) flood

and ensure food security. We should not

(~10% of yield loss, ~25% of the global rice

hesitate to use new technologies to develop

crop lands sees floods each year – with ~20

alternative options that should coexist with

million hectares of Asian rice land prone to

the established techniques.

flooding), (4) salinity (~10% of world's land

affected and ~12 million hectares in Asia

_**What are the other alternatives**_?

affected by increased soil salinity).

Certainly, GM crops are not the only option

to increase crop yield – they are an efficient

**SUSTAINABLE FOOD PRODUCTION**

and complementary tool to the established

**STRATEGIES**

tools at our disposal. It is recognized that

efficient crop management will improve

There are several ways to increase crop

yield to some extent. These include the use

yield. Four of the prominent options are

of right amount of chemical fertilizers and

mentioned

here.

(1)

Expanding

the

pesticides, because excess use of such

cultivated area: land expansion can account

agricultural chemicals leads to yield loss as

for ~ 20% increase in yield. (2) Increasing

well as air and water pollution. Ecological

the cropping intensity can contribute about

engineering is another concept being tested

10% increase. (3) Reducing pre- and post-

with positive effect. For example, the use of

harvest loss and wastage: 20 to 30% increase

beneficial insects for pest control by

if all wastage is prevented, (4) Improving

growing flowering plants around the fields is

crop yields using latest knowledge and

one such ecological modification tool. This

technologies. Therefore, about 40% increase

method helped to reduce pesticide use by

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 5

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_Global Food Security and Sustainability_ Kumar

~21% in Vietnam according to a study

can design a new variety on the computer

coordinated by IRRI. Alternate Wetting and

which combines plant lines with highest

Drying (AWD) method is another crop

scores (using the predictive models built).

management tool. AWD can lead to ~35%

One of the more recent GWAS studies with

less water requirement for rice growing

impressive results is on oil palm (Teh _et al_.,

compared to the traditional farming method,

2016). Also, several improved crops

with ~10% yield increase and attendant

generated using these techniques are being

reduction in greenhouse gas (methane)

released and more will be coming in the near

emission from the fields. The reduction in

future. The gene that facilitates deep rooted

methane emission is due to the reduction in

phenotype beneficial to develop drought

methanogenic bacteria growing in the root

tolerant crops, e.g., _DEEP ROOTED1_ gene

zones of rice plants when cultivated under

or _DRO1_ of rice was identified by GWAS

permanent

submergence

(leading

to

approaches while as mentioned above, the

anaerobic soil favoring the methanogenic

SUB1 rice was developed by marker

bacteria). The AWD management practice

assisted breeding.

leads to reduced growth of such bacteria

along with reduced water needs.

_**Genetically modified (GM) crops: to use or**_

_**not**_?

Marker-assisted breeding, genome-wide-

Based on ample evidence from the use of

association study (GWAS) leading to

GM crops during the past couple of decades

Genomic Selection (GS) are established as

it is safe to conclude that GM food can save

non-GM alternatives for crop improvement

millions of kids from dying due to

in the past decade or so (Meuwissen _et al_.,

malnutrition. The use of GM crops can also

2001, Desta and Ortiz, 2014). Genomic

help to reduce pesticide usage and

Selection is based on the powerful tools of

environmental pollution. In this connection

genome-wide

prediction

in

plant

it is worth remembering that technological

improvement. GWAS helps to generate

advances

are

continually

occurring.

statistical models to predict how a plant will

Accordingly, new genome editing methods

perform before conducting a genetic cross

may be seen as good alternative tools to

and field-testing of actual crops. This

generate the new generation, advanced crop

involves the combined use of genomic

plants. Besides GM, several non-GM

information, statistics, bioinformatics tools,

strategies are being developed and some of

and prediction-based breeding for crop

the novel strategies for crop breeding by

improvement. Of course, this requires that

genome editing include: (a) TALENs (b)

the scientists establish robust association

Zinc Finger Nucleases (ZFN) (Petolino and

between DNA seqence (SNPs) and specific

Kumar, 2016) and (c) CRISPR-Cas9 system

traits for a large number of breeding lines

(Chen and Bailey, 2016; Kleinstiver _et al_.,

(1000s of individual plants). This will then

2016).

be used to estimate Genomic Breeding

Values (GEBV). New plants to be used for

TALEN stands for Transcription Activator-

breeding will then be sequenced; for each

like

Effector

Nucleases;

while

new breeding line a DNA-profile will be

CRISPR/Cas9

stands

for

Clustered

determined and using such profiles from

Regularly Interspaced Short Palindromic

each line scientists will estimate GEBV for

Repeats/CRISPR-associated(Cas)

system.

every trait of interest. Based on the genetic

CRISPR/Cas9 was first named as Short

potential for the different traits the breeder

Regularly Spaced Repeats (SRSR) in 2000.

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_Res. Highl. 4Bs (2016)_

_Global Food Security and Sustainability_ Kumar It was renamed as CRISPR in 2002.

direct beneficiaries until now. But, when we

CRISPR/Cas9 induces double-strand break

carefully examine it there are already plenty

(DSB), generates deletion and/or insertion of

of indirect benefits for all. GM papayas

short sequences at the break leading to

(ring-spot virus resistant) saved the papaya

altered gene of interest. In fact, the common

industry in Hawaii, GM crops will play an

feature of all these genome editing tools is

important role in protecting soil and water

that they induce DSBs in the genomic DNA

resources, minimizing crop diseases and

at specific locations (gene of interest) – with

attendant loss of produce, and responding to

or without short insertions. The DSB will

the pressures of climate change. Cultivation

then be repaired by one of two repair

of Bt-cotton has reduced the amount of

processes, namely, Non-Homologous End

neurotoxic insecticide use by millions of

Joining (NHEJ), or Homology Dependent

kilograms every year in many countries

Repair (HDR). This results in gene editing.

where it is grown. Hence, one way of

looking at it is that the concept of organic

**FUTURE PERSPECTIVES**

crops and GM crops coexist! GM crops do

not pose any conflict with traditional or

Gene-edited CRISPR mushroom has been

organic farming. A book published by

generated recently and it was exempted from

Oxford University Press in April 2008 by

having to go through US Regulatory

authors

Pamela

Ronald

and

Raoul

process; because, such organisms do not

Adamchak (UC, Davis) advocates a food

harbor foreign genes and are similar to

system that is organic and genetically

induced

mutants.

The

white

button

engineered! They argue that a judicious

mushrooms generated have their polyphenol

blend of GM and organic farming

oxidase gene edited, which helps to prevent

representing two important strands of

browning

(Waltz,

2016).

Scientific

agriculture is key to helping feed the world's

American (14 April 2016) also indicated that

growing population in an ecologically

this is one of almost 30 genome edited

balanced manner.

organisms to avoid regulatory requirement

currently. These represent the first few

Even newer GM crops that will directly

improved crops and more such crops will be

benefit the consumers are coming, for

released by various companies in the years

example, the purple tomatoes produced by

to come (Wolt _et al_., 2016).

Prof Cathie Martin's lab, John Innes Centre,

UK. These GM tomatoes have high amounts

_**If they are safe why is there hesitation to**_

of plant nutrients (e.g., anthocyanins found

_**use GM crops**_?

in some berries), which are super foods that

One of the possible contributing factors is

will confer tremendous health benefits to the

that the general public and most of the

consumers. The purple tomatoes were grown

opponents of GM crops do not fully

in controlled greenhouses in Canada and

understand the underlying process. Perhaps

juice from these GM tomatoes is undergoing

equally importantly, consumers are not

tests and evaluations. Clearly, this represents

direct beneficiaries of the GM technology so

a 'super food' that could not be otherwise

far. GM crops of the past have been

made available to us in plentiful quantities.

designed to enhance the productivity of

They have already shown that inclusion of

industrial farming – so only the farmers who

these high-anthocyanin GM tomatoes in the

adopted the GM crops and the seed

diet of cancer-prone mice can extend their

companies that sell them have been the

healthy life-span by 30% - a most welcome

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 7

_Res. Highl. 4Bs (2016)_

_Global Food Security and Sustainability_ Kumar prospect indeed! It is a firm belief of the

**FAO paper (2009).** How to Feed the World

author that people will line up for this type

in 2050? Document available at the

of GM superfoods and demand that more be

FAO

web

site.

supplied!

http://www.fao.org/fileadmin/template

s/wsfs/docs/expert_paper/How_to_Fee

From the foregoing discussion it is clear that

d_the_World_in_2050.pdf

the future global food supply can benefit

greatly by the use of new initiatives in

**James, C. (2013).** Global status of

research. We should be open to employ a

commercialised biotech/GM crops:

multidisciplinary

approach

involving

2013,

ISAAA

Brief

No.

46.

molecular genetics, cell and developmental

International

Service

for

the

biology, systems biology, as well as

Acquisition

of

Agri-Biotech

ecology. GM crops have been grown and

Applications, Ithaca, NY. ISBN 978-

consumed for over 20 years now and the

1-892456-55-9.

www.

isaaa.

underlying technology is continually being

org/resources/publications/briefs/46,

improved. Hence, the emerging novel tools

2014. ****

such as genome editing will help us alleviate

food scarcity in the future decades. We

**Kleinstiver BP, Pattanayak V, Prew MS,**

should be mindful to develop 'safe

**Tsai SQ, Nguyen NT, Zheng Z,**

technology' and develop suitable alternative

**Joung**

**JK**

**(2016).**

High-fidelity

strategies such as GM, molecular marker

CRISPR–Cas9 nucleases with no

assisted breeding and genome editing.

detectable

genome-wide

off-target

Collectively, these will serve us well in

effects. _Nature_ **529, 490-495**.

ensuring future food security.

**Meuwissen THE, Hayes BJ, Goddard ME**

**(2001).** Prediction of total genetic

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value

using

genome-wide

dense

**ADB**

**(2009).**

Operational

plan

for

marker maps. _Genetics_ **157, 1819–**

sustainable food security in Asia and

**1829**.

the Pacific. Document downloadable

**Pamela C. R. and Raoul, W. A. (2008).**

from

ADB

web

site.

Tomorrow's Table: Organic Farming,

http://www.adb.org/publications

Genetics, and the Future of Food,

**Brookes G, Barfoot P (2009).** Global

Oxford University press (OUP).

impact of biotech crops: Income and

**Petolino JF, Kumar S (2016).** Transgenic

production

effects,

1996-2007.

trait

deployment

using

designed

_AgBioForum_ **12(2), 184-208**.

nucleases.

_Plant_

_Biotechnology_

**Chen H, Bailey S (2016).** Cas9, poised for

_Journal_ **14, 503–509**.

DNA

cleavage.

Structural

**Teh CK, Ong AL, Kwong QB, Apparow**

rearrangements explain how Cas9 cuts

**S, Chew FT, Mayes S, MohamedM,**

DNA. _Science_ **351, 811-812**.

**Appleton D, Kulaveerasingam H**

**Desta ZA, Ortiz R (2014).** Genomic

**(2016).**

Genome-wide

association

selection: genome-wide prediction in

study identifies three key loci for high

plant improvement. _Trends in Plant_

mesocarp oil content in perennial crop

_Science_ **19, 592-601**.

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 8

_Res. Highl. 4Bs (2016)_

_Global Food Security and Sustainability_ Kumar oil palm. _Scientific Reports_ **6, 19075** |

**Wolt JD, Wang K, Yang B (2016).** The

DOI: 10.1038/srep19075

regulatory status of genome-edited

crops. _Plant Biotechnology Journal_

**Waltz E (2016).** Gene-edited CRISPR

**14, 510–518**.

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_ 9

**Research Highlights in 4Bs**

__

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016), P10-19_

**Nano- and Bio-technological Advancement to assist in the Determination**

**of Halal Products**

****

Quamrul Hasan _1, 2,_ *

_1College 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 (immuno-

chromatography) 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

_Res. Highl. 4Bs (2016)_

_Determination of Halal Products_ Hasan **INTRODUCTION**

detect a species with PCR, adequate genetic

markers are chosen to develop the assay.

As processed and prepared ready-to-eat food

Either nuclear or mitochondrial genes can be

products with animal origins have become

targeted (Fajardo _et al_., 2008). However, the

increasingly available to consumers, due to

use of mitochondrial DNA (Mt DNA) offers

technological advances, the possibility of

a series of advantages over cell nucleus

fraudulent adulteration and substitution of

DNA. Mitochondrial DNA facilitates PCR

the expected species (source) with other

amplification, even in cases where the

sources has also increased. The same goes

availability of DNA template after its ex-

to confectionery and functional cosmetic

traction is insufficient for detection

products especially containing gelatin or

(Murugaiah et _al._ , 2009). This is attributed to

collagen peptides. Such practices pose a

the fact that Mt DNA is several fold more

substantial concern to consumers in terms of

abundant than that of nuclear genome; each

economic

loss,

allergies,

religious

mitochondrion is estimated to contain 2 to

observance, loss of traceability, and food

10 Mt DNA (Murugaiah _et al_., 2009).

safety. In many cases, porcine derivatives

Furthermore, Mt DNA evolves much faster

are used due to cheaper price and readily

than nuclear DNA and henceforth contains

available. For Muslim consumers, the major

more sequence diversity facilitating the

authenticity concerns are in meat and meat

identification of phylogenetically related

products include porcine gelatin, collagen,

species (Fajardo _et al_., 2010; Girish et

fat, and so on. The analytical methods used

al., 2005; Murugaiah _et al_., 2009). Among

for Halal authentication of meat and meat

the mitochondrial genes, cytochrome b (cyt

products

include:

Polymerase

Chain

b) (Aida et al., 2005; Murugaiah _et al_.,

Reaction

(PCR);

Enzyme

Linked

2009) and 12S rRNA (Chen _et al_., 2010;

Immunosorbent Assays (ELISA); Mass

Girish _et al_., 2005) are the most

spectrometry; Chromatography, Electronic

commonly used markers in the

nose and Spectroscopy. An overview of the

development of DNA methods for meat

currently available analytical methods or

species authentication.

techniques is given in Table 1. The

analytical methods currently in use to detect

_**Protein-based detection**_

the presence of porcine materials are mainly

Porcine protein, due to it is being cheap and

protein and DNA-based. These are described

readily available, might fraudulently be used

below.

to substitute other animal proteins. ELISA is

the most commonly used method to detect

_**DNA-based detection**_

animal proteins and a number of commercial

PCR is capable of amplifying very few

immunoassays are available. Chen and

copies of DNA and its detection limit is

Hsieh (2000) were the first ones to develop

much lower than what is observed with

the

immunoassay

(ELISA)

using

a

protein based assays. PCR amplification is

monoclonal

antibody

to

a

porcine

based on hybridization of specific oligo-

thermostable muscle protein for detection of

nucleotides to a target DNA and synthesis of

pork in cooked meat products. They

million copies flanked by these primers. The

observed no cross-reactivity with common

simplest PCR strategy applied to evaluate

food proteins. By employing this technology,

presence of any species in a meat product is

the first pork detection kit was developed in

the amplification of DNA fragments,

Japan by a Japanese company (Okamoto,

followed by agarose gel electrophoresis for

2016). This kit is immuno-chromatographic

fragment size verification. To successfully

employing

nano-sized

colloidal

gold

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 11

_Res. Highl. 4Bs (2016)_

_Determination of Halal Products_ Hasan particles to detect presence of pork in food

using any special equipment or requiring

samples. It can detect pork in both raw and

skill. The basic principle of immune-

cooked food. It allows rapid detection of

chromatographic kit is shown in Figure 1.

pork in food samples at low cost without

****

**Table 1** : Summary of analytical techniques applicable in the halal authentication of meat

products♦.

Authenticity issue

Analytical Techniques References

Pork adulteration

Murugaiah _et al_. (2009), Aida, Che Man, Raha,

Species identification

PCR-RFLP

and Son (2007), and Aida _et al_. (2005)

Martín _et al_. (2009), Kesmen, Gulluce, Sahin,

Real time PCR

and Yetim (2009), Tanabe _et al_. (2007),

Fumière, Dubois, Baeten, von Holst, and

Berben (2006), and López-Andreo, Garrido-

Pertierra, and Puyet (2006)

Species-specific PCR

Soares, Amaral, Mafra, and Oliveira (2010),

Alaraidh (2008), Che Man _et al_. (2007) and

Montiel-Sosa _et al_. (2000)

RAPD

Martinez and Malmheden Yman (1998)

PCR sequencing

Karlsson and Holmlund (2007)

Pork protein

ELISA

Chen and Hsieh (2000); Chen and Hsieh (2000)

Chromatography

Chou _et al_. (2007)

Peptide examination

Aristoy and Toldra (2004)

Isoelectric focusing

Hofmann (1985)

Pork fat (lard)

FTIR spectroscopy

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)

DSC

Marikkar, Ghazali, Man, and Lai (2003) and

Marikkar, Lai, Ghazali, and Che Man (2001)

Electronic nose

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)

Blood plasma

Isoelectronic focusing

Bauer and Stachelberger (1984)

ELISA

Church and Hart (1995)

Immunodiffusion

Price, Hart, and Church (1992)

LC – MS/MS

Grundy _et al_. (2007) and Grundy _et al_. (2008)

♦Source: Nakyinsige et al. (2012).

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 12

_Res. Highl. 4Bs (2016)_

_Determination of Halal Products_ Hasan **Figure 1** The basic principle of the immuno-chromatographic kit.

**MATERIALS AND METHODS**

****

****

**Table 2** Sequence of Porcine and Bovine

_**Detection of Porcine and Bovine DNAs**_

specific primers.

_**from**_

_**different**_

_**products**_

_**containing**_

Target

Primer

Sequence

_**gelatin/collagen from different sources**_

Porcine Forward

GAC CTC CCA

DNA extraction was performed using DNA

GCT CCA TCA

extraction kit from QIAGEN. About 0.25g

AAC ATC TCA

DNA per sample was extracted. The DNA

TCT TGA TGA

extraction was performed in duplicate for

AA

each sample. The conventional-PCR was

Reverse

GCT GAT AGT

performed

using

Thermal

cycler

AGA TTT GTG

(GeneAmp® PCR System 9700, Applied

ATG ACC GTA

Biosystems). Porcine and Bovine specific

Bovine Forward

GAC CTC CCA

primers for mitochondrial DNA were used.

GCT CCA TCA

Each primer sequence is shown in Table 2.

AAC ATC TCA

In addition, in order to check that DNA was

TCT TGA TGA

extracted successfully, the PCR using the

AA

primer for vertebrate detection was also

Reverse

CTA GAA AAG

performed.

The

PCR

condition

is

TGT AAG ACC

summarized in Table 3.

CGT AAT ATA

AG

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_Res. Highl. 4Bs (2016)_

_Determination of Halal Products_ Hasan

****

**Table 3** Condition of PCR

B. FAMIC method:

Temperature

Step

Time

Cycle

(°C)

Primer:FAMIC

Porcine

primer-

Initial

mitochondrial DNA

94

1 min

1

denaturation

DNA:

20 ng

Denaturation

94

30 sec

PCR steps:

Annealing

60

30 sec

30

Extension

72

30 sec

Temperature

Step

Time

Cycle

Final

(°C)

72

7 min

1

Extension

Initial

95

9 min

1

Hold

4



denaturation

Denaturation

92

30 sec

Annealing

60

1 min

45

_**Comparative study on the PCR-based**_

Extension

72

1 min

_**detection of the Porcine and Bovine genes**_

Final

72

5 min

1

_**using different primers**_

Extension

****

Hold

4



A. Neogen kit method:

Primer:

Neogen

Porcine

primer-

Primer:FAMIC

Bovine

primer-

genomic DNA

mitochondrial DNA

DNA:

20 ng

DNA:

20 ng

PCR steps:

PCR steps:

Step

Temperature Time

Temperature

Cycle

Step

Time

Cycle

(°C)

(°C)

Initial

94

10

Initial

1

95

9 min

1

denaturation

min

denaturation

Denaturation

94

15

Denaturation

92

30 sec

sec

Annealing

55

30 sec 45

Annealing

64

15

Extension

72

30 sec

30

sec

Final

72

5 min

1

Extension

72

15

Extension

sec

Hold

4



Final

72

3

1

Extension

min

Hold

4



****

****

****

****

**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

_Res. Highl. 4Bs (2016)_

_Determination of Halal Products_ Hasan **Table 4:** Summarized results of the

3. Two products (one marshmallow,

detection of porcine, bovine and vertebrate

one functional cosmetic) showed

DNA

positive result for vertebrate material.

Porcine

Bovine

Vertebrate

Only one of the two marshmallow products

Sample 1 Not

Not

Detected*

might contain fish material (gelatin) since it

detected

detected

was found porcine negative.

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).

**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

(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].

Assumptions on the detection of porcine,

**Figure 4** Result of PCR using the primer

bovine and vertebrate DNA from four

that common to a vertebrate; Lane 1: Marker

commercialized products are listed below:

(25bp ladder), 2: Negative control, 3: sample

1. Not a single product showed positive

1 (n=1), 4: sample 1 (n=2), 5: sample 2

result for bovine material.

(n=1), 6: sample 2 (n=2), 7: sample 3 (n=1),

2. Three products (one marshmallow,

8: sample 3 (n=2), 9: sample 4 (n=1), 10:

one Jelly, one functional cosmetic)

sample 4 (n=2), and 11: Positive control

showed positive result for porcine

[Microchip

electrophoresis

system

material.

(MultiNA), Shimadzu Corporation].

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 15

_Res. Highl. 4Bs (2016)_

_Determination of Halal Products_ 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/µl)

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** Porcine DNA; PCR product size:

Figure 5, no porcine DNA was detected in

380-bp (housekeeping fragment), 314-bp

any of the two samples tested in duplicates.

(porcine fragment); from left: marker, M-A,

M-B, T-A, T-B, positive control (380-bp,

B. FAMIC method: As shown in the Figure

314-bp).

6, Porcine DNA was detected for the two

samples though in single from duplicate (M-

B and T-A). No bovine DNA was detected

from the two samples tested.

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

**Figure 6** Porcine DNA; PCR product size:

methods for porcine and bovine materials in

126-bp; from left: M-A, M-B, T-A, T-B,

the processed food products (marshmallows

positive control.

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 16

_Res. Highl. 4Bs (2016)_

_Determination of Halal Products_ 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.

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

**Figure 7** Bovine DNA; PCR product size:

were

imported

from

other

countries.

126-bp; from left: M-A, M-B, T-A, T-B,

Moreover, one product (functional cosmetic)

positive control

claimed that it contained fish collagen from

Japan, which we found porcine positive.

and jelly containing gelatin) and functional

cosmetic

product

containing

collagen

Our results from this study proved that

peptides by employing conventional PCR

selection of the right primer is critical to

and two different primers (nuclear and

obtain the authentic result: the porcine DNA

mitochondrial genes). Earlier, two other

was detected positive only when the FAMIC

groups also reported about the successful

primer (mitochondrial DNA) was used and,

application of the conventional PCR in meat

in contrast, the result was negative using the

species detections including from the

NEOGEN kit primer (genomic DNA). This

processed foods (Tanabe _et. al_., 2007;

might be due to the higher sensitivity of

Matsunaga _et. al_., 1999). These conventional

mitochondrial gene target primer, which is a

PCR methods are simple and useful. That is

multicopy gene. In another study, we

why we have employed these. The choice of

attempted to develop a rapid method for

the target gene and the design of the primers

meat species identification based on the loop

have a great impact on the sensitivity and

mediated isothermal

amplification and

specificity of a detection system. It is well-

electrochemical DNA sensor (Ahmed _et al_.,

known that very sensitive PCR assays can be

2010).

established when the primer target is a

multicopy gene, such as a mitochondrial

In a separate effort, a protein-based-

gene (Holzhauser _et. al_., 2006). In this study,

immuno-chromatographic porcine detection

we chose both mitochondrial and nuclear

kit was successfully co-developed under a

(genomic) genes as the target to detect

collaborative effort between two universities

porcine and bovine materials for comparing

(in USA and Japan) and two companies in

the efficiency of these two primers while

Japan (the methods and results were not

using

the

processed

products.

DNA

available for this paper). This development

extraction was performed by using the

was owned and sponsored by a Japanese

commercial kit from Neogen Europe Ltd.,

company having a leading position in the

UK and following the manufacturer's

precious metal business including the

instructions. The DNA extraction was

expertise in nano-gold technology. This

carried out in duplicate for each sample

company

obtained

the

immune-

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 17

_Res. Highl. 4Bs (2016)_

_Determination of Halal Products_ Hasan chromatographic

know-how

from

a

Meat species identification based on

university in Japan. However, to develop a

the

loop

mediated

isothermal

porcine detection kit, the missing part was

amplification and electrochemical

the biotechnology: a monoclonal antibody to

DNA sensor. Food Control **21, 599-**

a porcine thermostable muscle protein for

**605**.

detection of pork in cooked meat products.

****

To address this issue, the author of this paper

**Aida, A. A., Che Man, Y. B., Wong, C. M.**

was approached by the Japanese company

**V. L., Raha, A. R. and Son, R.**

who successfully made the connection with a

**(2005).** Analysis of raw meats and

university in USA already reported on the

fats of pigs using polymerase chain

availability of this antibody and he

reaction for halal authentication.

coordinated the collaboration between the

Meat Science **69(1), 47–52**.

Japanese company and the US University.

****

Finally, the effort was paid-off. About 2

**Chen, F. C., and Hsieh, Y. H. P. (2000).**

years later, the Japanese company confirmed

Detection of pork in heat-processed

the successful development of porcine

meat

products

by

monoclonal

detection kit and announced it. Immediately,

antibody-based ELISA. Journal of

they were approached by a large US

AOAC International **83(1), 79–85**.

company (renowned analytical equipment

****

manufacturer) for this technology, which

**Chen, S. Y., Liu, Y. P. and Yao, Y. G.**

resulted

in

to

the

successful

**(2010).** Species authentication of

commercialization of this protein-based

commercial beef jerky based on

(immuno-chromatographic)

porcine

PCR–RFLP

analysis

of

the

detection kit in the global market.

mitochondrial 12S rRNA gene.

Journal of Genetics and Genomics 

## ACKNOWLEDGEMENTS

**37(11), 763–769**.

****

The author gratefully acknowledges the co-

**Fajardo, V., González, I., Martín, I.,**

operations from Professor Eiichi Tamiya at

**Rojas, M., Hernández, P. E.,**

Osaka University, Japan; Professor Emerita

**García,**

**T.**

_**et**_

_**al**_ **.**

**(2008).**

Peggy Hsieh at Florida State University,

Differentiation of European wild

USA; Mr. Masatoshi Watai at Japan Food

boar ( _Susscrofa scrofa_ ) and domestic

Research Laboratories, Japan; Dr. Hiro

swine ( _Susscrofa domestica_ ) meats

Haraguchi at FASMAC Co., Japan; Dr. Koji

by PCR analysis targeting the

Okamoto at Tanaka Precious Metal Co.,

mitochondrial D-loop and the nuclear

Japan; and the Ministry of industry and

melanocortin receptor 1 (MC1R)

primary

resources,

Brunei

Darussalam

genes. Meat Science **78(3), 314–322**.

without which this study would not have

****

been possible. The author is thankful to Mr.

**Fajardo, V., González, I., Rojas, M.,**

Haikal Ismail at STML, Universiti Utara

**García, T. and Martín, R. (2010).**

Malaysia for kindly assisting him in the

A review of current PCR-based

preparation of this manuscript.

methodologies for the authentication

of meats from game animal species.

**REFERENCES**

Trends

in

Food

Science

&

Technology **21(8), 408–421**.

**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

_Res. Highl. 4Bs (2016)_

_Determination of Halal Products_ Hasan **Girish, P. S., Anjaneyulu, A. S. R., Viswas,**

**Radu, S. (2009).** Meat species

**K. N., Shivakumar, B. M., Anand,**

identification

and

halal

**M., Patel, M. _et al_** **. (2005).** Meat

authentication

analysis

using

species identification by polymerase

mitochondrial DNA. Meat Science

chain reaction-restriction fragment

**83(1), 57–61**.

length polymorphism (PCR–RFLP)

****

of mitochondrial 12S rRNA gene.

**Nakyinsige, K., Che Man, Y.B., & Sazili, **

Meat Science **70(1), 107–112**.

**A.Q. (2012).** Halal authenticity

****

issues in mean and meat products.

**Holzhauser, T., Stephen O., and Vieths, S.**

Meat Science **91, 207-214.**

**(2006).** Polymerase chain reaction

(PCR) methods for the detection of

**Okamoto, K. (2016).** Development of

allergenic foods. In "Detecting

Porcine

Immunochromato.

Allergens in Food," S.J. Koppelman

Bioindustry, April 2016, **Vol. 33(4),**

and S.L. Hefle, edc. CRC Press,

**26-32,** CMC Books, Tokyo. (in

Boca Raton, **pp. 125-143**.

Japanese).

****

****

**Matsunaga, T., Chikuni, K., Tanabe, R.,**

**Tanabe, S., Miyauchi, E., Muneshige, A.,**

**Muroya, S., Shibata, K., Yamada,**

**Mio, K., Sato, C., and Sato, M.**

**J., & Shinmura, Y. (1999). ** A quick

**(2007).** PCR method of detecting

and

simple

method

for

the

pork in foods for verifying allergen

identification of meat species and

labeling and for identifying hidden

meat products by PCR assay. Meat

pork ingredients in processed foods.

Science **1, 143-148**.

Bioscience,

Biotechnology,

and

Biochemistry **71(7), 1663–1667**.

**Murugaiah, C., Noor, Z. M., Mastakim,**

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

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 19

**Research Highlights in 4Bs**

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_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

The European parliament has asked the

member states to specifically prioritize the

Antimicrobial

or

antibiotic

resistance

development

of

phage

therapy

(AMR/ABR) is an increasing concern world

(Parliamentary Assembly, 2014). Pathogenic

over. The World Health Organization in its

strains of _Salmonella_ spp. _, Campylobacter_

Global Surveillance Report on ABR states

spp. _,_ _E. coli, Listeria_ spp. _, Vibrio_ spp. and

that "The problem is so serious that it

many other foodborne bacteria are a

threatens the achievements of modern

significant cause of foodborne illnesses in

medicine. A post-antibiotic era - in which

humans worldwide and especially in the

common infections and minor injuries can

developing world. Such illnesses can be

kill – is a very real possibility for the 21st

treated with antibiotic chemotherapy but

century." The US President (Whitehouse,

over the years, there has been considerable

2014) and UK Prime Minister (O'Neill,

misuse of antibiotics. Many strains of

2014) have already asked their Governments

foodborne pathogens are becoming resistant

to prepare a roadmap to tackle this issue.

to antibiotics. The progressive resistance of

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 20

_Res. Highl. 4Bs (2016)_

_Phage Biocontrol of Foodborne Pathogens_ Bhandare

_Salmonella_ to the antibiotics has been

time is advisable and if successful another

reported (Fluit, 2005). The multiple drug

strain can be tackled. Eventually, a cocktail

resistance in zoonotic non typhoidal

of phages will give a broad-spectrum

_Salmonella_ from food animals and food

activity.

sources is of great concern all over the world

due to their entry into human food chain

Recently, the scientific community has

(O'Mahony _et al._ , 2005). Similar antibiotic

witnessed the sudden surge of interest in

resistance is noticed in _Campylobacter_

bacteriophage

research.

Bacteriophage

species (Bester and Essack, 2008), _E. coli_

therapy is a promising and natural way of

(Diarrassouba _et al._ , 2007 and Saenz _et al._ ,

reducing _Salmonella_ (Atterbury _et al._ ,

2001) and _Listeria_ species (Li _et al._ , 2007).

2007) _, Campylobacter_ (Atterbury _et al._ ,

Drug resistance emerges through excessive

2005) __ and _E. coli_ (Raya _et al._ , 2006) __ as a pre

usage of antimicrobials in humans and in

harvest treatment. While, post harvest

food animals, which imposes a selection

application of phages to reduce _Listeria_

pressure for bacteria that are resistant to

_monocytogenes_ (Leverentz _et al._ , 2003) __

antibiotics (Huges and Heritage, 2006).

contamination in later stages of food

Particularly, in fish farming antibiotics are

production is logical. Phages can help to

given to the whole population with

reduce specific bacterial load in food

inaccurate doses and thus healthy fishes

animals through proactive ante mortem

along with sick ones are exposed to

interventions rather than reactive end

antibiotics

and

thus

development

of

product testing or treatment of patients.

antibiotic resistance is inevitable (Sorum,

They can be applied directly to foods to

2008). In USA about 40% of total antibiotics

extend the shelf life and also as hygiene

produced are used on livestock and about

indicators in assessing food quality (Hudson

80% of that is used as growth promoters

_et_

_al.,_

2005).

The

isolation

and

(Hileman, 1999). Thus, considering the

characterization

of

bacteriophages

is

threat to public health the European

uncomplicated and is possible on large scale

Commission has imposed an EU wide ban

(Toro _et al.,_ 2005). Hankin in 1896 first

on the use of antibiotics as growth

noticed the bactericidal activity of phages

promoters in animal feed from January 1,

from the Ganges and Jumna river waters in

2006 (European Commission, 2006). The

India, which when filtered had antibacterial

development pathway for new antibiotics is

properties against _Vibrio Cholerae._ Phage

time consuming and very few new

therapy was pioneered by Felix D'Herelle,

antibiotics are discovered in recent years.

in early 19th century and his major

Hence, there is an urgent need for

contributions came from his work in India

alternatives to antibiotics and bacteriophage

and he could discover the phages in Ganges,

biocontrol is one of the viable alternatives to

the holy river in India (Summers, 2001). The

antibiotics especially for the multiple drug

former Soviet Union has been using phage

resistant

pathogens

(Barrow,

2001;

therapy since 1920s (Chanishvili _et al_.,

Matsuzaki _et al_., 2014). Lytic phages are

2001) but after the discovery of antibiotics

responsible for limiting bacterial numbers in

in early nineteenth century scientists stopped

an aquatic environment with up to 80 % of

working with phages until recently. The

mortality

evidenced

in

the

bacterial

reappraisal of phage therapy in the Western

population (Weinbauer, 2004). Phages are

world was done by H. Williams Smith and

strain specific and thus attempting a

his colleagues for oral _E. coli_ infection

biocontrol of single strain of bacteria at a

control in neonatal animals and for systemic

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_Phage Biocontrol of Foodborne Pathogens_ Bhandare

infection control of _E. coli_ in mice (Smith

_**Phage structure and morphology**_

and Huggins, 1982; Smith and Huggins,

They consist of a nucleic acid genome

1983). The concept of using bacteriophages

surrounded by a protein coat called as

as food safety tool is emerging rapidly

capsid. Many phages contain additional

(Hagens and Offerhaus, 2008) and they are

structures such as collar, tails, basal plate

becoming the logical agents for targeted

and spikes or fibers (Figure 1). Structure of

control of pathogenic foodborne bacteria to

phages may be icosahedrons, spherical

overcome

the

food

safety

concerns

shapes consisting of triangular faces, or

regarding the entry of such pathogens in

filamentous or complex structures consisting

food chain and thereby affecting the public

of icosahedral heads with helical tails. Their

health. There are regulatory concerns over

genomes can consist of either DNA or RNA,

the human phage therapy but their

single (ss) or double (ds) stranded, circular

application for food safety has relatively less

or linear (Nicklin _et al.,_ 1999).

regulatory concern owing to the presence of

phages already in the food environment.

****

**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.

**Figure 1:** Schematic representation of

Most phages are far more resistant to heat,

bacteriophage.

freezing, radiation, chemical disinfection

and natural inactivation than their host

bacteria (Jofre and Muniesa, 2000). They

_**Phage classification**_ ****

can infect and kill specific bacteria and do

The morphology of the phages helps in their

not affect other bacteria or cells, meaning

classification. The International Committee

the dysbiosis (imbalance of commensal gut

on Taxonomy of Viruses (ICTV) has

flora) often resulting from the use of broad

presently classified bacteriophages in to one

spectrum antibiotics can be avoided. They

order, ten families and forty genera (ICTV,

are obligate intracellular parasites that are

2011) based upon their symmetry, nucleic

capable of existing as phage particles

acid and other morphological features

outside the bacterial cell but can only

(Ackermann, 2006) (Table 1 and Figure 2)

reproduce inside the cell. ****

****

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_Phage Biocontrol of Foodborne Pathogens_ Bhandare **Table 1** : Classification of bacteriophages as per ICTV (Source: Bhandare, 2015).

**Order**

**Family**

**Genome**

**Genome size**

**Envelope** **Morphology**

**Virion size**

**(Kb)**

_Caudovirales_ _Myoviridae_

dsDNA linear

31-317

No

Icosahedral head with

Icosahedral heads: 60-45

tail

nm;

Elongated heads: 80-110

nm;

Tail: 16-20 × 80-455 nm

_Caudovirales_ _Podoviridae_

dsDNA linear

16-78

No

Icosahedral head with

Icosahedral heads: 60-70

short tail

nm;

Tail: 10-20 nm

_Caudovirales_ _Siphoviridae_

dsDNA linear

21-134

No

Icosahedral head with

Icosahedral heads: 40-80

long tail

nm;

Tail: 5-10 × 100-210 nm

Unassigned

_Corticoviridae_ dsDNA circular

10

No

Icosahedral

60 nm

supercoiled

Unassigned

_Plasmaviridae_ dsDNA circular

12

Yes

Quasi-spherical,

50-125 nm

supercoiled

pleomorphic

Unassigned

_Tectiviridae_

dsDNA linear

15

No

Icosahedral

66 nm

Unassigned

_Inoviridae_

ssDNA (+)

Inoviruses: 5.8-

No

Inoviruses: filamentous

Inoviruses: 7 × 700-3500

circular

12.4

Plectroviruses: rod

nm;

Plectroviruses: 4.5-

shaped

Plectroviruses: 15 × 200-

8.2

400 nm

Unassigned

_Microviridae_

ssDNA (+)

4.4-6.1

No

Icosahedral

25-27 nm

circular

Unassigned

_Cystoviridae_

dsRNA three linear

6.4-7.1;

Yes

Spherical

85 nm

segments

3.6-4.7;

2.6-3.2

Unassigned

_Leviviridae_

ssRNA (+)

3.5-4.3

No

Icosahedral

26 nm

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_Phage Biocontrol of Foodborne Pathogens_ Bhandare

****

****

**Figure 2:** Schematic representation of major phage groups. (Source: Ackermann, 2006)

Phages have binary (consisting of two

Phages possess the ability to infect a

parts), cubic, helical and pleomorphic

bacterium and redirect the cell to synthesize

symmetries. Most phages have double

phage components for replication. A typical

stranded (ds) DNA, but there are few phages

life cycle of a phage (Figure 3) starts by

with single stranded (ss) DNA, ssRNA or ds

adsorption of the phages on to a specific

RNA. Few phages contain lipid containing

receptor structures on the surface of the

envelope surrounding the capsid (the protein

bacterium. After attachment they inject their

coat covering viruses). The majority (96%)

genetic

material

inside,

which

re-

of the phages are tailed phages (binary

programmes the bacterium for synthesis of

symmetry), which are classified into the

phage nucleic acid and other molecules

order _Caudovirales_ and three very large

required for reproduction of complete viral

phylogenetically related families, while

particles and thereby stopping synthesis of

remaining 4% are other types. Taxonomic

host cell components. Then new phage

names of orders, families, and genera are

particles are assembled and when the new

typically constructed from Latin or Greek

viruses are mature, an enzyme is produced

roots and end in - _virales, -viridae_ and _–_

that digests the cell wall, allowing the phage

_virus,_ respectively (Ackermann, 2006).

to burst out of the cell. If bacteriophage

greatly outnumber their hosts, with many

_**Phage mode of action**_ ****

phages attached to each cell, the bacterium's

****

**Figure 3:** Replication cycle of Bacteriophage.

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_Phage Biocontrol of Foodborne Pathogens_ Bhandare

cell wall can become unstable and rupture,

viruses as different viruses have different

without replication of the viruses inside, this

latent periods and burst sizes. With many

phenomenon is called 'passive inundation'

bacterial viruses, the whole cycle may be

(Atterbury, 2006).

complete in 30-60 min (Madigan _et al.,_

2003). The burst size is the average yield of

The stages in the life cycle of the phages are

phage particles per cell, which is very

recognized when virus particles infect cells

important for determination of inoculum

in culture and are illustrated in Figure 4,

size in phage therapy. Many times small

which exhibits a one-step growth curve.

doses are not sufficient and even at large

This graph displays the results of a single

doses some times there is no active

round of viral multiplication in a population

replication and therapeutic benefits are

of cells. Following adsorption, the virus

obtained by using very large or repeated

particles undergo uncoating and viral

doses of phage (Payne and Jansen, 2001).

nucleic acid is injected into bacterial cell,

Large dosage may cause lysis from without

thus there is no growth observed and the

so that bacterial cells are destroyed without

phenomenon is called eclipse. During the

any phage replication if the phages are

latent period, replication of viral nucleic

applied at high (> 100) MOI i.e. Multiplicity

acid and proteins occurs. The maturation

of Infection, which means number of phage

period follows, when virus nucleic acid and

per bacterium. The latent period is the

protein are assembled in to mature virus

minimum length of time from the adsorption

particles. At this time, if the cells are

of phage to its host, until the release of

prematurely lysed (by the use of chloroform

newly formed phage particles, which is

for example), mature virus particles can be

crucial for phage therapy because the

detected. Finally **,** release occurs, either with

inoculations given in right time taking latent

cell lysis (e.g. lytic phage) or without cell

period in to account would be more

lysis (e.g. temperate phage). The timing of

effective.

the one-step growth cycle varies with the

**Eclipse**

**Rise**

**Latent period**

its

**Extracellular phage**

nu g

**Intracellular phage**

**Burst size**

in

rmofe u

Plaq

Time

****

**Figure 4:** One **-** step growth curve of bacteriophage replication (Source: Bhandare, 2015).

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**BACTERIOPHAGES**

**APPLICATION**

caused by three different _E. coli_ strains in

**AS**

**BIOCONTROL**

**AGENTS**

**TO**

calves, piglets and lambs and a mixture of

**CONTROL FOODBORNE BACTERIA**

two phages were given orally to protect

****

them. _E. coli_ infection was reduced to cure

There are several reports on successful

the diarrhea in all three species. The phage

usage of bacteriophages in controlling the

resistant mutants were emerged in the calves

foodborne pathogens and some of the

and piglets but they were less virulent than

important reports are discussed in this

their parent strains. A cocktail of phages

section. Phages can help to reduce

significantly reduced the numbers of _E. coli_

_Salmonella_ in poultry through proactive

O157:H7 in the intestinal tract of sheep

antemortem

interventions

rather

than

(Callway and colleagues, 2008). Even in the

reactive end product testing or treatment of

fisheries the successful phage therapy is

sick birds. Berchieri,

_et_

_al._

(1991)

carried out by Karunasagar _et al._ (2007).

successfully used bacteriphages to control

They could achieve biocontrol of pathogens

_Salmonella_ colonization in the poultry

in

shrimp

hatcheries

by

using

gastrointestinal tract. The mortality was

bacteriophages (Douglas, 1974). Also, the

reduced from 60% to 3% although large

Japanese researchers (Park _et al.,_ 2000; Park

numbers of phages were needed. Their study

and Nakai, 2003) studied the potential use of

revealed that the production of large

phages to control the fish infections caused

numbers of phages is practicable on farms. It

by _Lactococcus_ and _Pseudomonas_ by giving

was also noticed that phages spread readily

phages orally as phage impregnated feed.

between bacterially infected birds. Similarly,

Largely it is a win-win situation for the

_E. coli_ infection in the poultry can be

mankind if phage therapy is thoroughly

controlled by using phage therapy. Toro, _et_

scrutinized for applications in the food

_al._ (2005) stated that there is a beneficial

animals and then used because it will help to

effect of the phage treatment on weight gain

reduce the antibiotic usage to avoid multiple

performance

in

chickens

along

with

drug resistance along with the reduction in

reduction of _Salmonella_ colonization. Huff,

foodborne pathogen load.

_et al._ (2006) used aerosol spray of phages to

bring down the mortality of birds due to _E._

Rather than applying the phages in live

_coli_ infection. The mortality was brought

animals

before

their

slaughter

the

down to 7% in phage treated birds while it

application of phages after slaughter to the

was

48%

in

the

untreated

ones.

carcasses and to the food products during

_Campylobacter_ spp., the celebrity bug in

their processing prior to the consumption is

poultry as referred by Butzler (2004) is also

one more option to get rid of foodborne

being targeted by the phage therapy.

pathogens (Thorns, 2000). The significant

Wagenaar _et al._ (2005); Loc _et al._ (2005)

reductions in the pathogenic bacteria can be

and Atterbury _et al._ (2005) have carried out

achieved by their bacteriophage biocontrol

successful phage therapy trials against

in food processing. In number of studies the

_Campylobacter_ spp. in poultry.

poultry products are used for their surface

decontamination by phages. Atterbury _et al._

Amongst the phage therapy reports in large

(2006) achieved a reduction in _S. enteritidis_

food animals Smith and Huggins (1983)

and _S. typhimurium_ below the detectable

showed an effectiveness of phages in

levels by applying phages to the chicken

treating experimental _E. coli_ diarrhea in

skin. Similarly, Goode and colleagues

calves, piglets and lambs. Infections were

(2003) could reduce the _Campylobacter_

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_Phage Biocontrol of Foodborne Pathogens_ Bhandare

_jejuni_ by 1-1.3 log10 CFU within 24 hours

by application of phages to the surface of

**ADVANTAGES OF USING PHAGES**

chicken skin. The phage biocontrol of

**FOR FOOD SAFETY**

_Salmonella_ upon whole carcasses of broiler

****

chickens and turkeys was achieved by

♦ Phages are ubiquitous in the environment.

Higgins _et al._ (2005). Phages have also been

Every known bacterium in nature is

used to reduce the numbers of _Salmonella_

supposed to have its complementary phage

from chicken sausages (Whichard _et al.,_

and thus it is possible to use phage

2003).

biocontrol against any type of bacteria.

In case of beef the bacteriophage biocontrol

♦ Bacteriophages are strain specific to an

has been attempted to extend shelf life by

individual bacteria and they don't harm the

reducing spoilage organisms (Greer and

other beneficial bacterial flora of the gut as

Dilts, 2002). The numbers of _E. coli_

is the case with antibiotics which harm the

O157:H7 from the beef surfaces were

commensal gut flora. They can also be given

brought down to below the detectable levels

in cocktails for broad spectrum effect.

by O'Flynn _et al._ (2004). A 3 log10 CFU

reduction in the _Listeria_ counts on salmon

♦ Bacteriophages are self replicating and

was achieved by Hagens and Loessner

thus the given dosage can self amplify in

(2007) after applying high titres of phage. _L._

due course of the treatment so that they

_monocytogenes_ , is more likely to occur

effectively tackle the target bacteria. Also,

during food processing, and thus at this

they will only replicate till the target

point of time phage biocontrol of this

bacterium is present and thus they are

pathogen can be done successfully. Anti-

naturally self limiting.

Listeria phages as food additives have been

approved by the US FDA by according it a

♦ As there is resistance development in

Generally Recognized As Safe (GRAS)

bacteria for antibiotics, the phage resistance

status and such products are available in the

may also develop but it has been reported

market for use in food processing.

(Loc Carrillo _et al._ , 2005; Atterbury _et al._ ,

2007 and Smith and Huggins, 1983) that

For control of biofilms in the food

such bacteria would have low virulence or

processing

environment

the

phage

less survival rate owing to the 'fitness

application is being envisaged. Roy _et al._

penalty'. Also, the resistance to phage does

(1993) and Hibma and colleagues (1997)

not affect their sensitivity to antibiotics and

have showed that the formation of _Listeria_

the relevant phages naturally evolve

biofilms

in

the

food

processing

alongside as bacteria evolve resistance.

environments can be reduced by application

of phages.

♦ There are no reports of allergic reactions

to phage as in antibiotics and no serious side

A great deal of research is being carried out

effects have been described so far either in

for

non-thermal

ways

of

controlling

animals or humans. This may be due to the

foodborne pathogens during processing for

abundance of phages in our environment and

the want of maintaining nutritive values of

regular exposure of animals and humans to

food

and

phage-based

non-thermal

them.

intervention is an ideal way in such

situations.

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_Phage Biocontrol of Foodborne Pathogens_ Bhandare

♦ It is relatively cheaper and easier to

**I.F.**

**(2005).**

'Correlation

of

produce phage preparations especially as

Campylobacter bacteriophage with

topical

applications

for

surface

reduced presence of hosts in broiler

decontamination of foods.

chicken

ceca',

_Applied_

_and_

_Environmental_

_Microbiology_

**71,**

♦ As the human phage therapy is fraught

**4885-7.**

with the limitations of lack of public

confidence and more regulatory concerns,

**Atterbury, R.J., Van Bergen, M.A., Ortiz,**

the application of phages as biocontrol

**F., Lovell, M., Harris, J.A. and De**

agents in food animals and in food

**Boer, A. __**

**(2006).**

'Control of

processing is an ideal way of harnessing the

_Salmonella_

in

poultry

using

potential of this nature's weapon for food

bacteriophage', In _Proceedings of_

safety.

_the 13th_ _International Symposium_

_Salmonella Salmonellosis_. Colin, P.,

Though not totally eliminated, the foodborne

and Clement, G. (eds). Saint Malo,

pathogens can be reduced to the acceptable

France: 10–12 May, **pp. 579–580.**

levels by application of bacteriophages as

pre-harvest and post-harvest interventions.

**Barrow, P. A. (2001).** "The use of

Phages can be effectively used along with

bacteriophages for treatment and

other food safety tools to protect public

prevention of bacterial disease in

health.

animals and animal models of human

****

infection." _Journal of Chemical_

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_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 32

**Research Highlights in 4Bs**

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_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

_1Biomedical Electronic Engineering, School of Mechatronic Engineering, Pauh Putra Main_

_Campus, Universiti Malaysia Perlis, Arau, 02600 Perlis, Malaysia;_

_2School 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-α and commercial antibody _H. pylori_ urease-β. 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

established as the causative agent for acute

or chronic gastritis (Mitchell _et al_., 1999)

_Helicobacter pylori_ is one of the common

and could be further developed into peptic

bacterial infections in human and recognized

ulcer disease, gastric carcinoma and others

as the etiologic agent for majority of upper

upper gastro duodenal diseases (Kiesslich _et_

gastro duodenal diseases. _H. pylori_ has been

_al_., 2005; Ardekani _et al_., 2013). According

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 33

_Res. Highl. 4Bs (2016)_

_Cloning and Expression of the Urease_ Mohamad _et al_.

to the World Health Organization (WHO)

**MATERIALS AND METHODS**

statistic, _H. pylori_ infection is on the rise and

****

proportional to the progress of a country.

_**Escherichia coli**_ **_growth and maintenance_** ****

Almost 50% of the world's population is

The _E. coli_ strains (Merck Inc.) used in this

infected by _H. pylori_ (Sasidharan _et al_.,

study and their genotypes are shown in

2008). In Malaysia, _H. pylori_ infection is on

Table 1. _Escherichia coli_ strains were

the raise as well. From the year 2000 until

grown on LB broth at 37°C for 16 hours in a

2007, patients infected by _H. pylori_ were

shaker incubator (Shel Lab, UK). For

30.4% of the gastro duodenal cases reported

storage purposes, _E. coli_ strains were stored

(Sasidharan _et al_., 2008).

in LB broth containing 50% glycerol (50%),

then, kept at -80oC for long term storage.

Urease is one of the pathogenic factors that

help _H. pylori_ colonizes the epithelium in

**Table 1** : Genotypes of _E. coli_ strains.

the acidic environment of the stomach

**Strain**

**Genotype**

(Ardekani _et al_., 2013). _H. pylori_ urease

Nova Blue

_endA1 hsdR17_ ( _r_

_–_

_+_

_K12 mK12_ )

displays enzyme-independent effects in

_supE44 thi-1 recA1gyrA96_

mammalian

models,

mostly

through

_relA1 lac_ [F' _proA+B+_

lipoxygenases-mediated pathway (Uberti _et_

_lacIqZ_ Δ _M15_ ::Tn _10_ ] __

_al_., 2013). The urease would induce edema,

BL21 (DE3)

F _– ompT hsdS_

_–_

_–_

_B_ ( _rB mB_ )

neutrophil chemotaxis and shows apoptosis

_gal dcm_ (DE3)

inhibition reverted in the presence of the

lipoxygenase inhibitors esculetin (Uberti _et_

_**Helicobacter pylori**_ **J99 _growth and_**

_al_., 2013).

_**maintenance**_ ****

_Helicobacter pylori_ J99 (ATCC 700824)

In addition to its involvement in the

was grown on Eugon agar with 10% human

pathogenesis process of _H_. _pylori_ infection,

expired blood at 37°C, 10% CO2 and 100%

urease is also a target for vaccination

humidity in an incubator (Shel Lab, UK).

development (Voland _et al_., 2006) besides

Subcultured was performed every four to

being a suitable marker to use as a target

seven days to maintain fresh bacterium. For

protein for detecting presence of _H. pylori_

storage purposes, _H. pylori_ J99 strain was

infection among suspected gastrointestinal

stored in TSB containing 20% glycerol

patient.

(20%), then, kept at -20oC for short term

storage and -80oC for long term storage.

In this study, urease operon were isolate and

examined by colony PCR, restriction

_**Plasmid cloning vector**_

enzyme digestion and nested PCR with

The plasmid cloning vector used in this

specific primer to confirm orientation of

study

was

pET-32-Ek/LIC

(Merck,

urease operon was done before continue to

Germany). **** This vector contains 109aa

protein expression. The sodium dodecyl

Trx•TagTM which encodes for the 109 amino

sulphate polyacrylamide (SDS-PAGE) gel

acid of thioredoxin protein. The pET-32-

was performed to examine the immune

Ek/LIC vector was specially designed for

functioning assay of recombinant urease

cloning and high-level expression of target

operon of _Helicobacter pylori_ J99 and

protein. Thioredoxin protein and histidine

protein sequencing to make sure this protein

tag would be fused to the target protein to

was functioning.

enhance purification of the expressed

proteins.

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ 34

_Res. Highl. 4Bs (2016)_

_Cloning and Expression of the Urease_ Mohamad _et al_.

the PCR product was analyzed on 1%

_**Genomic extraction of H. pylori J99**_

agarose gel then stained with ethidium

Genomic of _H. pylori_ was extracted by

bromide (EtBr) and 1Kb DNA ladder

employing GENEAll ExGene Cell SV kit

(Promega, USA) was used as a marker.

(Intron, Korea). The extracted genomic

****

DNA was then stored at -20oC for further

_**Preparations of pET32 Ek/LIC insert**_

analysis. This genomic extraction of _H._

The purification PCR product of _H. pylori_

_pylori_ J99 was used as a template for

urease operon by employing PCR DNA

amplification of urease gene. Primer pair

Extraction System (Intron, Korea). After

(pET32UOHP-F & pET32UOHP-R) would

that, the annealing procedure between

amplify the urease operon. The specific PCR

purified PCR products (Ek/LIC insert) with

primers for urease operon with pET-32

pET32 Ek/LIC vector was follow according

Ek/LIC complimentary overhangs are shown

to manufacturing protocol (Merck Inc.).

in Table 2.

_**Transform recombinant into cloning host**_

**Table 2** : Specific PCR primers sequence

Three microliter (3 µl) of amplification

with vector-compatible overhang.

product was transferred into a new 1.5 ml

**Primer**

**Sequences**

Eppendorf tube which had been cooled on

pET32UOHP-F

5' **GAC GAC GAC**

ice and the remaining annealing product was

**AAG**

**AT** G

AAA

stored at -20°C. An uncut plasmid (0.1 ng)

CTC ACC CCA AAA

was used as a control for transformation

GAG 3'

efficiency

of

the

competent

cells.

pET32UOHP-R

5' **GA GGA GAA**

_Escherichia_ _coli_ NovaBlue competent cells

**GCC**

**CGG**

**T** TC

were thaw on ice before transformation

AAA CCT TTT GCG

procedure. Transformation involved mixing

TGG TGG 3'

50 µl of the competent cell with 2 µl of

annealing product and mixed gently. After

_**Amplification of urease gene**_

that, the mixture was incubated on ice for 5

The total volume for PCR reaction was 25

minutes, then, heat-shocked for 30 seconds

µl [1x PCR buffer, 0.1 mM dNTPs (NHK

at 42°C. Immediately after heat-shock, the

Inc), 0.4 pmol of each pET32UOHP-F,

tube was placed on ice for 2 minutes. Next,

pET32UOHP-R primers, 1 unit of i-Taq

80 µl of LB was added and the mixture was

DNA polymerase (NHK Inc) and 50 to 100

incubated at 37°C, shaker at 250 rpm for 60

ng of _H. pylori_ J99 DNA template].

minutes. The mixture was spread onto LB

Gradient temperatures for amplification of

agar

plate

containing

ampicillin

_urease operon_ were from 64oC to 74oC by

(100µg/mL). The plates were incubated

using

gradient

PCR

thermal

cycler

overnight (16 - 20 hours) at 37°C.

(Biometra, USA).

_**Screening of recombinant urease operon**_

PCR cycler was programmed for 30 cycles

Colonies that formed had the possibilities of

with 95oC for 1 minute, 57oC for 2 minutes

carrying the insert. Thus, the colonies were

for full length urease operon and 72oC for 1

screened for inserts by performing colony

minute. The final elongation was set at

PCR using specific primers as in Table 3.

72oC for 10 minutes. Negative control was

included consisting of all mixture except the

The potential clones were also subjected to

DNA template. After PCR ended, 1 µl of

restriction enzyme digestion to confirm the

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ 35

_Res. Highl. 4Bs (2016)_

_Cloning and Expression of the Urease_ Mohamad _et al_.

presence of the inserts. The used of

with ampicillin (100µg/mL) and incubated

restriction enzymes either as single ( _Sal_ 1) or

at 250 rpm, 37oC for 3 to 4 hours until

double

digestions

( _Eco_ R1/ __

_Pst_ 1

and

OD600 reached 0.4 to 1.0. Next, 2 ml of this

_Eco_ RV/ _Bgl_ II) followed the manufacturer

culture was aliquot into Mc Courtney bottles

protocol (Promega, Inc.).

and kept at 4oC overnight. The next day, 2

ml pre-culture from 4oC was centrifuged at

The orientation of the urease gene was

13 000xg for 15 seconds, the supernatant

determined so that it was in the correct

was discarded and the cell pellet was

reading frame by nested PCR. The specific

suspended with 2 ml LB broth with

primers cws1, cws2 and cws3 were used to

ampicillin (100µg/mL). The suspended cell

confirm the target insert as shown in Table

with fresh media then inoculated into a 250

3. Finally, the clones were verified using

ml flask containing 50 ml LB broth with

DNA sequencing which was carried out by a

ampicillin (100µg/mL). The cells were

service provider, NHK Bioscience Solution

grown at 37oC, 250 rpm until OD600 reached

Sdn Bhd. The selected recombinant plasmid

approximately 0.6 (~3 to 4 hours). One ml

was named as pET32 _UO_ A6 for urease

of this culture was removed as un-induced

operon recombinant for expression urease

sample for SDS-PAGE analysis. Cells were

UreA/UreB.

induced with 0.4mM IPTG at 37oC, 250 rpm

for 3 hours.

**Table 3** : Specific nested PCR primers used

for insert confirmation.

Subsequently, the culture was incubated on

Gene

Sequences

ice for 10 minutes and harvested by

cws-1-F

5-CGT TAT GTC CTT AAG

centrifugation at 5000 x g for 20 minutes at

GAA AAA AC-3'

4oC. The cell pellet was washed with 0.25

cws-1-R

5-CCC ATG AGC GAT CGC

culture volume of cold 20 mM Tris-HCl pH

TGG GTT AAT GG-3'

8.0, then centrifuged at 5000 x g for 20

cws-2-F

5'- CCA TTA ACC CAG CGA

minutes at 4oC. The supernatant were

TCG CTC ATG GG -3'

discarded and the cells pellet was kept at -

cws-2-R

5'- CTA TGG GGC ATG CTT

80oC for further analysis.

ACG GTT AAG -3'

cws-3-F

5-CAA AGC TGA ATT CCA

The expression of urease protein was

ACG ATC GCT TAA CCG

detected

by

Sodium-dodecyl-sulphate

TAA GCA TGC C-3'

polyacrylamide gel electrophoresis (SDS-

cws-3-R

5-GGT TAA AAA GAC TCG

PAGE). The preparation and the assembly

AGG GTT TTT TAA TC-3'

of SDS-PAGE electrophoresis was carried

****

out according to the manufacturer protocol

_**Transformation**_

_**of**_

_**pET32UOA6** _

_**into**_

(Bio-Rad, USA). Briefly, the resolving gel

_**expression host**_

was at 12.5% with standard 4% stacking gel.

The recombinant plasmids were sub-cloned

into an expression host, _E. coli_ BL21 (DE3)

Cell pellets were suspended with PBS before

following

the

manufacturer

protocol

5X SDS-PAGE sample buffers at the ratio

(Promega Inc.)

3:1 was added. Next, the sample was heated

at 95oC for 5 minutes. After heated, this

_**Expression of urease operon**_

sample became viscous and 29cc gouge

A single colony was inoculated into a

needle was used to reduce the viscosities of

universal bottle containing 5 ml of LB broth

the sample. Twenty microliter of sample

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ 36

_Res. Highl. 4Bs (2016)_

_Cloning and Expression of the Urease_ Mohamad _et al_.

was loaded per lane on the SDS-PAGE gels.

protein sequencing was carried out to ensure

The SDS-PAGE running buffer was filled

the expressed recombinant urease was

into the bottom and upper chambers and all

identical to _H. pylori_ J99 urease protein.

the bubble were removed from the wells of

****

the gels using a syringe. The gel was run at

**RESULTS AND DISCUSSION**

80V constant current for 35 minutes and

****

100V for 2 hours until the stacking dye had

_**Determine of recombinant urease operon**_

reached the end of the gel. After that, the gel

The urease operon was determined by their

was carefully removed from the glass plates

size based on urease gene sequence

and stained with Coomassie Blue stain for 1

information, accessed from NCBI database

hour and destained with destining solution

(Accession No. NC_000921.1). Complete

overnight (~16 hours). The expression of

urease operon that encode for urease

constructed recombinant urease operon was

enzymes and accessory proteins. As shown

important to measure the urease expression

in Figure 1, the presence of the urease

level and to know the biological active of

operon PCR amplified was detected with the

the recombinant urease.

expected size of 5974 bp.

****

_**Immune functioning assay**_

The immune functioning assay was used to

verify

the

recombinant

protein

of

pET32UOA6. The electrophoresis transfer

of proteins to 0.45 µm Polyvinylidene

fluoride (PVDF) membrane (Millipore Inc.)

was accomplished by a modification of the

method described by Towbin _et al_. (1979).

**Figure 1** : Amplification of urease genes

Protein was electro blotted from the gel onto

from ATCC 00824 _H. pylori_ J99. Lane 1:

PVDF membrane by electrophoresis transfer

1Kbp DNA ladder; Lane 2 to 7:

using a Mini Trans-bolt® Electrophoretic

amplification of urease genes; Lane 8:

Transfer Cell (Bio-Rad, USA). Lastly, the

Negative control (lack of i- _Taq_ DNA

membrane was put in a plastic wrap and the

polymerase).

image was captured within 1 minute using

Gene Genius Bio Imaging System.

_**Recombinant clone screening using PCR**_

The positive colonies were selected and

_**Protein sequencing**_

subjected to PCR reaction using specific

Recombinant urease clones were grown,

primers (Table 3) to confirm the presence of

induced with 0.4 mM IPTG, followed by

the inserts. All selected colonies was had

SDS-PAGE as described in Section 3.6.9.

inserts with the expected size that represent

After

SDS-PAGE,

the

band

which

the size of urease operon fragment.

represented the recombinant UreA and UreB

****

was purified and sent to 1st BASE, Inc. for

_**Restriction enzyme digestion analysis**_

protein sequencing. Finally, the amino acid

Two clones from each of the recombinants

sequence resulted from protein sequencing

were further selected for verification. The

was aligned using Protein Basic Local

plasmids from potential recombinant clone

Alignment Search Tool (NCBI BLASTp) of

pET32UO were digested with restriction

National

Center

for

Biotechnology

enzymes to further confirm the presence of

Information (Stephen _et al.,_ 1997) and this

urease gene fragments. **** A single digestion

( _Sal_ I) and double digestion ( _Eco_ RI & _Pst_ 1

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ 37

_Res. Highl. 4Bs (2016)_

_Cloning and Expression of the Urease_ Mohamad _et al_.

or _Eco_ RV & _Bgl_ II) produced different

Final verification of the recombinant

restriction enzyme patterns due to the unique

plasmids was made by DNA sequencing on

sites of these restriction enzymes on the

clone pET32UOA6. The DNA sequencing

plasmids. Table 4 shows the expected

was performed using specific primers as

fragment sizes resulted from the chosen

shown in Table 3. The results of the DNA

restriction enzymes. Furthermore, agarose

sequencing

showed

99%

nucleotide

gel electrophoresis verified the expected

similarity for urease operon to _H_. _pylori_ J99

fragment sizes from restriction enzyme

genome when analyzed using NCBI BLAST

digestions, as shown in Figure 2. All of the

program (Zheng _et al_., 2000). ****

selected clones indicated the presence of

recombinant plasmid harbouring urease

_**Expression of urease genes**_

genes.

Plasmid pET32 Ek/LIC carries IPTG

inducible T7 _lac_ promoter for protein

**Table 4** : Restriction enzyme digestions of a

expression (Merck, Inc.). The expressed

selected potential recombinant clone.

protein from this plasmid would be a fusion

Expected

protein of 109 amino acids thioredoxin to

Recombinant

Restriction

size

the

protein

of

interest.

Thus,

the

clones

enzyme

fragment

recombinant urease produced would be

(kbp)

slightly bigger than the native urease

pET32UO

_Eco_ RI &

1332 &

_Pst_ I

10559

_**Induction study of ureases production**_

In this study, the expression of recombinant

_Eco_ RV &

5865 &

urease was used 0.4 mM and/or 1.0 mM

_Bgl_ II

6026

IPTG and 2 and/or 3 hours induction time.

As shown in Figure 4, bands representing

_Sal_ I

11891

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 2** : Restriction enzyme analysis of

Figure 3, bands representing recombinant

potential recombinant clones. The restriction

UreA and UreB were detected indicating the

enzyme

digestion

of

urease

operon

clones were carrying functional urease

fragment. Lane 1: 1 Kb DNA ladder; Lane

genes.

2 and 6: undigested plasmids; Lane 3 and 7:

_Eco_ RI & _Pst_ I digestion; Lane 4 and 8:

_**Determination of immune functioning of**_

_Eco_ RV & _Bgl_ II digestion; Lane 5 and 9: _Sal_

_**the expressed ureases**_

I digestion and Lane 10: λ _Hind_ III DNA

The sizes of _H_. _pylori_ recombinant UreA

ladder.

and UreB (Figure 4) were bigger, more than

29 kDa and 62 kDa respectively due to the

_**DNA Sequencing**_

fused amino acids thioredoxin. Sometime,

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ 38

_Res. Highl. 4Bs (2016)_

_Cloning and Expression of the Urease_ Mohamad _et al_.

the presence of additional amino acids fused

ureases. These results confirmed the

to protein of interest could change the

recombinant UreA and UreB maintained

protein conformation. Therefore, it was

their immune properties.

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)

**Figure 4** :

Western blot analysis for

pET32UOA6 (B) pET32ureA3 and (C)

determination

of

recombinant

ureases

pET32ureB2. Lane 1: DGelTM Marker;

immune functioning in of UreA (A) and

Lane 2: Pre-culture urease gene; Lane 3 and

UreB crude cell (B). Lane 1, 10, 11 and 20:

6: uninduced culture; Lane 4: Induced

Kaleidoscope Prestained protein ladder;

culture with 0.4 mM IPTG at 2 hours;

Lane 2-3 and 12-13: pET32UOA6 cell

Lane7: Induced culture with 0.4 mM IPTG

crude; Lane 5-6: pET32ureA6 cell crude;

at 3 hours; Lane 5: Induced culture with 1.0

Lane 15-16: pET32ureB2 cell crude; Lane 4

mM IPTG at 2 hours and Lane 8: Induced

and 14: _Salmonella_ cell crude; Lane 7 and

culture with 1.0 mM IPTG at 3 hours.

17: _Pseudomonas_ cell crude; Lane 8 and 18:

_H. pylori_ J99 cell crude; Lane 9 and 19: _E._

Figure 4 shows immune functioning of

_coli_ cell crude.

UreA and UreB from cell crude of

recombinant urease clones compared with

Immune functioning assay verified the

other bacterial crude cells, as negative

recombinant UreA and recombinant UreB

controls and _H. pylori_ J99 crude cell as the

still maintained their antigenicity, equivalent

positive control. _Helicobacter pylori_ urease-

to native enzyme regardless of the presence

α and urease-β antibodies (Santa Cruz, Inc.)

of additional amino acids fused to them.

were very specific and sensitive to UreA and

These

were

supported

by

immune

UreB of _H. pylori_ J99 crude cell (lanes 8 and

functioning assay through Western blotting

18). Similar specificity and sensitivity were

and previous work by Hu _et_ _al_. (1992).

observed for recombinant UreA and UreB,

Purification of both recombinant ureases did

expressed in full operon unit (lanes 2-3 and

not affect the antigenicity, as evidence by

12-13). As suggested by the manufacturer,

Western blotting (Figure 4). Regardless of

crude cells from _Salmonella_ (lanes 4 & 14),

this condition, both recombinant ureases still

_Pseudomonas_ (lanes 7 & 17) and _E_. _coli_

maintained their antigenicity in immune

(lanes 9 & 19) gave negative results that

functioning assay. Purification process

indicate _H. pylori_ urease-α and urease-β

failed to separate UreA from UreB since _H_.

antibodies were very specific and sensitive

_pylori_ urease-α and urease-β antibodies

to the native, as well as, the recombinant

(Santa Cruz, Inc, USA) still detecting both

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ 39

_Res. Highl. 4Bs (2016)_

_Cloning and Expression of the Urease_ Mohamad _et al_.

of them in the immune functioning assay.

This study was supported by the Universiti

Thus, clone pET32UO expressed and

Malaysia Perlis for Academic Training

assembled the urease apoenzyme and

Scheme Funding (SLAI).

perhaps together with the accessory proteins

to form the holoenzyme.

**REFERENCES**

The confirmation of recombinant urease

**Ardekani, L. S., Gargari, S. L. M.,**

protein was demonstrated by protein

**Rasooli,**

**I.,**

**Bazl,**

**M.**

**R.,**

sequencing. The results of amino acid

**Mohammadi, M., Ebrahimizadeh,**

alignment based on BLASTp between

**W. and Zare, H. (2013).** A novel

recombinant urease against _H. pylori_ J99

nanobody against urease activity of

urease show high percent identities, 99-

_Helicobacter pylori_. _International_

100%. Thus, these results further confirmed

_Journal of Infectious Diseases_ **17(9),**

the authenticity of the recombinant ureases

**e723-e728**.

similar to _H. pylori_ J99 urease.

****

**Hu, L. T., Foxall, P. A., Russell, R. O. B.**

_**Confirmation of recombinant ureases by**_

**E. R. T. and Mobley, H. L. (1992).**

_**protein sequencing**_

Purification

of

recombinant

Figure 5 shows the results of protein identity

_Helicobacter_

_pylori_

urease

based on BLASTp between recombinant

apoenzyme encoded by _ure_ A and

urease and _H. pylori_ J99 ureases. These

_ure_ B. _Infection and Immunity_ _**60**_ **(7),**

results further confirmed the authenticity of

**2657-2666**.

the recombinant ureases expressed by

pET32UOA6.

**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),**

**2119-**

**2123**.

**Figure 5: __** The percentage identity of

recombinant ureases against _H. pylori_ J99

**Mitchell, H.M., Hazell, S.L., Bohane,**

ureases _,_ pET32UOA6.

**T.D., Hu P., Chen, M. and Li, Y.Y.**

**(1999).** The prevalence of antibody

**CONCLUSION**

to _cagA_ in children is not a marker

for specific disease. _Journal of_

In this study, the availability of a functional

_Pediatric_

_Gastroenterology_

_and_

replicon carries urease operon has potential

_Nutrition_ **28, 71 – 75**.

benefit related to _H. pylori_ pathogenesis. A

through and better understanding of _H._

**Sasidharan, S., Uyub, A.M. and Azlan,**

_pylori_ pathogenesis process could contribute

**A.A. (2008).** Further evidence of

to the improvement of diagnostic methods.

ethnic and gender differences for

_Helicobacter pylori_ infection among

**ACKNOWLEDGEMENT**

endoscoped patients. _Transactions of_

_the Royal Society of Tropical_

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ 40

_Res. Highl. 4Bs (2016)_

_Cloning and Expression of the Urease_ Mohamad _et al_.

_Medicine and Hygiene_ **102, 1226-**

**Uberti,**

**A.**

**F.,**

**Olivera-Severo,**

**D.,**

**1232**.

**Wassermann,**

**G.**

**E.,**

**Scopel-**

**Guerra,**

**A.,**

**Moraes,**

**J.**

**A.,**

**Stephen, F., Altschul, Thomas, L.,**

**Barcellos-de-Souza, P. and Carlini,**

**Madden, Alejandro, A., Schäffer,**

**C. R. (2013).** Pro-inflammatory

**Jinghui Zhang, Zheng Zhang,**

properties and neutrophil activation

**Webb, M. and David, J. L. (1997)** ,

by

_Helicobacter_

_pylori_

urease.

Gapped BLAST and PSI-BLAST: a

_Toxicon_ **69, 240-249**.

new generation of protein database

****

search programs. _Nucleic Acids_

**Voland, P., Zeitner, M., Hafsi, N. and**

_Research_ _**25**_ **, 3389-3402**.

**Prinz, C. (2006).** Human immune

response

towards

recombinant

**Towbin, H., Staehelin, T. and Gordon, J.**

_Helicobacter pylori_ urease and

**(1979).** Electrophoretic transfer of

cellular fractions. _Vaccine_ _**24**_ **(18),**

proteins from polyacrylamide gels to

**3832-3839**.

nitrocellulose sheets: procedure and

some applications. _Proceedings of_

**Zheng, Z., Scott, S., Lukas, W. and Webb,**

_the National Academy of Sciences_

**M. (2000).** A greedy algorithm for

_**76**_ **(9), 4350-4354**.

aligning DNA sequences. _Journal of_

_Computer Biology_ _**7**_ **(1-2), 203-214**.

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ 41

**Research Highlights in 4Bs**

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_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

_1Department 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

natural sources, synthesis by chemical

precursors and by biotechnological routes

Flavour is a combination of taste and

via _de novo_ or biotransformation.

aroma. It results from the perception of

odor-active volatile compounds. Food

The milk fat component of dairy source

flavours are mixtures of natural and/or

largely contributes to the release of

artificial aromatic compounds. They are

flavour. Butter possesses its own distinct

designed to impart, modify, or even mask

flavour. The flavour compounds usually

an undesirable flavour. Flavours along

are aldehydes, ketones and lactones of

with fragrances are highly prized in the

which diacetyl and acetoin play important

global market. Currently there are three

roles in parting the well-known 'buttery'

known methods of acquiring flavour

flavour. Lactones play important role in

compounds (Bicas _et al_., 2010). These

conferring the 'buttery' taste and sweet

include, extraction from pre-existing

aromas altogether (Hua _et al_., 2007).

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 42

_Res. Highl. 4Bs (2016)_

_Microbial Production of Butter Flavour_ Nadaraj _et al_.

Currently, diacetyl and acetoin, the flavour

**MATERIALS AND METHODS**

compounds are chemically synthesised at

The process flowchart in Figure 1

commercial scale. The inhalation of

highlights the major stages in the

intense synthetic butter flavour at the

production of butter flavour concentrate.

source of manufacturing and application

results

in

bronchiolitis

obliterans,

_**Isolation of Lactic Acid Bacteria (LAB)**_

commonly termed as 'popcorn lung'

_**from various samples**_

disease (Morris and Hubbs, 2009).

Raw milk was purchased from a local

market. Soured milk was prepared by

The increasing demand for natural butter

allowing the raw milk to sour for a day.

flavour has lead to the development of

Pasteurized milk (Marigold, Dutch Lady),

biotechnological processes. However, the

cheese (Emborg, Kraft), unsalted butter

fermentation processes which involve

(Devondale,

Tatura),

salted

butter

lactic acid bacteria in obtaining high yields

(Devondale,

Anchor), cultured drink

of desirable enantiomeric butter flavour

(Solivite, Nutrigen), yoghurt (Dutch Lady)

compounds from butter fat are yet to be

were purchased from TESCO supermarket

established. The development of solid state

in Alor Setar, Kedah Darul Aman,

fermentation

techniques

for

the

Malaysia. Soil, grass and water samples

bioconversion of butter fat and the

were collected from a nearby cattle farm.

recovery of butter flavour compounds are

Lactic Acid Bacteria **(** LAB) were isolated

important challenges for researchers in this

from these samples as per the method

field. Hence, this study was undertaken to

(Bettache _et al_., 2012). The isolates were

optimize the fermentation conditions for

stored in agar slants at 4°C. The individual

the

production

of

butter

flavour

isolates were tested for their ability to

concentrate from butter fat by lactic acid

ferment

butter

to

produce

flavour

bacteria in solid substrate fermentation.

concentrates.

**Figure 1** : Flowchart for the production of butter flavour concentrate. ****

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 43

_Res. Highl. 4Bs (2016)_

_Microbial Production of Butter Flavour_ Nadaraj _et al_.

_**ATCC strains of Lactic Acid Bacteria**_

_**Recovery of butter oil**_

_Lactobacillus acidophilus_ (ATCC 314)

Butter oil was extracted from fermented

and _Lactobacillus casei_ (ATCC 393)

butter fat samples for the sensory

strains were purchased from ATCC and

evaluation,

fatty

acid

analysis

and

also evaluated for the production of butter

dertermination of diacetyl and acetoin in

flavour.

the fermented samples.

_**Pasteurization and sterilization of butter**_

_**Microbial fermentation of butter fat by**_

_**fat**_

_**Solid Substrate Fermentation (SSF)**_

The recommended protocol by the

Ten ml of overnight culture in MRS broth

International Dairy Federation (Juffs and

with the isolated LAB bacteria was

Deeth,

2007)

was

used

for

the

prepared individually. Six g of butter fat

pasteurization of butter. Sterilization of

each in glass Perti plates were autoclaved

butter fat was by autoclaving at 121 ºC at

(121 ºC, 15 psi, 15 minutes). The melted

15 psi, for 15 minutes.

butter in the petri dishes was gently

swirled and cooled. Individual petri dishes

_**Standardization of butter oil extraction**_

were then inoculated with 2 ml of

_**methods**_

overnight culture of the Lactic acid

To standardize the butter oil extraction

bacteria. As a control, an uninoculated

method, three different protocols were

petri dish with only the sterilized butter fat

studied (Table 1). Based on the total

was used. At fixed time intervals (24th, 48th

amount of oil extracted, the percentage

and 72th hour) samples were drawn from

(%) of recovery was calculated based on

the petri dishes and evaluated by sensory

the following formula:

evaluation. A total of 10 evaluators were

selected to provide descriptions of the

% recovery = Amount of pure product

aroma from the samples such as odourless,

recovered (g) x 100 / Amount of crude

sweet, stale, sour, buttery or alcoholic.

material used (g)

Butter oil samples were then extracted

from the samples after scoring of the

**Table 1** : Different methods for the

aroma description. The samples were

extraction of butter oil from butter fat.

stored in the cold room for further

**Method**

**Conditions**

analysis.

1

a) Melting in a water bath at

40 ºC (±5ºC) for 10 minutes.

_**Supplementation of butter fat with**_

b) Centrifugation at 800 rpm

_**various carbohydrate sources**_

for 3 minutes

An

experiment

was

conducted

to

(Chongcharoenyanon _et al_.,

determine whether supplementation of

2012).

butter fat with various carbohydrates

2

a) Melting in a water bath at

sources such as galactose, lactose and skim

50 ºC (±5ºC) for 8 minutes.

milk

may

enhance

butter

flavour

b) Centrifugation at 3,700 rpm

production

upon

solid

substrate

for 12 minutes(Krause and

fermentation.

Initial

efforts

were

Gibson, 2008).

conducted in petri dishes where 6 g of

3

a) Melting in hot air oven at 70

butter was weighed and supplemented with

ºC (±5ºC) for 15 minutes.

10% (w/w) of galactose, lactose and skim

b) Centrifugation at 3,500 rpm

milk, individually. The supplemented

for 5 minutes (Miura _et al_.,

butter fat was then sterilized and uniformly

2004).

spread the butter fat in the petri dish. After

cooling, each Petri dish was inoculated

with 2 ml of overnight cultures of the

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 44

_Res. Highl. 4Bs (2016)_

_Microbial Production of Butter Flavour_ Nadaraj _et al_.

selected lactic acid bacteria and incubated

University. Prior to sampling, volunteers

at 37°C. One petri dish was designated as

were first requested of their health status

control. Sensory evaluation of samples

and free of any respiratory illnesses.

was performed at fixed time intervals (24th,

During inhalation of aroma, volunteers

48th and 72th hour) as reported previously.

were required to inhale the samples and

suspend breathing for 2-3 seconds and

_**Scale up studies of Solid Substrate**_

mark their preferences in the flavour

_**Fermentation (SSF)**_

survey form provided. Coffee powder was

Scales up studies were performed with

provided as a neutralizer to eliminate

increased quantities of butter fat. In order

traces of previous aromas. Samples

to increase the surface area for solid

indicating high preferences were selected

substrate fermentation, experiments were

for further analysis by GC-MS (Gas

carried out with various amounts of butter

chromatography-mass spectrometry).

fat taken in conical flasks (30 g in 250 ml

conical flask, 60 g in 500 ml conical flask

_**GC-MS analysis of butter oil for acetoin,**_

and 160 g in 1000 ml conical flask).

_**diacetyl and fatty acid**_

Overnight

cultures

were

prepared

Volatile compounds such as acetoin and

individually and the volume of overnight

diacetyl are generally analysed by GC-MS

inoculum used was 10 ml for 30 g, 20 ml

(Gokce _et al_., 2014). Extraction of diacetyl

for 60 g and 40 ml for 160 g substrates,

and acetoin from the treated butter oil was

respectively.

These

cultures

were

performed. Chromatographic analyses of

incubated at 37ºC under anoxic conditions.

the treated and control butter samples for

At specified time intervals of 24th, 48th and

aroma

compounds

and

fatty

acid

72th hour, samples were acquired for

components were performed using a split

sensory

evaluation

by

10

random

less injector system gas chromatograph

volunteers.

coupled

with

a

mass

spectrometer

(SHIMADZU

GCMS-QP2010).

The

_**Scale up studies of Solid Substrate**_

carrier gas used was ultra-pure helium with

_**Fermentation (SSF) with 10% galactose**_

a flow rate of 1.0 mL/min. The injection

Scale up studies was carried out with 30 g,

port was worked at 250°C in split less

60 g and 160 g of butter fat, supplemented

mode coupled with 1 minute split less

with 10% of galactose individually. The

time. A 1 µl injection volume was applied

flasks were sterilized and inoculated with

for each sample analysis and the syringe

overnight inoculum respectively. They

was washed with hexane upon completion

were incubated and samples were drawn

of injection. Separation was performed

and tested as previously.

using a DB-WAX (60 m x 0.25 mm x 0.15

µm) capillary column with a 0.15 µm

_**Sensory evaluation of butter oil samples**_

stationary film. The oven temperature

_**from different fermented butter samples**_

programme was set as follows: initial

Sensory evaluation of butter oil samples

temperature 40°C, increased by 7°C min-1

that were obtained from the fermented

to 200°C and held for 1 minute. Mass

butter samples were performed as per the

spectrometric parameters were set as

methods by Anvoh _et al_. (2009). A 5-point

follows: electron impact ionization with 70

hedonic scale was utilized where 1

eV energy and 250°C ion source. The

represents dislike the most, 3 represents

aromatic compounds and fatty acid

neither like nor dislike and 5 represents

profiles were detected and quantified

like the most. Sensory scores were

based on their retention time and peak

evaluated based on preferences of odour

areas on the chromatogram respectively.

by 120 students of Biotechnology from the

Faculty of Applied Sciences, AIMST

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 45

_Res. Highl. 4Bs (2016)_

_Microbial Production of Butter Flavour_ Nadaraj _et al_.

_**Butter powder formulation with**_

LAB are lactic acid production which

_**maltodextrin**_

results in the improvement of flavour,

Maltodextrin was used as a carrier

aroma, keeping quality and enhancement

material, as it is edible, slightly sweet, and

in nutritional content (Halász, 2009).

low in cost (Wandrey _et al_., 2010). For the

Butter is an important flavouring additive

production of butter oil powder, the

and creates the distinctive aroma and taste

extracted butter oil was mixed with

in bakery, dairy and other food products.

maltodextrin with different anti-caking

Diacetyl and acetoin are two important

agents and the entire mixture was then

constituents in butter fat that contribute to

treated with liquid nitrogen and mixed

unique butter flavour. Natural butter

evenly using a mixer. The addition of

flavour is expensive, hence artificial butter

liquid nitrogen was to imitate freeze drying

flavour

compounds

are

chemically

conditions. Formulation of butter powder

synthesized

from

petroleum-derived

from carrier material (maltodextrin) and

precursors. Synthetic

flavours

are

active material (butter oil) was performed

produced in bulk at low cost and it is a

individually by using 50 g of maltodextrin

mixture of racemic compounds. The

powder and varying amounts of butter oil

prolonged exposure to synthetic flavour

(5 g, 10 g, 15 g, 20 g, 25 g and 30 g). The

compounds to the line workers was

maltodextrin powder was added into a

reported harmful by National Institute for

plastic container, and butter oil of the

Occupational Safety and Health (NIOSH)

concerned weight was poured into the

(Kreiss, 2007).

container. Simultaneously, the mixture

was

added

with

liquid

nitrogen

Biotechnological approaches in producing

(approximately 100 ml) and manually

butter flavour compounds are being

stirred. The prepared powder was then

investigated as a replacement to chemical-

stored in air-tight glass containers and kept

based processes (Longo and Sanromán,

at room temperature away from any light

2006). This study was aimed at producing

source. Anti-caking agents are required in

butter flavour concentrate from butterfat

the formulation of powdered food or drug

by a microbial process. The investigation

preparations to either stop or postpone

included isolating LAB strains as well as

occurrences of caking (Lipasek _et al_.,

_Lactobacillus acidophilus_ (ATCC 314)

2011). For this, 50 g of the prepared butter

and _Lactobacillus casei_ (ATCC 393) in the

powder was placed in a plastic container

fermentation of butter fat by solid substrate

and supplemented individually with 2%,

fermentation (SSF). After fermentation,

4% and 6% (w/w) anti-caking agent that

removal of solids by centrifugation and

comprised

sodium

chloride,

sodium

recovery of butter oil from the treated

silicate, sodium bicarbonate, bentonite,

butter was performed. The butter oil was

mannitol and potato starch. The mixture

then subjected to sensory evaluation and

was manually stirred with intermittent

analysed for butter flavour compounds by

shaking to enable even dispersion of the

GC-MS.

anti-caking agent and the powder. The

prepared powder was then stored in a glass

_**Isolation of LAB from various samples**_

container that was sealed and stored in a

Raw milk, soured milk, cheese, unsalted

dry area.

butter, salted butter, yoghurt and grass,

soil, and water samples from cattle grazing

**RESULTS AND DISCUSSION**

areas were niches of LAB strains. A total

of ten colonies were isolated from the

Over the years lactic acid bacteria (LAB)

different samples.

have been used in various fermented food

preparations. The benefits conferred by

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 46

_Res. Highl. 4Bs (2016)_

_Microbial Production of Butter Flavour_ Nadaraj _et al_.

_**Pasteurization and sterilization of butter**_

reported that diacetyl may also contribute

_**fat**_

to the formation of undesirable, off-

Two

different

methods

namely

flavours as observed in spirits manufacture

pasteurization and sterilization of butter fat

(Krogerus and Gibson, 2013). The

were

evaluated.

Compared

to

fermented

butter

fat

samples

were

pasteurization (63ºC±5ºC for 30 minutes),

subjected to sensory evaluation by ten

the sterilization of butter fat samples was

volunteers.

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

****

**Figure 2** : Picture showing the recovery of

g of butter fat (Figure 2).

butter oil by using three methods.

_**Percentage recovery of butter oil by**_

**Table 2** : Percentage recovery of butter oil

_**different methods**_

by different methods. ****

On comparative analysis, it was found that

Butter oil recovery (g)

the percentage recovery of oil was high

M* Trial Trial Trial Average Recovery

(69.67%), when the butter fat was melted

1

2

3

%

at 50 °C for 8 minutes and centrifuged at

1 **4.14 4.11 4.12**

**4.12**

68.67

3,700 rpm for 12 minutes (Table 2). The

2 **4.17 4.19 4.18**

**4.18**

69.67

oil recovery was recorded low in the other

3 **3.79 3.81 3.77**

**3.79**

63.17

two methods. However, the clarity of

Values represent the mean of three

butter oil was found to be good in the

replicates; *Method 1: Melting of butter

second method when compared to methods

fat at 40 ºC (±5ºC) for 10 minutes and

1 and 3. This may be attributed to high

centrifugation at 800 rpm for 3 minutes

centrifugal forces for efficient separation

(Chongcharoenyanon

_et_

_al_.,

2012);

of

denser

materials

from

lighter

Method 2: Melting of butter fat at 50 ºC

counterparts (Anlauf, 2007).

(±5ºC) for 8 minutes and centrifugation at

3,700 rpm for 12 minutes (Krause _et al_.,

_**Solid Substrate Fermentation (SSF) of**_

2008); Method 3: Melting of butter fat at

_**butter fat in glass petri dishes (Figure 3)**_

70 ºC (±5ºC) for 15 minutes and

_**and sensory evaluation**_ ****

centrifugation at 3,500 rpm for 5 minutes

After 24 hours of incubation, there was a

(Miura _et al_., 2004).

significant improvement in the flavour

content in the fermented samples with CFS

_**Sensory evaluation of SSF (Solid**_

and CFG strains individually (Table 3).

_**Substrate Fermentation) samples with**_

Whereas, with prolonged incubation the

_**various concentrations of butter fat**_

off flavour production was recorded.

The fermented butter fat samples were

Butter flavour production is based on

recorded with preferred sweet and buttery

multi-step

reactions

which

involve

flavour after 24 hours of incubation with

lipolytic,

proteolytic

and

glycolytic

both the isolated strains (CFS and CFG) in

reactions (Smit _et al_., 2005). It was

Table 4. The increase in butter fat quantity

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 47

_Res. Highl. 4Bs (2016)_

_Microbial Production of Butter Flavour_ 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

substrate

CFS

CFG

_**Supplementation of butter fat with**_

fermentation

strain

strain

_**various carbohydrate sources**_

(SSF)

Lactic acid bacteria are able to ferment

(hour)

various hexose sugars, from simple

24

Buttery

Sweet

(galactose) to complex (lactose) sugars but

48

Sour,

Sour,

is

species-dependant

(Halász, 2009).

alcoholic

alcoholic

Galactose moieties present with lactose

72

Stale,

Stale,

molecules in milk when metabolized, may

odourless odourless

lead

to

intense

aroma

production

§Scores based on the aroma description by

(Hugenholtz _et al_., 2002). In order to

10 volunteers; CFS: bacterial strain

determine if addition of lactose, skim milk

isolated from cattle field soil sample;

and galactose would enhance the butter

CFG: bacterial strain isolated from cattle

flavour

production,

solid

substrate

field grass sample.

fermentation was carried with both the

strains. The butter fat (6 g) was

in the medium may have facilitated the

supplemented with 10 % (w/w) substrate

volunteers in scoring more for sweet and

individually and fermented with CFS and

buttery flavours. The increased amounts of

CFG strains. Table 5 summarizes sensory

raw material (fats and milk sugars) are the

evaluation results of the experiment by

intermediates for flavour compounds

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 48

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_Microbial Production of Butter Flavour_ Nadaraj _et al_.

**Table 4** : Sensory evaluation of SSF samples with varying concentrations of butter fat*. ****

Solid substrate

Sensory evaluation

fermentation

Butter fat 30 g

Butter fat 60 g

Butter fat 160 g

(SSF)

CFS

CFG

CFS

CFG

CFS

CFG

(hour)

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

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

Sensory evaluation

fermentation

CFS strain

CFG strain

(SSF) (hour)

Galactose Lactose Skim Milk Galactose Lactose

Skim Milk

24

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

cattle field soil sample; CFG: bacterial strain isolated from cattle field grass sample.

****

undergraduate students (Figure 4). The

_**Scale up studies of Solid Substrate**_

sensory evaluation of the samples by

_**Fermentation (SSF) with galactose**_

students is depicted in Table 6. _****_

Scale up studies were performed with

increased butter fat (30 g, 60 g and 160 g)

A sweet, butter flavour production was

supplemented

with

10%

galactose

enhanced with both the CFS and CFG

individually. Figure 5, indicates the scale

strains in 24 hours of incubation. Upon

up studies of 160 g butter fat supplemented

prolonged incubation (48 and 72 hours),

with 16 g (10%) galactose. After

stale and sour odour were recorded. The

fermentation, the butter oil was extracted

results

indicated

that

butter

fat

and subjected to sensory evaluation. Based

supplemented with 10 % galactose and

fermented specifically with the CFS strain

on the aroma description (sour, sweet,

was effective in producing butter oil with

buttery, milky, stale and rancid). Scoring

intense

butter

flavour.

Butter

fat

was done by 10 volunteers. Table 6,

supplemented with skim milk recorded a

indicates the sensory evaluation results of

more pronounced milk flavour, compared

the fermented sample.

to butter flavour. Therefore, scale up

****

****

studies were carried out with increased

_**Sensory evaluation of butter fat samples**_

amounts of butter fat (30 g, 60 g and 160

_**supplemented with galactose at scale up**_

g) supplemented with 10 % galactose

_**studies**_

individually.

Based on the sensory evaluation, the butter

fat samples fermented with strains CFS

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 49

_Res. Highl. 4Bs (2016)_

_Microbial Production of Butter Flavour_ 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

cattle field soil sample; CFG: bacterial strain isolated from cattle field grass sample.

and CFG individually were scored sweet

oil was extracted and subjected to sensory

and buttery within 24 hours of incubation

evaluation.

(Table 6). Extended incubation period (48

and 72 hours) resulted in formation of

_**Sensory evaluation of butter oil samples**_

undesired, foul (stale or rancid or

_**by 120 students**_ ****

alcoholic)

odours.

Therefore,

the

Compared to the control sample (BF), all

experiment was repeated and after 24

other samples were preferred (Figure 6).

hours

of

incubation

period,

the

Most individuals (38%) did not approve of

fermentation was arrested by freezing the

sample BF with 53% of the volunteers

samples at -20°C. The respective samples

being unsure of their preference. The least

were thawed at room temperature. Butter

amount (8.3%) of evaluators prefer this

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 50

_Res. Highl. 4Bs (2016)_

_Microbial Production of Butter Flavour_ 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

supplemented

with

galactose

and

analysis of unsupplemented butter fat

fermented with strain CFS) and from BF

fermented with isolated LAB, sample

(untreated butter as control) were analysed

BF+CFS was more preferred (45%) by

for diacetyl and acetoin content by GC-MS

students as compared to sample BF+CFG

method. The results were tabulated.

(19.2%). For supplemented butter fat with

****

galactose and fermented with isolated

_**Comparative analysis of diacetyl and**_

LAB, a large number of evaluators

_**acetoin content in the unfermented and**_

(56.7%) preferred sample BF+Gal+CFS as

_**fermented butter fat samples with CFS**_

compared to sample BF+Gal+CFG (45%).

_**strain**_ ****

Hence,

samples

BF+CFS

and

Acetoin content was highest (1321.2 ppm)

BF+CFS+Gal were selected for further

in the butter fat sample fermented with

GC-MS analysis of constituents with

CFS strain

(BF+CFS).

In contrast,

sample BF serving as the control.

untreated butter fat (BF) reported lowest

amount of acetoin (211.5 ppm) amongst all

_**Analysis of butter oil volatile constituents**_

3 samples (Figure 7). With regards to

It was inferred that the butter fat samples

diacetyl content, butter fat supplemented

fermented with CFS strain either with or

with galactose and fermented with CFS

without galactose was most preferred

strain had the highest concentration (511.4

based on the sensory analysis of five

ppm). Lowest content of diacetyl was

different samples by 120 volunteers.

found to be from untreated butter fat

Therefore, butter oil samples extracted

(161.7 ppm). Unlike acetoin, diacetyl

from BF+CFS (butter fat fermented with

strongly contributes to the buttery aroma

strain CFS), BF+Gal+CFS (butter fat

(Fuquay _et al_., 2011). Only in cohesion

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 51

_Res. Highl. 4Bs (2016)_

_Microbial Production of Butter Flavour_ 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,

caking agent. This was performed to

pleasant, mild, overall buttery aroma in

enhance flow capability and storage

addition to toning down diacetyl roughness

property of the butter powder.

(Bai _et al_ , 2014). The results of diacetyl

and acetoin analysis tallies with the

_**Standardization of butter oil and**_

preference of volunteers who preferred

_**maltodextrin ratio for butter concentrate**_

supplemented butter fat and fermented

_**powder formulation**_

with CFS strain the most (56.7%).

It was observed that the use of 25 g butter

Untreated butter fat was the least preferred

oil resulted in the formation of minor

(8.3%) and is reflected by the lowest

clumping with maltodextrin (Table 7).

concentrations of diacetyl and acetoin

Although lower amounts of butter oil did

content.

not result in clumping, desired intensity of

****

butter

aroma

was

not

imparted.

_**Butter powder formulation**_

Maltodextrin is a well-known carrier

Butter powder formulation was made with

material used for various preparations. It is

maltodextrin and to enhance its flow

digestible, lacks artificial colouring or

properties anti-caking agents (bentonite,

flavour, low in cost and miscible in liquids

sodium silicate, sodium chloride, potato

(Zuidam and Shimoni, 2010). In addition,

starch and mannitol) were evaluated

its good binding and high viscosity

(Figure 8). The samples were subjected to

capabilities enables spray or freeze drying

sensory evaluation. The recovered butter

of active ingredients to be made into

oil was then formulated to powder form by

powders easily (Akhilesh _et al_., 2012).

addition of a carrier material and anti-

From the various anti-caking agents tried,

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_Microbial Production of Butter Flavour_ 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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_Res. Highl. 4Bs (2016)_

_Microbial Production of Butter Flavour_ Nadaraj _et al_.

**Table 7** : Standardization of butter oil and

method for optimum recovery of butter oil

maltodextrin ratio for butter concentrate

from

the

butter

fat

samples

was

powder formulation. ****

standardized, which resulted in 69.67 %

Maltodz Butter

Quality attributes

recovery of butter oil. Upon preliminary

(g)

oil (g)

Texture

Aroma

screening of the LAB isolates, the CFS

50

5

No clumps

Faint

and CFG isolates were selected based on

50

10

No clumps

Faint

the sensory evaluation by 10 volunteers.

50

15

No clumps

Decent

50

20

No clumps

Decent

The solid substrate fermentation condition

50

25

Less clumps

Strong

by the two strains produced sweet and

50

30

Grainy clumps Strong

buttery notes within 24 hours. In the final

**z** Maltodextrin

phase, a scale up study of butter fat

supplemented with 10% galactose was

none are able to enhance the flow rate of

carried out by SSF. A large scale sensory

the

formulated

powder

at

various

evaluation was performed with 120

concentrations (2%, 4% and 6% w/w).

volunteers. The elevated concentration of

Such findings are in contrast to reports by

both diacetyl and acetoin was most

(Ganesan _et al_., 2008) who stated

preferred (56.7%). The formulation of

application of 2% anticaking agent is

butter powder with maltodextrin was

adequate for enhancing powder flow rates.

investigated for its flow properties.

A possible reason for failing to achieve the

Overall, the results obtained from this

desired powder texture might be due to

study will pave way for the future

structural collapse of the sample. This may

investigations for the development of a

be

attributed

to

decreased

product

microbial process for the production of

molecular porosity and overall volume, as

butter flavour concentrate from butter fat

a result of improper sample drying and

with lactic acid bacteria via solid substrate

storage (Bhadra _et al_., 2011).

fermentation.

****

**CONCLUSION**

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_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 56

**Research Highlights in 4Bs**

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_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

_1Electronics 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._

_3Faculty 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

been in use for recording these brain signals.

Electroencephalogram (EEG) recording is

Brain is the most complex part in the human

one of the most prominent among them

body and therefore studying and analysis of

(Teplan, 2002).

the same for understanding the features,

functioning and artifacts are difficult as

The brain reacts differently at different

compared to the other organs and or parts in

stages of time; hence, the brain signals will

the body. Brain waves are analyzed for the

be different accordingly. Brain signals

study of functioning and diagnosis of brain

consist of different frequency components

disorders. A number of techniques have

termed as EEG rhythms. For a proper

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_Spectral Decomposition of EEG Signals_ Biju _et al_.

analysis, these EEG rhythms have to be

inaccurate results. Use of Reconfigurable

extracted

separately.

For

spectral

Filter Banks helps solving this issue.

decomposition

and

analysis

lots

of

techniques like time-frequency analysis have

Various methods have been proposed for

been introduced earlier (Shayan _et al_.,

achieving the reconfigurability in the cutoff

2014). The various time-frequency analysis

frequency and bandwidths of filters. One

techniques in practice include Short Time

approach is to implement a variable cut off

Fourier

Transform

(STFT),

Wavelet

digital filter in which the cut off frequency

Transform (WT), Wavelet Packet Transform

could be controlled through a single

(WPT), Filter Banks, Moving Average

parameter which uses the principle of

filtering, Auto Regressive analysis, Auto

frequency transformation of linear phase

Regressive Moving Average model (Saeid

FIR filters (Oppenheim _et al_., 1976). In

and Chambers, 2007). The STFT, WT,

fractional delay method, each unit in delay

WPT,

Filter

Banks

etc.

enhance

operator in fixed coefficient FIR filter is

decomposition of signals into sub-bands and

replaced with second order FIR fractional

simultaneous analysis of the various

delay structure (Sasikumar _et al_., 2013).

(wanted) spectral components.

Cut-off frequency is changed by changing

the FD value which in turn changes the

Spectral analysis is performed on the signals

bandwidth. The frequency response masking

to extract various key-information (Bhagwat

(FRM) technique comprises the complete

and Vinod, 2013). For the spectral analysis

finest transition-band filter using many wide

lots and lots of transform techniques and

transition-band sub-filters (Lim, 1986; Lim

their variants have been proposed and are

and Boroujeny, 1992). In the modified

being used through decades. Since, in

frequency transformation technique the

particular, EEG is a non-stationary signal,

fixed-coefficient low-pass sub filter in the

transforms like DFT, FFT etc. cannot

first stage of a Fast Filter bank (FFB) is

describe it completely, and some transform

replaced by a modified second-order

technique localized in time and frequency as

frequency transformation (MFT) based low

well is needed (Bhagwat and Vinod, 2013).

pass variable digital filter (Filipe _et al_.,

The wavelet transform technique is in wide

2007).

practice for analysis of EEG signal for

decomposition into different bands of equal

In this paper the design of a reconfigurable

widths (Bhushan, 2013; Rafiee _et al_., 2011).

filter bank for EEG spectral decomposition

Wavelet Packets has been found better in the

is proposed. Here a uniform filter bank is

analysis of biological signals. At higher

used as a prototype filter bank and FRM

frequencies WT fails to localize time with

Technique

is

applied

for

achieving

required

accuracy.

Discrimination

of

reconfigurability in sub band bandwidths.

frequency is sacrificed at higher frequencies

for localization of time. WPT generalizes

**METHODOLOGY**

the time-frequency analysis of WT. Filter

The brain cells transfer the information by

Bank is also a candidate of sub-band

means of biochemical reactions across small

decomposition of EEG signals (Alfred,

spaces which are termed as synapses. The

1999; Chen _et al_., 2014; Darak _et al_., 2011).

nerve cells can be considered as a dipole

In all these cases the sub band bandwidths

with its own particular orientation as well as

are fixed. This leads to an inaccurate

polarity. Such dipoles with identical polarity

decomposition and analysis and in turn

will receive similar inputs and they add up

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together resulting in potentials. These

signals. Since each rhythm of EEG

signals are termed as brain waves and when

represents the activity of brain in different

recorded

using

Electroencephalogram

stages

of

activity

accurate

spectral

referred to as EEG signals (Alfred, 1999).

decomposition is of great importance (Saeid

EEG rhythms are different phenomena or

and Chambers, 2007). So far no work has

events in the EEG. The commonly used

been done on the design of a method for

terms for EEG frequency (𝑓) bands are as

accurate sub band decomposition. The

follows (Saeid and Chambers, 2007).

proposed reconfigurable filter bank is the

first attempt of its kind in the scenario of

EEG signal processing.

delta () ∶ 0.5 ≤ f < 4𝐻𝑧

In the proposed Reconfigurable Filter Bank,

theta () ∶ 4 ≤ f < 8𝐻𝑧

the center frequency and bandwidth of the

sub filters can be varied according to the

alpha (α) ∶ 8 ≤ f ≤ 13Hz

requirements. The design requirements

include sub band filters with sharp transition

beta(β) ∶ 13Hz  f

bands and considerably large stop band

In general, EEG signals do not reveal

attenuation.

information about any abnormalities or

_**Proposed design**_

disorders of brain. Time-frequency analysis

is required to extract such information, if

The proposed RFB consists mainly of two

any,

from

EEG

signals.

Spectral

stages: i) A perfect reconstruction Uniform

decomposition of EEG signals into EEG

Filter Bank and ii) Frequency Response

rhythms have been always a challenge since

Masking based masking filters. In section

the EEG rhythms are of low frequency and

3.1, the design and response of Uniform

accurate decomposition is very difficult

Filter Bank is described. In the following

(Shayan _et al_., 2014). The non-uniform band

section the principle of Frequency Response

widths of the sub bands also add up to the

Masking and the design steps of FRM

complexity in spectral decomposition. Lots

masking filters is discussed.

of methods like Wavelet Transform,

_**A.**_

_**Design of perfect reconstruction 16**_

Wavelet Packet transform, Filter Banks, etc.

_**channel filter bank**_

have been used for the same spectrum

The filter banks comprise two stages,

(Alfred, 1999; Shayan _et al_., 2014). The

analysis filter banks and synthesis filter

analysis using WT and WPT decomposes

banks filter banks (Vaidyanathan, 1993).

the finite energy signal in successive stages

Each stage consists of low pass, band pass

each with different scaling factor, Thus it

and high pass filters. In analysis filters the

gives the information in both time and

input finite energy signal is applied to M

frequency

domains.

But

at

higher

filters which will be decomposed into M

frequencies discrimination of frequencies is

uniform width sub-bands. The resulting

sacrificed for localization of time (Alfred,

signals are sub-sampled by a factor N to

1999). Wavelet Packet Transform mitigates

avoid redundancies. At the synthesis filter

this problem. But, the bandwidth of packets

bank stage, up sampling is done to recover

is multiples of two; hence, it is suitable only

the sampling rate of input signal. The up

for uniform sub band decomposition. The

sampled signals are applied to synthesis

existing methods have not succeeded in non-

filters which will compose the original

uniform sub band decomposition of EEG

signal. If number of filter stages equals the

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decimation factor it is referred to as critical

combination of the masking filters. This

sub-sampling, since above that factor perfect

method has very scanty coefficients and

reconstruction is not possible. The perfect

very low computation (Lim, 1986).

reconstruction filter bank returns the original

input signal with time shift and amplitude

Initially a low pass fir filter is selected

scaling (Vaidyanathan, 1990).

which is termed as the modal filter with

transfer function Ha(z). Now each delay of

Let c(n) be the input signal and r(n)

this filter is replaced by M delays to get a

represents the response, here the signals are

new filter with transfer function Hb(z) =

related as

Ha(zM). If Hb(z) is masked with another filter

H

R

c(z) we'll get a new impulse response

0(z2) = 0.5 [A0(z) C(z) + A0(-z) C(-z)] (1)

H

d(z) with transition width 1/M times that of

Ha(z). This method is used to obtain a sharp

R

transition band filter using Frequency

1(z2) = 0.5 [A1(z) C(z) + A1(-z) C(-z)] (2)

response masking. The consequences of the

masking technique is the transition width

C'(z) = [R0 (z2) S0(z) + R1(z2) S1(z)] (3)

reduce the by a factor of M for every M

delay. Which affect width of the pass band

Combining these equations, the

reduced by M factor (Sumedh _et al_., 2013;

input-output relation is given by

Lim and Lian, 1993). Hence, it is suitable

C'(z) = 0.5[A

only for a narrow-band design. Since our

0(z)S0(z) +A1(z)S1(z)]C(z) +

0.5[A

application requires narrow band filters only

0(-z)S0(z)+A1(-z) S1(z)]C(-z) (4)

FRM technique is optimum for the

Here A(z) represents the analysis filter bank

achieving reconfigurability in Filter banks

frequency response and S(z) represents the

for non-uniform sub-band decomposition of

synthesis filter bank frequency response.

EEG signals.

The first term and the second term represent

_**C.**_

_**Proposed method**_

transmission of the c(n) through the filters

As initial stage, a 16-channel perfect

and the aliasing component at the output of

reconstruction filter bank was designed. For

the

filter

bank

respectively.

Perfect

this initially, a two channel perfect

reconstruction is achieved if the transfer

reconstruction filter bank was designed. The

function for the aliasing component is zero.

order chosen was 90. This was chosen as the

_**B.**_

_**Design of masking filters**_

prototype filter bank for designing the M-

The reconfigurable Filter Bank was obtained

channel Uniform Filter Bank. Then, the tree

as a result of combining the uniform filter

type 16 channel filter bank was developed.

bank with masking filters generated using

Here, each filter impulse response is

the Frequency Response Masking technique

convolved with the interpolated impulse

(Mahesh and Vinod, 2011). The masking

response of the filters in previous stage to

filters of desired bandwidths are applied at

get the new impulse response.

the output of the uniform filter bank sub

Let ℎ

filters at the desired stages to extract the

𝐿𝑃 and ℎ

̅𝐿𝑃 are the low pass filter and

the complementary filter of the 2 channel

desired bands. In the masking filter method

perfect reconstruction model filter. Let 𝑔

the spectral analysis of complementary pair

𝐿𝑃

and 𝑔̅

filters are masked by the two suitable

𝐿𝑃 are represent their interpolated

versions respectively. Then the sub-band

masking filters. So the desired output is the

filters in the (i+1)th stage of the M stage

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 60

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_Spectral Decomposition of EEG Signals_ Biju _et al_.

filter bank are given by the following design

single hardware structure can be used for

equations.

mapping these sub-filters. Hence, just one

filter structure is needed and the number of

ℎ𝐿𝑃(𝑖 + 1)= ℎ𝐿𝑃(𝑖) * 𝑔𝐿𝑃(𝑖) (5)

adders and multipliers can be reduced. This

ℎ

̅ 𝐿𝑃(𝑖 + 1) = ℎ𝐿𝑃(𝑖)* 𝑔̅𝐿𝑃(𝑖 + 1) (6)

reduces the complexity in design.

ℎ𝐻𝑃(𝑖 + 1) = ℎ̅𝐿𝑃(𝑖) ∗ 𝑔̅𝐿𝑃(𝑖 + 1) (7)

**RESULTS AND DISCUSSION**

ℎ

̅ 𝐻𝑃(𝑖 + 1) =ℎ̅𝐿𝑃(𝑖) ∗ 𝑔𝐿𝑃(𝑖) (8)

The EEG signals were adopted from CHB-

The response shows minimum overlap in

MIT scalp EEG database prepared and

complementary bands and also the filter

hosted by Children's Hospital Boston (CHB)

bank has got fairly sharp transition band.

and

the

Massachusetts

Institute

of

The response of the 16-channel perfect

Technology (MIT). The signals are in the

European data format (.edf), which is a

reconstruction filter bank is shown in the

following Figure 1. As the next stage

standard file format used for the storage and

masking filters were designed using FRM

exchange of medical time series. The

sampling frequency is 256 Hz and the file

technique. This helps in perfect extraction of

EEG Rhythms. The cut off frequency and in

includes signals from 16 channels. Another

turn bandwidth of the required masking

source

of

database

was

that

from

filters were reconfigured by adjusting the

Department of Epileptology, University of

Bonn.

input pass band and stop band frequencies

without changing the order.

The procedure for simulation of the

Since the designed sub-filters are identical

reconfigurable filter bank as follows. The

**Figure 1:** Magnitude response of 16-channel uniform filter bank.

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 61

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_Spectral Decomposition of EEG Signals_ Biju _et al_.

input signals read in the .edf format were

5

x 10

Input Signal

applied to pre-processing which includes

2

low pass filtering for restricting to 64 Hz

1

followed. The signal was then applied to a

00

5

10

15

20

25

30

35

40

45

notch filter for removing power line

5

x 10

Delta Band

components. Pre-processed signal is applied

2

to uniform filter bank. The uniform filter

1

bank designed here is a 16-channel perfect

00

5

10

15

20

25

30

35

40

45

reconstruction filter bank which provides

Theta Band

accurate decomposition into bands of width

4000

4 Hz each. Masking filters are applied to

2000

0

sub filters for extracting the desired

0

5

10

15

20

25

30

35

40

45

Rhythms. Masking filters are designed

Alpha Band

according to the requirements. The first

40

20

masking filter for delta band was designed

0

such that it extracts the 0.5-4 Hz band. The

0

5

10

15

20

25

30

35

40

45

second masking filter for Theta band was of

Beta Band

bandwidth 4 Hz. The masking filter for

4000

2000

Alpha band was of frequency width 5 Hz

0

and was applied to the sub filter of third

0

5

10

15

20

25

30

35

40

45

stage. The Beta band was extracted using the

**Figure3:** Magnitude spectrum of Uniform

masking filter applied to the subfilter of

Filter Bank response.

fourth stage of uniform filter bank. The

output response and spectrum of uniform

Filter Bank is shown in the Figure 2 and

Figure 3 respectively. From the waveforms

Input Signal

it is clear that there is an overlap in the

500

spectra; hence, the obtained result is

0

inaccurate.

The

adjacent

spectral

-5000

5000

10000

15000

components are present in the desired

Delta Band

spectrum which will affect the accurate

500

diagnosis of diseases. Use of Reconfigurable

0

Filter Bank helps to mitigate this problem.

-5000

5000

10000

15000

The proposed RFB provides accurate

Theta Band

extraction of EEG rhythms with high

50

resolution. The waveforms in the Figure 4

0

and Figure 5 are magnitude spectrum of

-500

5000

10000

15000

healthy EEG signal and the abnormal

Alpha Band

(seizure) EEG signal respectively.

20

0

From the spectra it is clear that the extracted

-200

5000

10000

15000

rhythms have much less components form

Beta Band

adjacent spectra. Hence, it will be easier to

50

analyze different bands separately and to

0

-50

determine in which band abnormality

0

5000

10000

15000

occurs.

This

makes

the

spectral

**Figure 2** : Response of Uniform Filter Bank.

decomposition analysis for EEG signals and

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_Spectral Decomposition of EEG Signals_ Biju _et al_.

to detect the seizure activity which is

provides considerably narrow transition

dominating in the sub-bands.

band width without higher orders. Hence,

the circuit complexity is less. Since FRM

The main advantage of using FRM filters for

filters are FIR based, the design retains all

reconfiguring bandwidth is that no hardware

the advantages of FIR filters including

overhead is required for changing the

guaranteed

stability,

low

coefficient

bandwidth and centre frequency. Only thing

sensitivity, free of phase distortion etc.

required is the change in coefficients. Also it

5

x 10

Input EEG Spectrum

2

1

00

5

10

15

20

25

30

5

x 10

delta band

2

1

00

5

10

15

20

25

30

theta band

10

5

00

5

10

15

20

25

30

4

x 10

alpha band

2

1

00

5

10

15

20

25

30

beta band

10000

5000

00

5

10

15

20

25

30

****

**Figure 4:** RFB Magnitude Spectrum of healthy EEG signal.

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****

**Figure 5** : RFB Magnitude Spectrum of seizure EEG signal.

**CONCLUSION**

extraction of EEG Rhythms. Use of

reconfigurable filter bank is the first attempt

In this paper design of a reconfigurable filter

in EEG rhythm extraction. A perfect

bank for efficient and accurate spectral

reconstruction 2-channel filter bank was

decomposition of EEG signals is proposed.

used as modal filter bank and an M-channel

The existing techniques employed for

filter bank was designed using this modal

spectral decomposition such as Wavelet

filter bank. To mitigate the problem of

Transform, Wavelet Packet transform and

varied bandwidth EEG spectral extraction,

DFT Filter bank could only extract bands of

FRM based filters were applied as masking

uniform bandwidth. The proposed RFB

filters. Use of FRM technique for the

based on FRM technique provides accurate

generation of masking filters has provided

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 64

_Res. Highl. 4Bs (2016)_

_Spectral Decomposition of EEG Signals_ Biju _et al_.

sharp transition width narrow band low pass

International Conference. Acoustics,

and band pass filters to be used for

Speech

and

Signal

Processing,

extracting desired bands. Since the sub

Prague. **pp. 1629-1632.**

filters are identical they can be mapped into

a single hardware structure, it reduces the

**Filipe, C.C.B. D. IuriKothe, S. L. N. and**

complexity of hardware implementation.

**Luiz, W. P. B. (2007).** High

Since the reconfigurability is achieved

selectivity Filter Banks for spectral

without a change in hardware the technique

analysis of music signals. Advances

is found to be optimum for EEG rhythm

in Signal Processing **1-13.**

extraction.

****

**Lim, Y. C. and Lian, Y. (1993** ). The

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_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 66

**Research Highlights in 4Bs**

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_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 RIF-

induced 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

_al_., 2008; Chevalier _et al_., 2010). Several

medications are known inducers of acute

Oxidative stress plays a major role in the

interstitial

nephritis

which

includes

induction and progression of renal failure

commonly

used

non-steroidal

anti-

both acute and chronic. Several conditions

inflammatory drugs (NSAIDS), interferon

like hypertension, diabetes, infection,

and

antibiotics

such

as

penicillins,

obstruction

in

the

urinary

tract,

cephalosporins and anti-tubercular drugs

autoimmune disorders such as lupus

such as rifampicin (Rossert, 2001). Studies

erythematosus, genetic disorders such as

exploring the sub-cellular mechanisms of

polycystic kidney disease, drugs such as

renal injury causing AIN have been carried

antibiotics, diuretics and anti-inflammatory

out recently (Mitchell _et al_., 1977; Servais

drugs induce oxidative stress in renal

_et al_., 2007; Chevalier _et al_., 2010). It has

tissues (Schattner _et al_., 2000; Crispín _et_

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_Coenzyme Q10 Dietary Supplementation_ Udhaya and Sabina

been proven that interstitial inflammation

**MATERIALS AND METHODS**

in renal tissue might be due to the

predominant infiltration of local B-cells,

_**Chemicals and drugs**_

eosinophils and mononuclear leucocytes in

Synthetic coenzyme Q10 was purchased

the interstitium (Heller _et al_., 2007). In

from Sigma Aldrich, India. INH and RIF

addition,

the

development

of

anti-

were

purchased

from

Lupin

Ltd.,

rifampicin antibodies occurs rarely in

Aurangabad, India and the standard

patients on antitubercular therapy. Studies

hepatoprotective drug silymarin from

have

shown

that

these

rifampicin-

Quality Pharmaceuticals Ltd., India. INH

dependent

antibodies

cause

acute

and RIF were dissolved in normal saline

haemolysis and renal failure (van der

while silymarin was dissolved in sterile

Meulen _et al_., 2009; Beebe _et al_., 2015).

distilled water. Coenzyme Q10 was

Several case studies have reported the

dissolved in 0.2 ml corn oil. All the other

occurrence of acute interstitial tubulopathy

chemicals and reagents used were of

in pulmonary tuberculosis patients on

analytical grade procured from SD Fine

antituberculosis regimen (Muthukumar _et_

Chemicals Pvt. Ltd., Mumbai, India.

_al_., 2002; Rosati _et al_., 2013). Drug-

induced ARI is a major adverse effect due

_**Animals**_

to

rifampicin

though

uncommon.

30 female Wistar albino rats of body

Isoniazid (INH) and rifampicin (RIF) co-

weight 143.26±12.81 g were procured

administration causes injury in the

from the Animal House, VIT University,

hepatocytes leading to hepatotoxicity

Vellore, Tamilnadu. The rats were divided

(Baskaran and Sabina, 2015).

into 5 groups and treated as follows for 28

days: group I was normal control; group II

Coenzyme Q10 is a fat soluble vitamin-

was treated with INH and RIF (50 mg/kg

like compound with significant antioxidant

b.w. each/day); group III was INH and

potential.

It

plays

key

role

in

RIF-induced rats co-administered with

mitochondrial bioenergetics. Though the

coenzyme Q10 (10 mg/kg b.w./day); group

compound is synthesised within the body,

IV was INH and RIF-induced rats co-

there are certain conditions that lead to low

administered with silymarin (25 mg/kg

levels of coenzyme Q10 (CoQ10) such as

b.w./day); group V was treated with

vitamin B deficiency, use of statins for

coenzyme Q10 (10 mg/kg b.w./day) alone.

hypercholesterolemia,

old

age,

The animals were sacrificed after the study

cardiovascular disorders and oxidative

duration using ether anaesthesia. Blood

stress (Gempel _et al_., 2007; Quinzii _et al_.,

and kidneys were procured for further

2007;

Quinzii

and

Hirano,

2011).

analysis.

Renal

homogenates

were

Recently, it has been found that this

prepared using 5 % phosphate buffered

antioxidant also plays significant role in

saline (PBS).

the regulation and alteration of genes

involving cell signalling and metabolic

_**Estimation of serum markers of renal**_

pathways (Groneberg _et al_., 2005). Our

_**function**_

previous

study

showed

significant

Serum levels of renal functional markers

protective effects of CoQ10 against INH

such as creatinine, urea, uric acid and acid

and RIF induced hepatotoxicity (Baskaran

phosphatase

were

estimated

using

and Sabina, 2015).

commercial diagnostic kits obtained from

AutoSpan Diagnostics Ltd., India.

Current study was an attempt to evaluate

the occurrence of renal injury due to the

_**Assessment of antioxidant profile**_

co-administration of INH and RIF and the

Renal homogenates were used to assess the

role of CoQ10 as a nephroprotective agent

activities

of

superoxide

dismutase

in Wistar albino rats.

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_Coenzyme Q10 Dietary Supplementation_ Udhaya and Sabina

(Marklund and Marklund, 1974), catalase

_**Effect of coenzyme Q10 on serum**_

(Sinha, 1972), glutathione peroxidase

_**markers of renal function in INH and**_

(Rotruck _et al_., 1973) and Glutathione-S-

_**RIF induced rats**_

transferase (Habig _et al_., 1974); levels of

INH and RIF induced rats showed

total reduced glutathione (Moron _et al_.,

significant increase in the levels of urea,

1979) and lipid peroxidation (Ohkawa _et_

creatinine, uric acid and acid phosphatase

_al_., 1979).

while

concomitant

administration

of

coenzyme Q10 caused significant (P<0.05)

_**Histopathological analysis**_

reduction in the elevated levels of the

A portion of the kidneys were fixed in 10

aforementioned parameters (Figures 1-4).

% formalin after thorough washing with

This particular effect of coenzyme Q10 in

ice-cold 5 % PBS. The tissues were then

minimizing INH and RIF-induced changes

dehydrated with descending grades of

in serum markers of renal function was

isopropanol. After treating with xylene, the

comparable with that of silymarin treated

tissues were embedded in molten paraffin

rats. The oxidative stress parted by the

wax and tissue sections (5 µm) were cut.

toxic metabolites of isoniazid on renal

The

sections

were

stained

with

tissue might lead to oxidative cellular

hematoxylin and eosin and examined

damage. In addition, evidence from

microscopically for pathological changes.

literature suggest that the generation of

anti-rifampicin

antibodies

causes

**RESULTS AND DISCUSSION**

**Figure 1** : Effect of coenzyme Q10 on serum urea in INH and RIF induced rats. Each value

represents the Mean±sd 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-keul's test.

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_Coenzyme Q10 Dietary Supplementation_ Udhaya and Sabina

formation

of

drug-antibody

immune

are adverse effects of RIF reported in

complexes which result in glomerular

tuberculosis patients (van der Meulen _et_

endotheliosis and cell damage thereby

_al_., 2009). A case study has reported the

leading to tubular injury and reduced renal

evidence of acute renal failure (ARF) in a

function (Muthukumar _et al_., 2002). Acute

38 year old tuberculosis patient (Beebe _et_

tubular necrosis and interstitial nephritis

_al_.,

2015).

****

****

****

****

****

****

****

****

****

****

****

****

****

****

****

****

**Figure 2** : Effect of coenzyme Q10 on serum creatinine in INH and RIF induced rats. Each

value represents the Mean±sd 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-keul's test.

****

****

****

****

****

****

****

****

****

**Figure**

**3** :

Effect

of

coenzyme

Q10 on serum

uric acid in

INH and RIF

induced rats.

Each value represents the Mean±sd 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-keul's test.

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 70

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_Coenzyme Q10 Dietary Supplementation_ Udhaya and Sabina

**Figure 4** : Effect of coenzyme Q10 on serum acid phosphatase in INH and RIF induced rats.

Each value represents the Mean±sd 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-keul's test.

_**Effect of coenzyme Q10 on antioxidant**_

DNA fragmentation and apoptosis in

_**profile in INH and RIF induced rats**_

organs that are vulnerable to oxidative

There was significant (P<0.05) reduction

damage. The mechanism that underlies

in the activities of antioxidant enzymes

mitochondrial

membrane

damage

is

such as superoxide dismutase, catalase,

oxidative stress exerted due to the

glutathione peroxidase and glutathione-S-

production of ROS.

transferase in renal homogenates of INH

and RIF induced rats (Table 1). Also, there

_**Effect of coenzyme Q10 on renal tissues**_

was significant (P<0.05) reduction in the

_**in INH and RIF induced rats**_

levels

of

reduced

glutathione

and

ARI is an uncommon and less known

significant (P<0.05) increase in the levels

adverse

effect

arising

due

to

of lipid peroxidation on treatment with

antituberculosis regimen which includes

INH and RIF. Co-administration of

rifampicin (Topping _et al_., 2012). Case

coenzyme Q10 was able to restore these

studies in elderly population have shown

parameters to near normal levels which

evidence of ARF (De Vriese _et al_., 1998;

were compared with that of silymarin

Chang _et al_., 2014). There are also

treated INH and RIF induced rats. The

mounting evidences on the occurrence of

reduction in the activities of the enzymic

ARI in patients undergoing intermittent or

and non-enzymic antioxidants and elevated

interrupted administration of rifampicin

lipid peroxidation levels reflect the extent

giving rise to the development of anti-

of oxidative stress in the INH and RIF

rifampicin antibodies (Pereira _et al_., 1991;

treated rats. It has been proven that

Rosati _et al_., 2013). Histopathological

mitochondrial membrane damage induces

examination of renal tissue architecture in

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_Coenzyme Q10 Dietary Supplementation_ Udhaya and Sabina

the current study reveals damage to

(Ly _et al_., 2003). It has been shown that

glomerular cells and karyolysis in the INH

CoQ10 prevents MMP collapse by

and RIF treated rats. Apart from ARI

preventing mitochondrial depolarization

caused by rifampicin, there could have

and hence may possess antiapoptotic

been significant damage caused due to

activity (Papucci _et al_., 2003). This

oxidative stress and depletion of cellular

particular activity of CoQ10 along with its

antioxidants

parted

by

the

toxic

antioxidant and free radical scavenging

metabolites of isoniazid (Chowdhury _et_

activities might have prevented tubular

_al_.,

2006).

Mitochondrial

membrane

injury.

potential (MMP) collapse causes apoptosis

****

**Table 1** : Effect of concomitant administration of coenzyme Q10 on antioxidant parameters in

renal tissue homogenates of INH and RIF induced rats.

Parameters

Group 1

Group 2

Group 3

Group 4

Group 5 (CoQ10

(Control)

(INH+RIF 50

(INH+RIF &

(INH+RIF &

10 mg/kg b.w.)

mg/kg b.w.)

CoQ10 10mg/kg

silymarin 25

b.w.)

mg/kg b.w.)

SOD

(units/min/mg

protein)

180.14±3.42 95.04±3.40a* 165.11±2.29a*b* 168.05±5.21a*b* 186.03±3.84b*c*d*

Catalase

(units/min/mg

60.14±2.85

35.12±2.84a*

55.04±2.00b*

53.05±2.28a*b*c*

64.32±3.13b*c*

protein)

Glutathione

peroxidase (μg

of GSH

38.08±2.28

18.02±2.28a*

32.07±1.42a*b*

32.64±3.49a*b*

37.05±1.42b*c*

utilized/min/

mg protein)

Reduced

glutathione

(nmol/mg

46.14±1.45

28.04±2.00a*

42.04±4.00b*

41.14±2.02b*

47.09±2.01b*d*

protein)

Glutathione-S-

transferase

(nmol of CDNB-

GSH conjugate

20.09±1.43 11.142±1.45a*

16.00±1.41b*

16.51±3.78b*

20.01±2.00b*

formed/min/mg

protein)

TBARS

(mM/TBARS/100

0.80±0.20

1.80±0.14a*

1.00±0.14b*

1.10±0.14b*

0.70±0.14b*d*

g of wet tissue)

Each value represents the Mean±sd 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-keul's test.

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**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**

and RIF treated rats. The determination of

serum renal function markers and kidney

Current study shows that CoQ10 was able

histopathological alterations reveal that

to restore normal antioxidant status in INH

CoQ10 minimizes damage to renal tissue

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 73

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_Coenzyme Q10 Dietary Supplementation_ Udhaya and Sabina

due to INH and RIF. Free radical

**Chowdhury,**

**A.,**

**Santra,**

**A.,**

scavenging property of CoQ10 might have

**Bhattacharjee, K., Ghatak, S.,**

reduced the extent of damage to cellular

**Saha, D.R. and Dhali, G.K.**

membranes and macromolecules thereby

( **2006** ). Mitochondrial oxidative

rendering protection against oxidative

stress and permeability transition in

stress. In addition, the anti-apoptotic

Isoniazid and Rifampicin induced

property of CoQ10 might have minimized

liver injury in mice. _Journal of_

cell death due to mitochondrial membrane

_Hepatology_ **45** , **117–126**.

depolarization thereby reducing injury to

tubular interstitium which might have

**Crispín, J.C., Oukka, M., Bayliss, G.,**

aided

the

maintenance

of

renal

**Cohen,**

**R.A.,**

**Beek,**

**C.A.V.,**

histoarchitecture. Further studies would

**Stillman, I.E., Kyttaris, V.C.,**

explore the molecular mechanisms of the

**Juang, Y.-T. and Tsokos, G.C.**

effect of CoQ10 in INH and RIF induced

( **2008** ). Expanded Double Negative

renal injury.

T Cells in Patients with Systemic

Lupus Erythematosus Produce IL-

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_Cases Journal_ **2** , **6643**.

**Servais, H., Ortiz, A., Devuyst, O.,**

**Denamur, S., Tulkens, P.M. and**

****

**Mingeot-Leclercq, M.-P.** ( **2007** ).

****

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 76

**Research Highlights in 4Bs**

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_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 financing 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**

where nearly 1.0 billion people lack fresh

****

water for daily uses (Aho _et al.,_ 2012).

Access to safe drinking water is the

fundamental need for healthy human life and

Although, approximately 71% of earth's

water has been shown linked to the

surface is covered with water and more than

development of all areas including health,

97% of the earth's water is salty sea water,

nature,

urbanization,

industrialization,

while 2% is stored in glaciers, ice caps, and

energy production, food security, equality

snowy mountain ranges (Yongabi, 2010);

and

other

aspects

of

community

thus, less than 1.0% of the earth's water is

development (Colwell _et al_., 2003; Huq _et_

surface water, of lakes, rivers, streams,

_al_., 2010). In all these areas, not only water

ponds (0.022%) and groundwater (0.397%)

but also clean, pure, safe and quality of

available for humans for their daily needs

water are required to ensure proper

(Yongabi, 2010). However, all these

development. According to United Nations

accessible surface water is not necessarily

(UN), to meet up daily necessities like

safe for drinking, and depending on the

drinking, cooking, and personal hygiene,

location and sources, clean water is exposed

approximately 20 to 40 liters of clean water

to a variety of contaminants, many arising

is required by each individual. In today's

from human and animal wastes, debris from

world, scarcity of safe water is a burning

homes, chemicals from industries, fertilizers

issue; especially in developing countries

and pesticides from agricultural lands, and

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 77

_Res. Highl. 4Bs (2016)_

_Safe Water as the Key to Health_ Bari environmental pollutants as well as other

programme (2014), about 28 million

contributing factors (Figure 1) (Zeenath _et_

Bangladeshis, or just over 20% of the

_al_., 2015). In addition, insanitary practices

population, are living in harsh conditions in

of people have greatly contributed to the

receiving clean water, and **** safe clean water

deterioration of the quality of surface water

supply situation is going to become worst in

sources (Zaman _et al_ , 2013). On the other

coming decades to meet the need of

hand, groundwater is the most important

predicted 200 million people by 2020 (Silvia

source of water supply in most of the

_et al_., 2011).

countries including Bangladesh.

**CHALLENGE**

**RELATED**

**TO**

**CLIMATE CHANGE**

****

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 flow which can

mobilize pathogens and other contaminants.

Increased frequency and magnitude of flood

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 identified as water

temperatures,

increased

precipitation

**Figure 1:** Important drivers that impacted

intensity and longer periods of low flows

health.

will exacerbate many forms of water

pollution, with impacts on ecosystems and

Physically groundwater is clear, colorless

human health as shown in Figure 2.

with little or no suspended solids and has a

relatively

constant

temperature.

The clean water scarcity usually exists in

Groundwater is also free from disease-

developing countries due to: 1) lack of

producing

micro-organisms

which

are

capacity, 2) community capacity and

normally present in large numbers in surface

engagement, 3) technological capacity, 4)

waters (Bari and Yeasmin, 2014). However,

institutional capacity, and 5) inadequate

the groundwater for drinking purposes has

financial support. New global financing

become a problem because of: 1) the

initiative within the water and sanitation

presence of arsenic, 2) excessive dissolved

sector is highly required before being

iron, 3) salinity in the shallow aquifers in the

advocating food safety issues in these

coastal areas, 4) lowering of groundwater

countries (UNU-INWEH, 2010). As the

level (water table), and 5) rock/stony layers

accessibility of clean drinking water is

in hilly areas (USEPA, 2015). Among these

directly related to, poverty, social and

problems arsenic in groundwater has

economic development, and human health;

become a great concern for water supply in

thus, the benefits of water and sanitation

Bangladesh. According to a study by the

program can be made in terms of improved

World

Bank's

water

and

sanitation

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ 78

_Res. Highl. 4Bs (2016)_

_Safe Water as the Key to Health_ 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,

benefits 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

**CHALLENGES RELATED TO FOOD**

income level, diets, local conditions, and

**SAFETY**

government infrastructures. Figure 3 shows

****

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

_Res. Highl. 4Bs (2016)_

_Safe Water as the Key to Health_ Bari **Figure: 3:** Foodborne cases per year in percentages of population in different countries.

should follow good-sense of practices when

changes in animal husbandry practices, 2)

considering the source, treatment and

changes in agronomic process, 3) changes in

intended use of water in food production to

lifestyle and consumer demands, 4) increase

ensure the quality and safety of the food

in susceptible populations, 5) increased

products.

travel, and 6) increasing international food

trade etc., (Bari and Ukuku, 2016). Thus, it

Food

safety

concerns

in

developing

appears that each developing countries

countries

typically

include:

1)

the

limitations are more basic in nature, which

inappropriate use of agricultural chemicals,

are recoverable than the developed countries

2) the use of untreated or partially treated

limitation

that

are

non-recoverable.

wastewater, 3) the use of sewage or animal

Therefore, greater emphasis on capacity

manure on crops, 4) the absence of food

building needs to be built internally to a

inspection, including meat inspection, 4) a

country

and

through

South-South

lack of infrastructure, such as adequate

collaborations and partnerships, rather than

refrigeration, and finally 5) poor hygiene,

depending on consultants from developed

including a lack of clean water supplies.

countries. Capacity is a flexible concept and

However, food safety concerns in developed

encompasses the public sector, academia;

countries are mainly associated with: 1)

community based organizations, private

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ 80

_Res. Highl. 4Bs (2016)_

_Safe Water as the Key to Health_ Bari sector, which ranges from the individual to

institutions to society as a whole.

****

**CREATING FOOD SAFETY CULTURE**

****

People's 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).

**Figure 4:** Building a food safety culture.

Maintaining a food safety culture means that

operators and staff know the risks associated

with the products or meals they produce,

**CONCLUSION**

know why managing the risks is important,

and effectively manage those risks in a

It is very clear that water-related diseases

demonstrable way. In an organization with a

are responsible for a significant proportion

good food safety culture, individuals are

of the global health burden. On the other

expected to enact practices that represent the

hand, it is equally clear that adequate

shared value system and point out where

sanitation measures have not been managed

others may fail. By using a variety of tools,

to keep up with population growth.

consequences and incentives, businesses can

Sanitation is not only critical for dignity and

demonstrate to their staff and customers that

health but it is the most basic form of source

they are aware of current food safety issues,

that they can learn from others' mistakes,

water protection also. This realization is not

new, and focus on clean water alone does

and that food safety is important within the

not necessarily result in improved access to

organization (Jack _et al._ , 2012). Thus,

sanitation. Thus, each gap needs to be taken

creating a culture of food safety requires

into account and adequate investment in

application of the best science with the best

terms of education, capacity and financing

management and communication systems,

to be done to improve the food safety and

including

compelling,

rapid,

relevant,

sanitation status. A strong moral, civil, and

reliable and repeated food safety messages

political will is essential to bring in an

using multiple media.

adequate sanitation for the global population

to improve health and welfare.

Training employees on food safety practices

****

has been shown to be one of the most

**REFERENCES**

important programs that food service

****

establishments can implement. However,

**Aho, I. M. and Lagasi, J. E. (2012).** A new

numerous study reports suggested that

water

treatment

system

using

traditional approaches used to educate and

_Moringa oleifera seed._ _American_

train employees (such as Serve Safe) may

_Journal of Scientific and Industrial_

not be particularly effective and new

_Research Science Huβ_ _**3 (6),**_ **487-**

behavior-based approaches that include food

**492**.

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_ __ 81

_Res. Highl. 4Bs (2016)_

_Safe Water as the Key to Health_ Bari **Colwell, R. R., Huq, A., Islam, M. S.,**

**Githeko, A., Scheraga, J.D. and**

**Aziz, K. M. A., Yunus, M., Khan,**

**Woodrow,**

**A.**

**(Eds.).**

**(2003).**

**N. H., Mahmud, A., Sack, R. B.,**

Climate Change and Human Health:

**Nair, G. B., Chakraborty, J., Sack,**

Risks and Responses. WHO, WMO

**D. A. and Russek-Cohen, E.**

and

UNEP.

Available

from:

**(2003).** Reduction of cholera in

http://www.who.int/globalchange/pu

Bangladeshi villages by simple

blications/climchange.pdf (Accessed

filtration.

_Proceedings_

_of_

_the_

June 9, 2016) ****

_National Academy of Sciences_ **100,**

****

**1051-1055**.

**Silvia, B., Elena, B., Sara, B., Osvaldo, C.,**

****

**Franca, P. and Elisabetta, C.**

**Bari M. L. and D. O Ukuku (2016).**

**(2011).** Development of a PCR

Foodborne

Pathogen and Food

protocol

for

the

detection

of

Safety (Eds) Md. Latiful Bari and

_Escherichia_

_coli_

O157:H7

and

Dike O Ukuku **,** Published by CRC

_Salmonella_ spp. in surface water.

Press **(** Taylor and Francis Group),

Environmental

Monitoring

and

6000 Broken Sound Parkway NW,

Assessment **177, __****493–503**.

Suite 300, Boca Raton, FL 33487,

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USA. **pp 1-37**.

**UNU-INWEH (2010).** Sanitation as a Key

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to Global Health: Voices from the

**Bari, M.L. and Yeasmin, S., 2014.** Water

Field. United Nations University

Quality

Assessment:

Modern

Institute for Water, Environment and

Microbiological

Techniques.

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Health. **pp 2-45**.

Batt, C.A., Tortorello, M.L. (Eds.),

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Encyclopedia of Food Microbiology,

**USEPA.**

**(2015).**

Drinking

Water

vol 3. Elsevier Ltd, Academic Press,

Contaminants,

**pp. 755–765**.

http://www.epa.gov/dwstandards

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regulation (Accessed on April 19,

**Huq, A., Yunus, M., Sohel, S.S., Bhuiya,**

2016).

**A., Emch, M., Luby, S. P., Russek-**

****

**Cohen, E., Nair, G. B., Sack, R. B.**

**[WHO] World Health Organization.**

**and Colwell, R. R. (2010).** Simple

**(2001).** Water Quality: __ Guidelines,

sari cloth filtration of water is

Standards and Health. Chapter 6

sustainable and continues to protect

and 13. __ Edited by L. Fewtrell and J.

villagers from cholera in Matlab,

Bartram.

Published

by

IWA

Bangladesh. mBio **1(1), 1 e00034-**

Publishing, London, UK. ISBN 1

**10**.

900222 28 0.

****

****

**Jack, A., Neal, M. B. and Henroid, D.**

**Yongabi, K. A. (2010).** Biocoagulants for

**(2012).**

Assessing

Factors

water and wastewater purification: A

Contributing to Food Safety Culture

review. _International Review of_

in Retail Food Establishments. Food

_Chemical Engineering_ **2(3), 444-**

Protection Trends **32(8), 468–476**.

**458**.

****

**McMichael, A.J., Campbell-Lendrum,**

**Zaman, S., Yeasmin, S., Inatsu, Y.,**

**D.H., Corvolan, C., Ebi, K.L.,**

**Ananchaipattana, C. and Bari, M.**

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ 82

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_Safe Water as the Key to Health_ Bari **L. (2013).** Low-Cost Sustainable

**K., Rahman, Md. A., Uddin, M. A.**

Technologies for the Production of

**and Bari, M.L. (2015).** Assessment

Clean Drinking Water—A Review.

of Heavy Metals, Minerals and

_Journal of Environmental Protection_

Pesticides in different Branded

**5, 42-53**. ****

Drinking

Bottled

Water

of

****

Bangladesh and their impact on

**Zeenath,**

**F.,**

**Md.**

**Harunur,**

**R.,**

human health. Online publication (25

**Chowdhury, M. A. Z., Alam, Md.**

Jan 2015).

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ 83

**Research Highlights in 4Bs**

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_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

_1Faculty 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.)]

Kratom leaves (Figure 1) are known to

is indigenous to Thailand and Southeast

produce complex stimulant and opioid-like

Asia. This plant is also known as Biak or

analgesic effects (Adkins _et al_., 2011; Babu

Ketum in Malaysia. It is a member of the

_et al_., 2008). In Malaysia, Kratom has been

Rubiaceae family, and grows in tropical and

used to starve off fatigue by enhancing

subtropical regions of Asia (Puff _et al_.,

tolerance for hard work and to manage pain,

2005; Hassan _et al_., 2013). It is well

diarrhea, and opioid withdrawal (Hassan _et_

distributed in Southeast Asia, especially

_al_., 2013). Recently, Kratom has become

Malaysia in general and the northern region

widely available in the Malaysia, as people

bordering Thailand in particular (Hassan _et_

are allowed to keep the plants for

_al_., 2013; Suwanlert _et al_., 1975).

individual's personal use. In Malaysia,

commercial planting of this plant is banned

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 84

_Res. Highl. 4Bs (2016)_

_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.

consequences are becoming an important

Analyses of medical literature indicate that

issue for health authorities of Malaysia.

individuals in Malaysia, especially in the

north peninsular Malaysia are increasingly

There were studies performed to observe the

using Kratom.

effect of mitragynine several years ago

(Grewal, 1932; Macko _et al_., 1981). The

Kratom contains pharmacologically active

acute toxicities studies in rates include

constituents, most notably mitragynine and

increasing blood pressure, hepatotoxicity,

7-hydroxymitragynine. Kratom possesses

and nephrotoxicity (Harizal _et al_., 2010).

dose-dependent pharmacological effects,

The recent research findings of Kamal _et al_.

with stimulant effects in lower doses and

(2012) suggest that in very high doses,

opiate-like effects in higher doses (Babu _et_

Kratom did not cause death and or any

_al_.,

2008).

Thus,

the

potential

for

significant toxicity. In humans, its adverse

youngsters' addiction and adverse health

effects include dry mouth, changes in

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 85

_Res. Highl. 4Bs (2016)_

_Kratom Plant: Beneficial or Detrimental?_ Low _et al_.

urination,

nausea,

vomiting,

anorexia,

performance. Some of them use it as a

weight loss, constipation, nystagmus, tremor

replacement for expensive drugs if they do

and seizures (Babu _et al._ , 2008; Boyer _et al._ ,

not have the cash to buy drugs.

2008; Ulbricht _et al._ , 2013; Nelsen _et al._ ,

2010; Trakulsrichai _et al_., 2013). Primary

From interviewed villagers, we gathered that

hypothyroidism (Sheleg and Collins, 2011)

Kratom is also used as a folk medicine by to

and intrahepatic cholestasis (Kapp _et al_.,

treat several diseases or as an opioid

2011) are also reported in several cases

alternative by chewing fresh leaves and

related to Kratom. For chronic toxicity,

brewing as tea or concoction. Some villagers

impairment of cognitive behavioral function

claim that Kratom is useful for lowering the

is found in mice fed with mitragynine for a

blood pressure, treating diarrhea, and as

long period (Apryani _et al_., 2010). However,

anxiolytic and antidiabetic.

in a study of chronic Kratom administration

****

showed that it does not significantly impair

Previously, Kratom was not considered to be

social functioning of users in a natural

a drug of abuse, but as a supplement for

context in Malaysia (Singh _et al_., 2014). In

better health; because, it is known to help in

humans,

anorexia,

weight

loss,

lowering the blood pressure, as an

hyperpigmentation over skin and cheeks on

antidiarrheal, anxiolytic, antidiabetic and

face, and psychosis are also described in

antinociceptive (Hassan _et al_., 2013;

chronic abusers (Babu _et al_., 2008).

Suwanlert,

1975).

Villagers' strongly

believe

that

Kratom

is

a

powerful

Kratom is well known and consumed as a

antidiarrheal property. An antidiarrheal

norm in the northern peninsular region of

property of plant was confirmed when there

Malaysia. Based on prior reports which

was an outbreak of food poisoning after a

suggest that several drug addicts and some

wedding reception, where majority of those

villagers from 'Tanjung Dawai' village as

who had consumed Kratom drink did not

well as youngsters from adjacent areas come

suffer from food poisoning while others who

here [at 'Tanjung Dawai' village] on a

did not were affected.

regular basis for Kratom drinks and or

products. We visited the village to collect

Currently, Kratom is causing concern as it

primary information. We carefully selected

appears as a potential drug of abuse (Hassan

some residents from 'Tanjung Dawai'

_et al_., 2013). It was noted by Boyer _et al._

village and also those grew them in their

(2008) and Vicknasingam _et al_. (2010) that

backyard

and

interviewed

them.

An

Kratom is often used as opioid replacement

interview with the village head was also

to alleviate opioid withdrawal symptoms

carried out and comments were noted.

and it is an easily accessible and serves as an

****

economical alternative to other opiod-

The information we received from villagers

replacement medications. Kratom is often

suggest that the majority of Kratom users

used as a drug of abuse alone or in

are adult males, mostly those are manual

combination with other substances, has been

laborers. Addicts consume Kratom drinks to

shown to cause dependence and withdrawal

enhance their physical endurance, boost

symptoms following repeated consumption

their energy, productivity, elicit euphoric

(Hassan _et al_., 2013; Babu _et al._ , 2008;

effects, and to improve their sexual

Vicknasingam _et al._ , 2010; McWhirter and

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 86

_Res. Highl. 4Bs (2016)_

_Kratom Plant: Beneficial or Detrimental?_ Low _et al_.

Morris, 2010; Saingam _et al._ , 2013;

**Apryani, E., Hidayat, M.T., Moklas, M.A.,**

Saingam _et al.,_ 2014; _Singh et al_., 2014).

**Fakurazi, S. and Idayu, N.F.**

However, based on the animal studies,

**(2010)**. Effects of mitragynine from

Kratom is only slightly toxic (Macko _et al_.,

_Mitragyna speciosa_ Korth leaves on

1981).

In

mice,

repeated

7-

working

memory _. J_

hydroxymitragynine administrations elicit

_Ethnopharmacol_ **. 129(3), 357–360**.

tolerance, cross-tolerance to morphine, and

naloxone-precipitated withdrawal symptoms

**Babu, K.M., McCurdy, C.R. and Boyer,**

(Matsumoto _et al_., 2005). Few poisoning

**E.W. (2008).** Opioid receptors and

cases related to Kratom consumption has

legal highs: Salvia divinorum and

been reported in the past (Kapp _et al._ , 2011;

Kratom. _Clin Toxicol (Phila)_ **46(2),**

Neerman _et al._ , 2012; Tungtananuwat and

**146–152**.

Lawanprasert, 2010). However, people

including youngsters are using it; hence, its

**Boyer, E.W., Babu, K.M., Adkins, J.E.,**

(Kratom) usage is causing concern as it

**McCurdy, C.R. and Halpern, J.H.**

emerges as a potential drug of abuse. Most

**(2008).** Self-treatment of opioid

recently, Malaysian police have seized 2.8

withdrawal

using

Kratom

tonne of Ketum powder worth MYR

( _Mitragynia_

_speciosa_

235,000 which also indicates that Kratom

korth). _Addiction_ **103(6), 1048–1050.**

(Ketum) is smuggled for the international

****

market (Star, 2016).

**Grewal, K.S. (1932).** Observations on the

pharmacology

of

mitragynine _. J_

In a conclusion, although Kratom has been

_Pharmacol Expr Ther._ **46(3), 251–**

associated with drug abuse and as a

**271.**

dangerous drug in recent years, it possesses

****

certain beneficial properties. Currently, we

****

do not have enough scientific information to

**Harizal, S.N., Mansor, S.M., Hasnan, J.,**

weigh the health benefits of this medicinally

**Tharakan, J.K. and Abdullah, J.**

important

plant.

The

systematic

**(2010).** Acute toxicity study of the

pharmacological study needs to be done in

standardized methanolic extract of

order to understand the beneficial properties

_Mitragyna_

_speciosa_

Korth

in

of this plant. Hence, further research is

rodent _. J Ethnopharmacol_. **131(2),**

required to explore the potential applications

**404–409**.

of this plant.

**Hassan, Z., Muzaimi, M., Navaratnam, V.,**

**REFERENCES**

_**et al**_ **. (2013).** From Kratom to

****

mitragynine and its derivatives:

**Adkins, J.E.** **, Boyer, E.W. ****and McCurdy,** physiological and behavioural effects

**C.R. (2011).** _Mitragyna speciosa_ , a

related

to

use,

abuse,

and

psychoactive tree from Southeast

addiction. _Neurosci Biobehav Rev_.

Asia with opioid activity. _Curr Top_

**37(2), 138–151.**

_Med Chem_. **11(9), 1165-75.**

****

****

**Kamal, M.S.A., Ghazali, A.R., Yahya,**

**N.A., Wasiman, M.I. and Ismail Z.**

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 87

_Res. Highl. 4Bs (2016)_

_Kratom Plant: Beneficial or Detrimental?_ Low _et al_.

**(2012).** Acute toxicity study of

exposure. _J Med Toxicol_. **6(4), 424–**

standardized _Mitragyna speciosa_

**426.**

Korth aqueous extract in Sprague

****

Dawley rats. _J Plant Stud_. **1(2), 120–**

**Puff,**

**C.,**

**Chayamarit,**

**K.**

**and**

**129.**

**Chamchumroon,**

**V.**

**(2005).**

Rubiaceae of Thailand; **** a pictorial

**Kapp, F.G., Maurer, H.H., Auwärter, V.,**

guide to indigeneous and cultivated

**Winkelman, M. and Hermanns-**

genera Bangkok: _Forest Herbarium,_

**Clausen, M. (2011).** Intrahepatic

_National Park, Wildlife and Plant_

cholestasis

following

abuse

of

_Conservation Department._

powdered

Kratom

( _Mitragyna_

__

_speciosa_ ). _J Med Toxicol._ **7(3), 227-**

**Saingam,**

**D.,**

**Assanangkornchai,**

**S.,**

**31.**

**Geater, A.F. and Balthip, Q. (2013).**

****

Pattern and consequences of krathom

**Macko, E., Weisbach, J.A. and Douglas, B.**

( _Mitragyna speciosa_ Korth.) use

**(1981).** Some observations on the

among male villagers in southern

pharmacology of mitragynine. _Arch_

Thailand: a qualitative study _. Int J_

_Int Pharmacodyn Ther_. **145–161.**

_Drug Policy_. **24(4), 351–358.**

****

****

**Matsumoto, K., Horie, S., Takayama, H.,**

**Saingam,**

**D.,**

**Assanangkornchai,**

**S.,**

**Ishikawa, H., Aimi, N., Ponglux, D.,**

**Geater, A.F. and Lerkiatbundit, S.**

**Murayama, T. and Watanabe. K.**

**(2014).**

Validation

of

Krathom

**(2005).** Antinociception, tolerance

( _Mitragyna_

_speciosa_

Korth.)

and withdrawal symptoms induced

Dependence 
Scale

(KDS):

a

by

7-hydroxymitragynine,

an

dependence

screen

for

alkaloid from the Thai medicinal

internationally

emerging

herb _Mitragyna speciosa_. _Life Sci._

psychoactive substance. _Subst Abus_.

**78(1), 2-7.**

**35(3), 276–283.**

****

****

**McWhirter, L. and Morris, S. (2010).** A

**Sheleg, S.V. and Collins, G.B. (2011).** A

case report of inpatient detoxification

coincidence

of

addiction

to

after Kratom ( _Mitragyna speciosa_ )

"Kratom" and severe primary

dependence. _Eur Addict Res_. **16(4),**

hypothyroidism. _J_

_Addict_

**229–231.**

_Med_. **5(4):300–301.**

****

****

**Neerman, M.F., Frost, R.E. and Deking, J.**

**Singh, D., Müller, C.P. and Vicknasingam,**

**(2012).** A drug fatality involving

**B.K. (2014).** Kratom ( _Mitragyna_

Kratom. _J Forensic Sci._ **58(S1),**

_speciosa_ ) dependence, withdrawal

**S278-S279.**

symptoms and craving in regular

****

users. _Drug Alcohol Depend._ **139,**

**Nelsen, J.L., Lapoint, J., Hodgman, M.J.**

**132–137.**

**and Aldous, K.M. (2010).** Seizure

****

and

coma

following

Kratom

**Star (Newspaper),** **March 11 2016** : "Cops

(Mitragynina

speciosa

Korth)

smash ketum plants". ****

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ 88

_Res. Highl. 4Bs (2016)_

_Kratom Plant: Beneficial or Detrimental?_ Low _et al_.

****

**Windsor, R.C. (2013).** An evidence-

**Suwanlert S. (1975).** A study of Kratom

based systematic review of Kratom

eaters in Thailand. _Bull Narc_. **27(3),**

( _Mitragyna speciosa_ ) by the Natural

**21–27.**

Standard Research Collaboration. _J_

****

_Diet Suppl._ **10(2), 152–170.**

**Trakulsrichai, S., Tongpo, A., Sriapha, C.,**

****

_**et al**_ **. (2103).** Kratom abuse in

**Vicknasingam, B., Narayanan, S., Beng,**

Ramathibodi

Poison

Center,

**G.T. and Mansor, S.M. (2010).** The

Thailand **:** A five-year experience. _J_

informal use of ketum ( _Mitragyna_

_Psychoactive Drugs_. **45(5), 404–408.**

_speciosa_ ) for opioid withdrawal in

****

the northern states of peninsular

**Tungtananuwat, W. and Lawanprasert, S.**

Malaysia and implications for drug

**(2010).** Fatal 4x100: Homemade

substitution therapy. _Int J Drug_

Kratom juice cocktail. _J Health Res_.

_Policy._ **21(4), 283–288.**

**24(1), 43-7.**

****

****

****

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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 region's 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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) gold-

deposited 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. Antibody-

antigen 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 300−1000 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 gold-

capped 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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._ 2 _AIMST 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 _tcp_ A gene, virulence genes namely _ace_ , _zot_ and _ctx_ A genes and _tet_ A 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 resource-

limited settings.

_**Keywords**_ : Biosensors; Cholera; Diagnostics; DNA; PCR; _Vibrio cholera_.

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ __ 92

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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 WHO's 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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 / +91-9524818862, 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 "World's 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 "World's 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 surface‐area‐

to‐volume ratio and compatibility with flexible substrates of these materials make them as

unique candidate for various applications. Surface Plasmon Resonance (SPR) of nano-

metallic surfaces is the incoming light results in a collective oscillation of the electrons at the

metal's 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Highly Sensitive Detection of DNA Hybridization and Immunoassay Based**

**on Nanomaterials**

Werasak Surareungchai*

_King Mongkut's 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 high-

throughput 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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 one's 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

compounds;

Biodiagnotics;

Biosynthesis;

Drugs;

Fungi;

Phytochemicals.

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ __ 101

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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 25th

minutes. **Methods:** _Salmonella_ bacteria was used here as a model organism.The magnetic

beads were used as an alternative method for detection of DNA. Whatman®papers 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 LAMP-

MBs 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/µL 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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 human-

adapted 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 Pan _-_

_Salmonella_ gene _(inv_ A) 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Isoluminol-functionalized gold nanoparticles and graphene oxide**

**nanoribbons composite for development of enzyme-based**

**electrochemiluminescence biosensors**

Nur Syakimah Ismail1* and Eiichi Tamiya2

_1School 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 (ABEI-

AuNP) 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Disposable Screen-Printed Electrodes Modified With Nanoparticles for**

**Sucrose Sensor**

Detpisuttitham W.a, Rijiravanich P.b,c*, and Surareungchai W.d

_aPilot 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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._

_2Department 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_ chalcone-

flavanone isomerase ( _Eo_ CHI) enzyme encoding cDNA. This _Eo_ CHI is an important enzyme.

Therefore, to understand more about it this study was undertaken. The objective of this study

was to elucidate _Eo_ CHI gene cDNA sequence and to predict the three-dimensional (3D)

structure of deduced protein. **Methods:** Positive and negative strands of the _Eo_ CHI cDNA

clone were sequenced using M13 forward and M13 reverse primers to elucidate the

nucleotide sequence. The _Eo_ CHI 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 _Eo_ CHI protein were predicted by using the PHYRE

automatic fold recognition server. **Results:** The sequencing results analysis showed

that _Eo_ CHI 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 _Eo_ CHI. **Conclusion** : The annotation of _Eo_ CHI showed that 942

bp long _Eo_ CHI cDNA encodes for 234 amino acid long protein that contains conserved

domains required for biological functions of CHI. The predicted deduced _Eo_ CHI 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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_

_flexneri_ a 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Herbal Based Stabilizers of Native and Misfolded State of Nuclear Co-**

**repressor (N-CoR)**

Matiullah Khan1*, Thunga Pandurangan2, Sridevi Visvanathan3, **** Sam Annie Jeyachristy3,

Mahes Maheswaram4

__

_1Department 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 N-

CoR 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Molluscicidal Effect of Poly herbal Extracts on Golden Apple Snail,**

_**Pomacea maculata**_

****

Guruswamy Prabhakaran1*, Thambirajah, J.J2, Subhash Janardhan Bhore1 and Manikam

Ravichandran1

_1Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling_

_Campus, 08100 Bedong, Kedah Darul Aman, Malaysia._

_2Faculty 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± 2°C) 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

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

****

Guajardo-Yévenes C. F.1,4*, Issaragkul P.2,4, Sae-Tang C.2,4 and Surareungchai W.3,4

_1Pilot Plant, Development and Training Institute_

_2Department of Chemistry, Faculty of Science_

_3School of Bioresources and Technology_

_4King 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 double-

stranded 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

_1Department of Biotechnology, Lahore College for Women University Lahore, Pakistan._

_2Quality Operational Laboratory, University of Veterinary and Animal Sciences Lahore,_

_Pakistan; *corresponding author, e-mail: rashidasbs@yahoo.com, _ _Ph No: 0992-03024680187_

__

****

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Umami Tasting Detection Based Electrochemical Sensor**

****

Promphan R.a, Chaiboon T.b, Lertanantawong B.b,* and Surareungchai W.c

__

_aDepartment of Chemical Engineering, King Mongkut's University of Technology Thonburi,_

_ThungKhru, Bangkok 10140, Thailand._

_bPilot Plant Development and Training Institute,King Mongkut's University of Technology_

_Thonburi, Bang KhunThian, Bangkok 10150, Thailand._

_cSchool 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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*

_1Institute 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;_

_3Communicable 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**The Fabrication of Membrane-Based Pneumatic Microvalves in**

**Microfluidic System**

Suppaso L.1, Boonpakdee D.1, Ngamchana S.2*, Guajardo-Yévenes C. F.3 and Surareungchai

W.4

__

_1Department of physics, Faculty of Science, KMUTT, Bangkok 10150 Thailand._

_2Biochemical Engineering and Pilot Plant Research and Development Unit, National Center_

_for Genetic Engineering and Biotechnology at King Mongkut's University of Technology_

_Thonburi (KMUTT), Bangkok 10150 Thailand._

_3Pilot Plant Development and Training Institute, KMUTT, Bangkok 10150 Thailand._

_4School 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 µm and 172.42 – 31.08 µm 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

__

_1Quality 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 Koch'spostulates. 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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.: +60−4−4293006._

**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 37°C, 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 175.71±7.05 to 761.32±6.01 g Ascorbic Acid

Equivalent/mg extract, 254.44±5.36 to 1451.67±27.54 g Gallic Acid Equivalent/mg extract,

and 12.22±9.62 to 615.00±30.05 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 pro-

oxidant properties at lower concentrations. Further studies in this respect are highly

warranted to explore their pharmacological activities based on their antioxidant and pro-

oxidant 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Sensitivity Analysis of Graphene Based Surface Plasmon Resonance**

**Biosensor**

Toloue H.1,2*, Centeno A.1, Tamiya E.2, Kuwano N.1

_1Malaysia-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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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 olanzapine's 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

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

**Sudan I contamination in chili**

****

Phanthong C.1 and Khownarumit P.2*

__

_1Biochemical Engineering and Pilot Plant Research and Development Unit, National Center_

_for Genetic Engineering and Biotechnology at King Mongkut's University of Technology_

_Thonburi (KMUTT), Bangkok 10150, Thailand; e-mail: chatuporn.pha@biotec.or.th; 2Pilot_

_Plant Development and Training Institute, King Mongkut's 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 × 10-

6, 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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,*

_1Faculty of Plantation and Agrotechnology, Universiti Teknologi MARA, Shah Alam,_

_Selangor, Malaysia;_

_2Integrative 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 {1-

Naphthaleneacetic 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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 (Φ2 and Φ3) 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; Φ2 and Φ3

could lyse 4.3% and 62.6% of the total 91 _Vibrio_ _cholerae_ strains, respectively. The latent

period and burst size of Φ2 were 14 ± 1.6 m and 06 ± 01 PFU/cell; while that of Φ3 were 13

± 4.1 m and 54 ± 26 PFU/cell, respectively. Electron microscopy revealed that Φ2 was of the

_Myoviridae_ family while Φ3 was of the _Siphoviridae_ family. Phage Φ2 had a genome of less

than 48.5 kb; while Φ3 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 
**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

_1Faculty 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 eco-

friendly 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Mutiplex Isothermal Amplification for Detection of Melioidosis**

Jilien Michelle Wong Tzelinga, Chan Yean Yeana,b*

_aDepartment of Microbiology & Parasitology, School of Medical Science, Universiti Sains _

_Malaysia, 16150 Kubang Kerian, Kelantan._

_bInstitute 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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 µm to 5 µm). 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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.a, Carlos S.a, Suresh V. C.b, Zaidah A. R.c, and Phua K. K.a*.

_aInstitute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains_

_Malaysia (USM), 16150 Kubang Kerian, Kelantan, Malaysia;_

_bFaculty 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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*

_1Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia,_

_11800, USM, Pulau Pinang, Malaysia; 2Department of Biotechnology, Faculty of Applied_

_Sciences, AIMST University, 08100, Bedong, Kedah, Malaysia;_ * _corresponding author, e-_

_mail: 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 37˚C 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. StyR-

9 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

_1Department 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

_1Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia,_

_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 5'end 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 63°C 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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;_ 2 __

_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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

_**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

__

_aSchool of Bioresources and Technology, King Mongkut's University of Technology_

_Thonburi, Bang Khun Thian, Bangkok 10150, Thailand._

_bBiochemical Engineering and Pilot Plant Research and Development Unit, National Center_

_for Genetic Engineering and Biotechnology, King Mongkut's University of Technology_

_Thonburi, Bang Khun Thian, Bangkok 10150, Thailand._

_cNational 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 MB-

loaded 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Electrochemical Characterisation and Determination of _Mycobacterium_**

_**tuberculosis**_ **by Voltammetry at Polymer Nanocomposite modified Platform**

****

Himkusha Thakur1, Navpreet Kaur1, Dipti Sareen1, Priyanka2, Nirmal Prabhakar*1

****

_1Department of Biochemistry, Panjab University, Chandigarh, India._

_2Institute of Nano Science & Technology, Mohali, India; *corresponding author, e-mail: _

 _nirmalprabhakar@pu.ac.in_ __

****

****

**Abstract: Background:** Tuberculosis, caused by _Mycobacterium tuberculosis_ , produces ill-

health 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 PEDOT-

CNT 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Abstracts (Poster Presentations)**

****

**Cloning, Over-expression, and Purification of Hfq Protein from _Klebsiella_**

_**pneumoniae**_ ****

****

_Devarubini K.1, Kishan S.1, Suresh V.C.1_ *

_1AIMST 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

_**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, e-_

_mail: 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. torvum_ a 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

_1Faculty of Medicine, AIMST University, Semeling, Bedong, Malaysia_

_2Faculty of Applied Sciences, Department of Biotechnology, AIMST University, Semeling,_

_Bedong, Malaysia_

_3School 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 HPLC-

PDA 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 790µg/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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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 *

__

_1Integrative Pharmacogenomics Institute (iPROMISE), Level 7, FF3, Universiti Teknologi_

_MARA Selangor, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia._

_2Faculty 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 (F0-

C). 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 F0-

H 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;

Heroin addiction; Opiates;

Opioid;

Paternal

addiction;

Transgenerational; .

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ __ 140

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Optimization of Cryopreservation Using Different Cryoprotective Agents**

**and Differential Temperatures on Freeze Dried Probiotics**

Hassan Pyar1, 2 and K.K Peh*3

_1Faculty of Pharmacy, AIMST University, Semeling, 08100, Bedong, Kedah Darul Aman,_

_Malaysia; 2Hadramout University of Science and Technology, Mukalla, Hadramout, Yemen;_

_3School 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 (28°C) 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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 non-

fouling surface for electrochemical biosensor applications. **Methods:** A combination of aryl

diazonium

salt

with

zwitterions

such

as

sulfanilic

acid

and

(4-

aminophenyl)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 antigen-

antibody 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 (25ºC). The good blocking of Fe(CN) 3-/4-

6

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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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._

_2Advanced 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 pET-

28b(+) 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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* __

__

_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:** _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-80°C 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Understanding the Host-Pathogen Interaction in _Klebsiella pneumo_** **niae**

**Infected Rat Model via Metabolomics Approaches**

Mohd Izwan Mohamad Yusof1,2, Mohd Salleh Rofiee2, Teh Lay Kek2, Mohd Zaki Salleh*2

_1Integrative Pharmacogenomics Institute (iPROMISE), Level 7, FF3 Building, Universiti_

_Teknologi MARA Selangor Branch, Puncak Alam Campus, 42300 Puncak Alam, Selangor,_

_Malaysia._

_2Faculty 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

_1Integrative Pharmacogenomics Institute (iPROMISE), Level 7, FF3 Building, Universiti_

_Teknologi MARA Selangor Branch, Puncak Alam Campus, 42300 Puncak Alam, Selangor,_

_Malaysia._

_2Faculty 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 "Lipinski's

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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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._

_2 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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 meta-

analysis. **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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Biocontrol of Macergen Infestation on Plants using Bacteriophage Cocktail**

Sasireigga J.*, Ravichandran M., and Kurunathan S.

__

_Department of Biotechnology, Faculty of Applied Sciences, AIMST University, 08100_

_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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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 ****

_1Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong_

_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 community's 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 sub-

gingival plaques samples suggest that 325 types of bacteria were embedded in the sub-

gingival 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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-specific 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 (SPB-

S 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 λ phage-

like 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Transcriptome Analysis for the Identification of Novel ncRNAs in**

_**Acinetobacter baumannii**_ ****

Saw H. S., Sumitha S., Kishan Raj S., Xavier R., Suresh V.C.*

__

_Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong_

_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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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, e-_

_mail: 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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, e-_

_mail: 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 anti-

inflammatory 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Arduino Microcontroller Based Heart Rate Monitor using Fingertip**

**Sensors**

Srilahari Namani1,*, Manickam Ramasamy1, Sunitha Namani2, Satyanarayana Namani2

_1 Faculty of Engineering and Computer Technology, AIMST University, Malaysia._

_2 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 doesn't 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

__

_1AIMST 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Safety and antiobesity effect of _Garcinia atroviridis_**

Suriati Mohd Nasir1, Teh Lay Kek1,2, Mohd Zaki Salleh*1,2

_1Integrative 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Development of Ochratoxin A Detection Based on Electrochemical Sensor**

**by Using Au-ball Labels**

Suttiporn. P.*1, Patsamon. R2 and Werasak. S3

_1Food 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 Mongkut's 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 275±17 nm. After coated with Au layer,

size of Au-ball was 280±14 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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.*

__

_AIMST University, Department of Biotechnology, Faculty of Applied Sciences, 08100_

_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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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.*

_****_

_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 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 0.22µm and 0.45µm 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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* 
__

_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:** _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

doesn't 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Computational Analysis of Common Bean ( _Phaseolus vulgaris_** **L.) S-**

**adenosyl-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

_1Department 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 ( _Pv_ SAM MTase)

gene and to understand more about it this study was undertaken. The objective of this study

was to annotate _Pv_ SAM MTase cDNA and deduced amino acid sequence using

computational tools. **Methods:** Both strands of _Pv_ SAM 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 _Pv_ SAM 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 _Pv_ SAM MTase protein is closely related to its counterpart from _Vigna_

_radiata_ L. The _Pv_ SAM MTase protein structure was successfully predicted and will be

discussed. **Conclusion** : The cDNA was annotated and primary analysis of the _Pv_ SAM

MTase deduced protein sequence suggested that it contains catalytic sites essential for its

function. Further study is required to validate the predicted structure of _Pv_ SAM 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Biochemical Changes in African catfish, _Clarias gariepinus_** **Exposed to**

**Buprofezin**

Gobinath R., Marimuthu K., Xavier R., Suresh C.V.*

__

_AIMST University, Department of Biotechnology, Faculty of Applied Sciences, 08100_

_Bedong, Kedah, Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com, _ _Ph._

_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 catfish, _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 sub-

lethal 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Haematological Changes in African catfish, _Clarias gariepinus_** **exposed to**

**Buprofezin**

Gobinath R., Marimuthu K., Xavier R., Suresh C.V.*

__

_AIMST University, Department of Biotechnology, Faculty of Applied Sciences, 08100_

_Bedong, Kedah, Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com, _ _Ph._

_No: +604-4298165_

****

****

**Abstract: Background:** The study was carried out to investigate the haematological changes

in a freshwater African catfish, _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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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.*

_1AIMST University, Department of Biotechnology, Faculty of Applied Sciences, 08100_

_Bedong, Kedah, Malaysia; 2AIMST University, Faculty of Dentistry, 08100 Bedong, Kedah,_

_Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com, _ _Ph. No: +604-4298165_

__

****

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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.

_1Center 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, e-_

_mail: 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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 energy-

dependent 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 "Bergey's 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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*

_1Medical 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._

_3Stempeutics Research Sdn Bhd, Technology Park Malaysia, 57000 Bukit Jalil, Kuala_

_Lumpur, Malaysia; 4Britannia Women and Children Specialist Centre, Selangor, Malaysia;_

_5Department 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. hAF-

MSCs 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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*

_1Institute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains_

_Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia._

_2School 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 PacBio's 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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*_

__

_1AIMST University, Department of Biotechnology, Faculty of Applied Sciences, 08100_

_Bedong, Kedah, Malaysia._

_2Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia,_

_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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**Identification of Novel Non-coding RNA (ncRNA) from _Bacillus_**

_**thuringiensis**_ ****

Shereenrani S., Saw H.S., Sumitha S., Maheswaran S., Xavier R., Suresh C.V.*

_AIMST University, Department of Biotechnology, Faculty of Applied Sciences, 08100_

_Bedong, Kedah, Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com, Ph. _

_No: +604-4298165_

__

****

**Abstract: Background:** _Bacillus thuringiensis_ (Bt) is a Gram-positive, rod-shaped, spore-

forming 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 species-

specific 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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*

__

_1Medical 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 dichloromethane-

methanol 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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, Bedong-_

_Semeling 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 37°C 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

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**An Alternative Method of Screening the _M2/ANXA5_** **Haplotype for**

**Repeated Pregnancy Loss**

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

Roslani, 3Krishna Kumar, 5Bavanandan Naidu, 2Ridzuan bin Abdullah, 6Nadja Bogdanova,

1Narazah Mohd Yusoff, 7Wan Zaidah Abdullah, 6Arseni Markoff, 1Thean-Hock Tang*

_1Advanced 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.

****

****

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ __ 179

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

__

_1Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong-_

_Semeling 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 Oil-

palm, _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 Oil-

palm, 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.

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ __ 180

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

_**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

__

_1Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong-_

_Semeling 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** : 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 ( _Pv_ O6FAD) gene cDNA sequence and to

predict the three-dimensional (3D) structure of deduced protein. **Methods:** Positive and

negative strands of the _Pv_ O6FAD cDNA clone were sequenced using M13 forward and M13

reverse primers to elucidate the nucleotide sequence. Deduced _Pv_ O6FAD 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 _Pv_ O6FAD

protein were predicted by using the PHYRE automatic fold recognition server. **Results:** The

sequencing results analysis showed that _Pv_ O6FAD 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 _Pv_ O6FAD 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 _Pv_ O6FAD 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.

****

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ __ 181

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

_1Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong-_

_Semeling 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:** 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.

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ __ 182

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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

__

_1Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong-_

_Semeling 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** : 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.

****

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ __ 183

**Research Highlights in 4Bs** __

__

_**B** iosensors, **B** iodiagnostics, **B** iochips and **B** iotechnology _

_Res. Highl. 4Bs (2016)_

**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, Bedong-_

_Semeling 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.

****

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ __ 184

_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 Bartholomew's 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-Chancellor's post at Universiti Sains

Islam Malaysia and the fifth woman to be appointed as Vice-Chancellor of a public

university.

Prof. Asma's 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 University's 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 country's 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 Asma's 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 Bio-

diagnostics 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 high-

impact 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 Bison-

SeqTech 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 Kumar's 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 USM's 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 Malaysia's 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),

USM's 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 Women's Day Award of

St.Annes College.

She serves, as Editorial Board Member in Nanoscience and Nanotechnology (ISSN:2163-

2588). Her publications have been cited 211 times which shows the high impact / value. Her

nanotechnology based innovations "World's 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

World's lowest priced graphene" & "World's 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.

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_**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. Gopinath's

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 Sudesh's 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.

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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 Amine's 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 (1994-

2001).

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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 ( _Febreze-_

_Allergen 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

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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.

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 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. Bari's 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 biochemical's 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

_Keynote and plenary speakers_

_who delivered their talk in 3rdRC4Bs-2016_

_**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 2009-

2012 and 1997. Dr. Hossain served as Visiting Professor,

Department of Cultural Fisheries, Faculty of Agriculture, Kochi University, Japan and Rhein-

Waal 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 People's 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, books-

chapters, 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**

**Time**

**Programme**

**Venue**

Medical/ Dental

08.00-09.00

Registration

Building

Ground Floor

**Workshop-1** : Drug-Protein Interaction and Personalized Medicine.

Computer Lab,

_Speaker: Ms. Monisha Hajra, ScientiaBio, Bangalore, India_

Medical Building,

AIMST University

09.00-17.00

**Workshop-2** : Basic Statistics using Microsoft Excel and SPSS' &

Third Floor

'Basic Concept of Effective Scientific Communications.

Computer Lab,

_Speakers: Snr. Assoc. Prof. Dr. K. Marimuthu and Snr. Assoc. Prof._

Medical Building,

_Dr. P. Balakumar, AIMST University, Malaysia_

AIMST University

**Registration of Participants (Main Conference)**

**Time**

**Programme**

**Venue**

Medical Building,

15.00-19.00

Registration

AIMST University

Guest House /

Apartment /

15.15-19.00

Check-in (Pre-booked participants only)

Hostels of AIMST

University

**Day 2: 21/04/2016 (Thursday)**

**Time**

**Programme**

**Venue**

07.30 - 08.45

**Registration**

**Great Hall**

09.00 - 10.45

**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_

**Great Hall**

## 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

Tea break

**Foyer**

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__

_Scientific programme of the 3rdRC4Bs-2016_

**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**

_Associate Prof. Bent Petersen, Technical University of Denmark,_

_Denmark_

Relevance of Biotechnological Applications for Global Food

Security and Sustainability

12.00-12.30

**P3**

_Prof. Prakash P. Kumar, National University of Singapore,_

**Great Hall**

_Singapore_

12.30-12.40

Summary and conclusion of the session

**Cafeteria,**

12.45-14.00

Lunch

**1st Floor**

**Medical**

13.15-14.00

Poster evaluation

**Building**

**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

14.00-14.30

Diseases for Low Resources Settings __

**P4**

_Prof. Dr. Phua K. K, Universiti Sains Malaysia (USM), Malaysia_

Paper-based visual detection of Salmonella bacteria using

Isothermal DNA amplification and magnetic beads aggregation

**Lecture**

14.30 -14.45

OP1

**Theatre- 1,**

_Dr. Ahmed M.U., Universiti Brunei Darussalam, Brunei_

**Medical**

**building**

Development of a Reverse Hybridization Assay (RHA) for

Simultaneous Identification of _Salmonella_ Serotypes Causing

14.45 -15.00

OP2

Enteric Fever

_Carlos S., Universiti Sains Malaysia (USM), Malaysia_

Decrypting the Evolutionary Path of Antimicrobial Resistance of

_Acinetobacter baumannii_ via Next-Gen Sequencing

15.00 -15.15

OP3

_Mohamad Izwan Ismail, Universiti Teknologi MARA (UiTM)_

_Puncak Alam, Malaysia_

__

15.15- 15.25

Summary and conclusion

**Medical**

15.25-16.00

Tea Break

**building**

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__

_Scientific programme of the 3rdRC4Bs-2016_

**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 __

**Lecture**

_Nur Syakimah Ismail, Universiti Malaysia Perlis, Malaysia_

**Theatre- 2,**

**Medical**

Disposable Screen-Printed Electrodes Modified With Nanoparticles

**building**

for Sucrose Sensor __

14.45 -15.00

OP5

_Detpisuttitham W., King Mongkut's University of Technology,_

_Thailand_

Cloning and expression of the urease operon from _Helicobacter_

_pylori_ J99

15.00 -15.15

OP6

_Mohamad CWSR, Universiti Malaysia Perlis, Malaysia._

15.15- 15.25

Summary and conclusion

**Medical**

15.25-16.00

Tea Break

**building**

**Session 2C**

**Chair:** Assoc. Prof. Dr. Bent Petersen, Technical University of Denmark, Denmark

**Co-chair:** Dr. Ramesh Kumaresan, AIMST University, Malaysia

Aptasensors: Bench to Bedside and Beyond

**Lecture**

_Dr._

_Gopinath_

_S.C.B,_

_Universiti_

_Malaysia_

_Perlis_

14.00-14.30

**P6**

**Theatre- 3,**

_(UniMAP),Malaysia_

**Medical**

**building**

Analysis of chalcone-flavanone isomerase (CHI) gene cDNA

isolated from American oil-palm ( _Elaeis oleifera_ ) mesocarp tissue

14.30 -14.45

OP7

cDNA library

_Yu Chuen Leow, AIMST University, Malaysia_

Non-Protein coding RNA genes as novel diagnostic markers to

detect pathogenic bacteria __

14.45 -15.00

OP8

_Suresh C.V., AIMST University, Malaysia_

**Lecture**

Herbal based stabilisers of native and misfolded state of nuclear

**Theatre- 3,**

co-repressor (N-CoR)

15.00 -15.15

OP9

**Medical**

_Matiullah Khan, AIMST University, Malaysia_

**building**

15.15- 15.25

Summary and conclusion

**Medical**

**building**

15.25-16.00

Tea Break

****

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ 200

__

_Scientific programme of the 3rdRC4Bs-2016_

**Session 3**

**Session 3A**

**Chair** : Prof. Dr. Tang Thean Hock, University Sains Malaysia

**Co-chair:** Dr. Yu Chye Wah, AIMST University, Malaysia

Recent progress in the production of biodegradable plastics from

16.00-16.30

**P7**

palm oil in Malaysia

_Sudesh K., Universiti Sains Malaysia, Malaysia_

**Lecture**

Hepatoprotective effect of methanol extract of _Polygonum minus_

**Theatre-1,**

16.30 -16.45

OP10

leaves in carbon tetrachloride-induced liver damage in rats

**Medical**

_Christapher V, AIMST University, Malaysia_

**building**

Molluscicidal Effect of Poly herbal Extracts on Golden Apple

16.45 -17.00

OP11

Snail, _Pomacea maculata_

_Guruswamy Prabhakaran, AIMST University, Malaysia._

Production of Butter Flavour Concentrate from Butter fat with

Lactic Acid Bacteria by Solid Substrate Fermentation

17.00 -17.15

OP12 ****

_Thambirajah_

_J.J,_

_Department_

_of_

_Biotechnology,_

_AIMST_

_University, Malaysia._

17.15- 17.30

Summary and conclusion

**Medical**

17.30-18.00

Poster evaluation

**Building**

**Session 3B**

**Chair: Assoc.** Prof. Dr. Chan Yean Yean, University Sains Malaysia ****

**Co-chair:** Dr. Sawri Rajan, AIMST University, Malaysia ****

Recent advances in biosensors based on enzyme inhibition

16.00-16.30

**P8**

_Prof. Aziz Amine, Hassan II University of Casablanca, Morocco_

__

Design and characterization in time of an on-off DNA biosensor

16.30 -16.45

OP13

_Guajardo-Yévenes C. F, King Mongkut's University of Technology_

**Lecture**

_Thonbury, Bangkok, Thailand._

**Theatre-2,**

__

**Medical**

Optimization of PCR for rapid detection of _CTX-M_ gene in ESBL

**building**

producing _Klebsiella pneumoniae_ clinical isolates

16.45 -17.00

OP14

_Rasheeda B, Lahore College for Women University Lahore,_

_Pakistan_

__

Coenzyme Q10 dietary supplementation during antitubercular

therapy prevents renal damage in rats

17.00 -17.15

OP15 ****

_Evan Prince Sabina, School of Biosciences and Technology, VIT_

_University, Vellore, India_

__

17.15- 17.30

Summary and conclusion

****

****

17.30-18.00

Poster evaluation

**Medical**

**Building**

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ 201

__

_Scientific programme of the 3rdRC4Bs-2016_

**Session 3C**

**Chair:** Dr. Gopinath S.C.B, Universiti Malaysia Perlis (UniMAP),Malaysia ****

**Co-chair:** Dr. Subhash J. Bhore, AIMST University, Malaysia ****

**Lecture**

**Theatre- 3,**

Nano- and Bio-technological Advancement to assist in the

**Medical**

Determination of Halal Products ****

**building**

16.00-16.30

**P9**

_Prof. Quamrul Hasan, Universiti Utara Malaysia (UUM),_

_Malaysia & Chairman, Japan Halal Research Institute for _

_Products and Services (JAHARI), Japan_

Umami Tasting Detection Based Electrochemical Sensor

16.30 -16.45

OP16

_Chaiboon T, King Mongkut's University of Technology Thonburi,_

_Bang KhunThian, Bangkok 10150, Thailand._

Detection of _Salmonella enterica_ serovar Typhi Form Water

**Lecture**

Samples and Its Association with Geographical Clustering of

16.45 -17.00

OP17

**Theatre- 3,**

Enteric Fever

**Medical**

_Ismail Aziah, Universiti Sains Malaysia, Malaysia_

**building**

The fabrication of membrane-based pneumatic microvalves in

microfluidic system __

17.00 -17.15

OP18

_Ngamchana S, King Mongkut's University of Technology_

_Thonburi (KMUTT), Bangkok 10150 Thailand._

17.15- 17.30

Summary and conclusion

**Medical**

17.30-18.00

Poster evaluation

**Building**

**19.00-22.30**

**Cultural Programme and Conference Dinner**

**Great Hall**

**Day 3: 22/04/2016**

Introduction of Key note Speaker by Snr. Prof. M. Ravichandran, VC &

09.00-09.10

CEO, AIMST University, Malaysia

**Keynote Address**

**Lecture**

Tamiya E., Osaka University, Japan

**Theatre- 1,**

09.10-09.55

Nanotechnology oriented biosensors and biomedical application

**Medical**

_Tamiya E., Professor, Department of Applied Physics, Graduate School of_

**building**

_Engineering, Osaka University, Japan_

09.55-10.10

Tea Break

**Session 4**

**Session 4A**

**Chair** : Snr. Assoc. Prof. Dr. P. Balakumar, AIMST University, Malaysia

**Co-chair** : Dr. Annie Jeyachristy, AIMST University, Malaysia

**Lecture**

**Theatre- 1,**

Genomics of the endangered Orang Asli: disease susceptibility

**Medical**

and sustainability

**building**

10.10-10.40

**P10**

_Dato' Prof. Dr. Mohd Zaki Salleh., UiTM, Malaysia_

__

Role of Outer member proteins (OMP) and lipopolysaccharides

(LPS) in antibody response against _Pasteurella multocida_ type B-

**Lecture**

2 in bovines

**Theatre- 1,**

10.40 -10.55

OP19

_Imran A, University of Veterinary and Animal Sciences_

**Medical**

_(UVAS),Pakistan_

**building**

__

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ 202

__

_Scientific programme of the 3rdRC4Bs-2016_

Exploration of Novel Endophytic Bacterial Isolates for Their

10.55 -11.10

OP20

Antioxidant and Pro-oxidant Properties

_Monowar T, AIMST University, Kedah, Malaysia_

Sensitivity analysis of graphene based surface plasmon resonance

biosensor

11.10 -11.25

OP21

_Toloue H., University Teknologi Malaysia, Kuala Lumpur,_

_Malaysia_

Ameliorative Effect of Curcumin on Olanzapine Induced Obesity

11.25 -11.40

OP22

in Sprague Dawley Rats

_Parasuraman. S, AIMST University, Malaysia_

11.40- 11.55

Summary and conclusion

12.00-14.30

Lunch

**Cafeteria**

**Medical**

13.10-14.30

Poster evaluation

**building**

**Session 4B**

**Chair:** Prof. Md. Latiful Bari, University of Dhaka ****

**Co-chair:** Dr. **** Suresh C.V., AIMST University, Malaysia ****

Highly Sensitive Detection of DNA Hybridization and

**Lecture**

Immunoassay Based on Nanomaterials

**Theatre-2,**

10.10-10.40

**P11**

**Medical**

_Werasak Surareungchai, King Mongkut's University of_

**building**

_Technology Thonburi, Thailand_

Study of Nanoparticle-Modified Screen-Printed Electrodes for

detection of Sudan I contamination in chili __

10.40 -10.55

OP23

_Phanthong C, King Mongkut's University of Technology_

_Thonburi (KMUTT), Thailand._

Bioengineering of _Tacca integrifolia_ (Bat flower): effects of

hormones on in vitro rooting and production of Taccalonolides

10.55 -11.10

OP24

_Fatimah A.L, UniversitiTeknologi MARA, Shah Alam,_

_Selangor,Malaysia_

**Lecture**

Isolation,

characterization

and

potential

application

of

**Theatre-2,**

11.10 -11.25

OP25

bacteriophages for phage therapy

**Medical**

_Bhandare, S.G, Universiti Malaysia Kelantan, Kelantan, Malaysia_

**building**

Reconfigurable Filter Bank for Accurate Spectral Decomposition

11.25 -11.40

OP26

of EEG Signals

_Girish Kumar C., AIMST University, Malaysia_

11.40- 11.55

Summary and conclusion

12.00-14.30

Lunch

**Cafeteria**

****

****

13.10-14.30

Poster evaluation

**Medical**

**building**

****

**Session 4C**

**Chair:** Prof. Dr. Pandurangan, AIMST University, Malaysia ****

**Co-chair:** Dr. Leela A. J, AIMST University, Malaysia ****

**Lecture**

**Theatre-3,**

P12 - Fungal Secondary Metabolites - A Pharmaceutical Chemist

**Medical**

Perspective

**building**

10.10-10.40

**P12**

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_ __ 203

__

_Scientific programme of the 3rdRC4Bs-2016_

Eco-friendly Biosynthesis of Atrocarpusaltilis Mediated Silver

Nanoparticles - Characterization and Evaluation of its

10.40 -10.55

OP27 ****

Antimicrobial and Antioxidant Potential

_Dr. Ravichandran V, AIMST University, Malaysia_

Mutiplex Isothermal Amplification for Detection of Melioidosis

10.55 -11.10

OP28

_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 __

11.10 -11.25

OP29

_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

**Lecture**

11.25-11.40

OP30

Typhi Based on Single-Gene Target __

**Theatre-3,**

_Goay Y. X., INFORMM, Health Campus, Universiti Sains_

**Medical**

_Malaysia, Kelantan, Malaysia._

**building**

Assessment of Biodiesel Properties From the FAME Composition

of a Malaysian Rhodophyte ( _Kappaphycus_ sp.) __

11.40-11.55

OP31

_Md. Sayedur Rahman, Aimst University_

11.55- 12.10

Summary and conclusion

12.10-14.30

Lunch

**Cafeteria**

**Medical**

13.10-14.30

Poster evaluation

**building**

**Session 5**

**Session 5A**

**Chair:** Snr. Assoc. Prof. Dr. K. Marimuthu, AIMST University, Malaysia ****

**Co-chair:** Dr. Kazi A Selim, AIMST University, Malaysia ****

Safe Water as the Key to Food Safety & Global Health

14.30-15.00

**P13**

**Lecture**

_Associate Prof. Md. Latiful Bari, University of Dhaka, Bangladesh_

**Theatre-1,**

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

**Medical**

15.00 -15.30

**P14**

Bangladesh

**building**

_Prof. M Anwar Hossain, University of Dhaka, Bangladesh_

Generation of RNA aptamers against _Mycobacterium tuberculosis_

secretory protein ESAT-6 - a preliminary study

15.30 -15.45

OP32

_Bakhtiar B, AMDI, Universiti Sains Malaysia, Kepala Batas,_

_Malaysia._

An Expression Analysis of _Salmonella_ Pathogenicity Island (SPI)-

15.45 -16.00

OP33

Derived Non-Protein Coding RNAs in _S_. Typhi Biofilm formation

_Anbalagan, INFORMM, Universiti Sains Malaysia, Malaysia._

**Lecture**

**Theatre-1,**

**Medical**

**building**

Summary and conclusion

16.00- 16.15

_ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1_ __ 204

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_Scientific programme of the 3rdRC4Bs-2016_

**Session 5B**

**Chair:** Snr. **** Prof. Dr. M. Ravichandran, AIMST University, Malaysia ****

**Co-chair:** Assoc. Prof. Dr. Matiullah Khan, AIMST University, Malaysia ****

Quantitative, Single-Step Measurement of Hemoglobin A1c in

14.30-14.45

OP34

Whole Blood for Personalized Medicine

_Khor S. M., University of Malaya (UM), Malaysia_

Development of rapid diagnostic detection for _Salmonella enterica_

subspecies _enterica_ serovar Paratyphi A using cross priming

14.45 -15.00

OP35

amplification

_Roziana, M.H, INFORMM, Universiti Sains Malaysia, , Kelantan,_

**Lecture**

_Malaysia_

**Theatre-3,**

Conversion of rice husks to polyhydroxyalkanoate (PHA) __

**Medical**

OP36

**building**

15.00 -15.15

_Heng K.S, Universiti Sains Malaysia, Penang, Malaysia_

_Salmonella typhimurium_ Detection based on Electrochemical

Immunoassay using Methylene blue/MWNTs/Magnetic Particle

15.15 -15.30

OP37

_Ngoensawat, U, King Mongkut's University of Technology,_

_Bangkok 10150, Thailand._

Electrochemical

Characterisation

and

Determination

of

_Mycobacterium Tuberculosis_ by Voltammetry at Polymer

15.30- 15.45

OP38

Nanocomposite modified Platform __

_Himkusha Thakur, Panjab University, Chandigarh, India_

15.45- 16.00

Summary and conclusion

**Valedictory function**

 Welcome

15.45-16.15

**Lecture**

 Prizes and awards

**Theatre- 1,**

 Vote of thanks

**Medical**

**building**

16.15- 17.00

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_

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_WITH BEST COMPLIMENTS_

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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

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**Research Highlights in 4Bs**

_**Biosensors, Biodiagnostics, Biochips and Biotechnology**_

**Published by AIMST University**

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