Research Highlights in 4Bs Biosensors

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

Proceedings of the 3rd Regional Conference on Biosensors, Biodiagnostics, Biochips and Biotechnology 2016 (3rdRC4Bs-2016) held on the campus of AIMST University, Kedah, Malaysia, in April 2016

Editor Subhash Bhore

2016

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:

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: [email protected] / [email protected]

Front Cover Design Mr. Mahes D.S. AIMST University, Malaysia Edition First; July 26, 2016

Dedication This book is dedicated to all speakers, participants and organizing committee members of the 3rd Regional Conference on Biosensors, Biodiagnostics, Biochips and Biotechnology 2016 (3rdRC4Bs-2016) in recognition of their contributions for making event.

this

conference

a

successful

Conference Organizing Committee

Chairman Dr. Subhash J. Bhore Co-chairman Dr. Matiullah Khan Secretary Ms. Kalaiselvee Rethinam Scientific Committee Snr. Prof. Dr. M. Ravichandran Prof. Dato' Dr. Mohd Zaki Salleh Prof. Dr. Uda Hashim Prof. Dr. Teh Lay Kek Dr. Subhash J Bhore Dr. K. Marimuthu Dr. Lee Su Yin Dr. V. Ravichandran Dr. Werasak S. Dr. P. Balakumar Dr. Benchaporn L. Dr. S. Kathiresan Dr. Kazi Selim Anwar Dr. Sivakumar Pendyala Dr. Ramesh Kumaresan Secretariat Dr. Annie Jeyachristy Dr. Heera Rajandas Treasurer Mr. Arvinth Murugiah Mr. Vijayan Krishnan Logistics Committee Ms. Musalinah Buzri Mr. V. Krishnan Ms. Yoganandhini Mr. Christapher V. Mr. Mahes D.S. Mr. G. Prabhakaran Dr. D. Jawahar

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Publicity & Exhibitions Committee Dr. Sivachandran P. Mr. Siventhiran B. Mr. M. K. Faiz Mr. Dhanaraj R. Mr. P. K. Karuna Reception Committee Ms. Sridevi Visvanathan Ms. Elil Suthamathi Dr. Tahmina Monowar Ms. Azdlina First Aid and Safety Committee Dr. Sawri Rajan Dr. Leela A. J. Mr. Maheswaran S.

iv

Foreword

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

Senior Professor Dr. M. Ravichandran Chief Executive & Vice-Chancellor, AIMST University, Malaysia

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Preface

The 21st century is the age of biological sciences and a special emphasis on biotechnology is clearly noticeable woldwide. Biotechnology do have a tremendous potential not only to address the challenges in agriculture and health care sectors but also to generate the new jobs and wealth (bio-economy). In fact, biotechnology has become an integral part of the knowledge-based economy of developing and developed countries. In biotechnology industry and health care sector, biosensors, biodiagnostics and biochips based technologies are playing an important role in providing the most innovative products and services. In April 2016, the 3rd Regional Conference on Biosensors, Biodiagnostics, Biochips and Biotechnology 2016 (3rdRC4Bs-2016) was held at the AIMST University which provided a platform for scientists, researchers, academicians, students and stakeholders to share their knowledge, challenges, recent advances and future perspectives in the multidisciplinary areas of biosensors, biodiagnostics, biochips and biotechnology (4Bs). The scientific programme of the conference was rich and wide-ranging with 2 keynote talks, 14 invited plenary talks, 38 technical papers and about 50 posters’ presentation. Prof. Asma (Ministry of Higher Education, Malaysia) delivered a keynote address and highlighted various aspects of ‘Moving the Regional Biotechnology and Bioeconomy Forward’. By giving examples of policies and long term vision plan implemented by Chinese Government for moving their national bio-economy forward, she highlighted that most of the Asian countries including Malaysia have a great potential and resources to boost regional bio-economy; however, the lack of alignment of the numerous policies, strategies and initiatives makes it a challenge to coordinate implementation. In a keynote address, Prof. Eiichi Tamiya (Osaka University, Japan) highlighted the achievements, challenges and latest trends in ‘nanotechnology oriented biosensors and biomedical applications’. In a plenary talk, Prof. Prakash Kumar (NUS, Singapore) highlighted that we need to use modern biotechnological approaches to enhance the agricultural productivity for the global food security and sustainability. All 14 plenary talks were very comprehensive and highlighted the latest trends, achievements and challenges in various domain of biosensors, biodiagnostics, biochips and biotechnology. This book contains full-length articles, talk abstracts of all invited speakers, and abstracts of all technical papers presented in oral and poster sessions. I wish to thank and acknowledge the support of all co-organizers, supporters, and AIMST University. Nevertheless, the success of the conference was possible because of the hard efforts of dedicated and committed members of the organizing committee team as well as volunteers and they deserve the appreciation for it. Subhash Bhore July 26, 2016 ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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

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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

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Exploration of Novel Endophytic Bacterial Isolates for Their Antioxidant and Prooxidant Properties .................................................................................................. 119 Sensitivity Analysis of Graphene Based Surface Plasmon Resonance Biosensor 120 Ameliorative Effect of Curcumin on Olanzapine Induced Obesity in Sprague Dawley Rats ........................................................................................................... 121 Study of Nanoparticle-Modified Screen-Printed Electrodes for detection of Sudan I contamination in chili ............................................................................................ 122 Bioengineering of Tacca integrifolia (Bat flower): Effects of Hormones on in vitro Rooting and Production of Taccalonolides ............................................................ 123 Isolation, characterization and potential application of bacteriophages for phage therapy.................................................................................................................... 124 Eco-friendly Biosynthesis of Atrocarpus altilis Mediated Silver Nanoparticles Characterization and Evaluation of its Antimicrobial and Antioxidant Potential . 125 Mutiplex Isothermal Amplification for Detection of Melioidosis ......................... 126 Effective Pulmonary Therapeutic Delivery via Surface Acoustic Waves Nebulization and Phononic Crystal Structures ...................................................... 127 Development of a Novel Duplex PCR Assay for Specific Detection of Salmonella enterica subspecies enterica serovar Typhi Based on Single-Gene Target ........... 128 Assessment of Biodiesel Properties From the FAME Composition of a Malaysian Rhodophyte (Kappaphycus sp.) ............................................................................. 129 Generation of RNA Aptamers Against Mycobacterium tuberculosis Secretory Protein ESAT-6 - a Preliminary Study .................................................................. 130 An Expression Analysis of Salmonella Pathogenicity Island (SPI)-Derived NonProtein Coding RNAs in S. Typhi Biofilm formation ........................................... 131 Quantitative, Single-Step Measurement of Hemoglobin A1c in Whole Blood for Personalized Medicine ........................................................................................... 132 Development of Rapid Diagnostic Detection for Salmonella enterica Subspecies enterica Serovar Paratyphi A using Cross Priming Amplification ........................ 133 Conversion of Rice Husks to Polyhydroxyalkanoate (PHA) ................................. 134 Salmonella typhimurium Detection Based on Electrochemical Immunoassay using Methylene blue/MWNTs/Magnetic Particle .......................................................... 135 Electrochemical Characterisation and Determination of Mycobacterium tuberculosis by Voltammetry at Polymer Nanocomposite modified Platform ...... 136 Abstracts (Poster Presentations) ............................................................................ 137 Cloning, Over-expression, and Purification of Hfq Protein from Klebsiella pneumoniae ............................................................................................................ 137

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

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

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16S Ribosomal DNA Sequence Based Identification of Bacterial Endophytes Isolated from Seeds of Starfruit (Averrhoa carambola L.).................................... 178 An Alternative Method of Screening the M2/ANXA5 Haplotype for Repeated Pregnancy Loss ...................................................................................................... 179 Computational Analysis of Class IV Chitinase Gene cDNA Isolated from American Oil Palm (Elaeis oleifera) Fruit Mesocarp Tissue cDNA Library......................... 180 In Silico Analysis of Omega-6 Fatty Acid Desaturase Gene cDNA Isolated from Common Bean (Phaseolus vulgaris L.) Pod Tissue cDNA Library ...................... 181 Computational Analysis of Common Bean (Phaseolus vulgaris L.) Palmitoyltransferase Gene cDNA Isolated from Bean Pod Tissue cDNA Library ................................................................................................................................ 182 Annotation of Elaeis oleifera CAP cDNA and its Deduced Amino Acid Sequence using Computational Tools .................................................................................... 183 Comparison of Methods for Pure Quality RNA Isolation from Polysaccharide Rich Averrhoa carambola L. Fruits (Starfruits) ............................................................. 184 Appendices ................................................................................................................ 185 Appendix I: Brief biography of keynote and plenary speakers who delivered their talk in 3rdRC4Bs-2016. .......................................................................................... 185 Appendix II: A copy of scientific programme of the 3rdRC4Bs-2016................... 198

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Men love to wonder, and that is the seed of science. --- Ralph Waldo Emerson

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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: [email protected]

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 The use of genetically modified (GM) crops has been made into a controversial topic that elicits quite polarized views. This article is

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based on a plenary talk delivered at a conference. Based on scientific evidence and first-hand experience with GM plant production, the intended take-home message of this talk was that we should not hesitate to

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Res. Highl. 4Bs (2016)

Global Food Security and Sustainability use modern technologies for crop improvement. It is acknowledged that having adequate tests to verify the general safety of the GM crops is important, but once proven safe (or not significantly different from the conventional crop variety), the GM crop should be used as a valuable alternative. With judicious use of technology and crop management practices, we can achieve global food security. GM FOOD What is Genetic Modification (GM) and is it vastly different from conventional plant breeding in its principle? Crop modification has been ongoing for thousands of years. The use of everchanging technologies has been a constant theme in this as with all other modern human endeavors. We recognize that use of any novel technology involves risks and benefits, it is up to us to evaluate them and decide to adopt them if the benefits vastly outweigh risks. Also, human ingenuity is such that technology can be constantly refined to minimize the initial risks to negligible levels as was done with the minimization of radiation emission in the present day cell phone or mobile phone technology. The yield enhancement as a result of the Green Revolution has reached its limit now. Although it is known that crop plants have not reached their biological upper limits for yield increase, any further increases in crop yield will have to rely on novel tools. One of the relatively recent novel technologies is genetic modification or genetic engineering (also known as biotechnology, recombinant DNA technology, etc.). Two main approaches in GM plant production are: a) Modifying existing genes in plants - e.g., fruit ripening by modifying ethylene biosynthesis or ethylene action, and ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Kumar b) Expression of introduced (foreign) gene to confer a new/modified trait to the crop plant, e.g., carotenoid production in the endosperm tissue of the Golden Rice grains. The main examples of GM plants The main examples of GM plants that have been commercially cultivated for the last 20 or so years include herbicide resistant and insect resistant plants. The major crops with such GM varieties include corn, soybean, canola and cotton. In 2013, GM crops were grown in about 175 million hectares of land by 18 million farmers in 27 countries (James, 2013). The global market value of GM crops for 2013 was estimated to be about US$15.6 billion. Some of the other traits being introduced to crop plants include drought tolerance, disease tolerance (both fungal and viral), resistance towards root nematodes, modified nutritional parameters such as altered lipid profiles in oil crops and fortified grains. Although the Golden Rice has been fully developed and safety tested – it has gone through the so called deregulation process mandatory for GM crops – it has not yet received the necessary Government approvals. The Golden Rice has been touted as a staple grain that will lead to vast improvements in the nutritional status (with respect to vitamin A and iron) of millions of poor people in Asia and Africa. Nevertheless, it has not been released to the growers. Scientists at the International Rice Research Institute, Philippines (IRRI) have developed Golden Rice varieties suitable for several rice growing regions with the help of generous funding from philanthropic organizations such as the Bill and Melinda Gates Foundation. Therefore, when approved, the Golden Rice seeds can be distributed to growers without profiteering. The IRRI has established a highly positive and remarkable reputation for successfully 2


Global Food Security and Sustainability releasing the flood tolerant SUB1 rice that was developed by non-GM methods (marker assisted breeding). However, it must be highlighted that this could not have been achieved without data from GM tools. Prior studies using molecular biological tools helped to identify the flood tolerant variant of the Sub1a gene in rice. Experimental GM rice plants demonstrated that when this resistant form of the gene was introduced to flood sensitive rice varieties they could be turned into flood tolerant forms. Therefore, without GM tools scientists could not have so easily developed the marker assisted SUB1 rice varieties in such a short time span. These varieties dubbed ‘Scuba Rice’ are being grown for the past several years by thousands of happy farmers in millions of hectares of land, for example, in the flood prone areas of India and Bangladesh. OTHER GM FOOD Besides the examples cited above, there are several other minor successes in GM plants including the virus resistant GM papaya in Hawaii, the delayed ripening tomatoes, as well as some ornamentals such as long-vaselife carnation flowers and the Blue Rose marketed by Suntory Biotechnology company. The use of GM products such as recombinant proteins, mainly enzymes in food industry is noteworthy development. GM or ‘Recombinant proteins’ from bacteria and fungi are used in cheese making. Cheese production requires the use of a protein called chymosin (also known as rennin or rennet). Chymosin was previously obtained only from calf stomachs, which had several implications including health concerns, conflict with nutritional preferences due to religious restrictions or personal choices of various groups of people. The use of chymosin from GM microbes has become ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Kumar common in cheese making now, with estimated 70% to 90% of cheeses in the USA manufactured using such microbial GM rennet now. Also, the US Food and Drug Administration (FDA) has approved over 30 other recombinant enzymes for use in food production (including α-amylase, used to make glucose or fructose syrups). Another noteworthy development in the recent years is the FDA approval of the first transgenic animal for food use, namely, GM salmon. After an exhaustive and rigorous review that lasted nearly twenty years, the FDA has approved the use of fast growing GM salmon (AquAdvantage) in November 2015. FDA determined that the AquAdvantage salmon is as safe to be consumed by humans as the commercial non-GM Atlantic salmon. This was a longawaited positive decision welcomed by biotech scientists around the world. AGRICULTURAL ECONOMY How important are GM crops to the agricultural economy? An economic assessment of the impact of commercial agricultural biotechnology on global agriculture was done (Brookes and Barfoot, 2009). The study highlighted the significant impacts on the production of soybeans, corn, cotton and canola crops. According to this study, GM technology led to net economic benefits at the farm level of about US$10.1 billion in 2007. The other benefits associated with the use of GM had a positive impact to the tune of 25% of the total direct farm income benefit in the USA. Biotech crops contributed to increasing global production levels of the four main crops examined. For example, the use of GM soybeans corresponded to adding 68 million tons and GM corn added 62 million tons to global production levels in 2007. Their use was equivalent to 172 million kg 3


Global Food Security and Sustainability reduction in pesticide use (which corresponds to 14% reduction in the environmental footprint associated with pesticide use). In addition, other factors from growing these biotech crops significantly reduced greenhouse gas emissions from agriculture equivalent to removing 5 million cars from the roads (Brookes and Barfoot, 2009). THE NEED TO INCREASE FOOD PRODUCTION Why not just stick to old ways of producing food? Why resort to new tools? According to a UN study, the world population is expected to grow to ~9.7 billion by 2050. According to that study, Africa and Asia would be adding 1.14 billion more people to their population. By 2050, Africa (2478 million) and Asia (5267 million) collectively will have a population size larger than the current world population.

Kumar Further complications are already evident from the global climate change. The predicted impacts of climate change include, reduced wheat production in South Asia by 2030, and rice production in Southeast Asia, particularly in the Greater Mekong Subregion (ADB, 2009). These will almost certainly lead to increased food prices. The ADB estimates that rice, wheat, and soybean prices could increase by 10%–50%, while corn price may double by 2050. This should be recalled in connection with the global food and energy price surge in 2007–2008, which exposed the vulnerabilities of households and of the international food and nutrition insecurity. Increase in extreme weather events (floods, droughts, typhoons) will have serious consequences for agriculture, food, and forestry production in the coming decades. Therefore, it is imperative that crop yield has to be raised to ensure food security. FOOD PRODUCTION AND WATER

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

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


Global Food Security and Sustainability world water use (www.adb.org/publications/asian-waterdevelopment-outlook-2013). Currently, ~40% of this water is lost due to inefficient agricultural practices (crops and farm animals). The water required for production of some of the key agricultural produce illustrate the vastness of water needed in agriculture. Liters of water necessary per kg of produce (indicated in parentheses): for rice (2,500), other grains in a favorable climate (2,000), grains in arid climate (5,000) and beef (15,000). Therefore, the total world water use is 7,450 to 8,160 km3/year, and the water used for food production is estimated to be 6,390 to 7,200 km3/year, with domestic needs accounting for 180 to 344 km3/year (IRRI, and various other sources). Some of the major challenges for rice and grain production include abiotic and biotic stresses. The major abiotic stresses are (1) cold (a climatic factor that affects ~66% of land worldwide), (2) drought, (3) flood (~10% of yield loss, ~25% of the global rice crop lands sees floods each year – with ~20 million hectares of Asian rice land prone to flooding), (4) salinity (~10% of world’s land affected and ~12 million hectares in Asia affected by increased soil salinity). SUSTAINABLE FOOD PRODUCTION STRATEGIES There are several ways to increase crop yield. Four of the prominent options are mentioned here. (1) Expanding the cultivated area: land expansion can account for ~ 20% increase in yield. (2) Increasing the cropping intensity can contribute about 10% increase. (3) Reducing pre- and postharvest loss and wastage: 20 to 30% increase if all wastage is prevented, (4) Improving crop yields using latest knowledge and technologies. Therefore, about 40% increase ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Kumar in crop yield has to be through plant breeding and biotechnology. In view of the above, the answer to the question of whether we should use novel technologies (e.g., GM technology) for crop improvement should be obviously affirmative! I cite the example of breast feeding v/s infant formula in support of this view. We all know breast feeding is the best nutritional option for infants. But, global market for baby food is huge (over US$10 billion/year) with 50% to 70% of it as the value of infant formula! North America and Europe constitute 33% of this market, while Asia forms 53% of the infant food market. Over 120 companies manufacture and sell infant formula! What has happened here is the need for convenience (in most cases, necessity in some) has led to the adoption of an alternative to breast feeding as an acceptable alternative for our infants. In a similar rational manner, we need to develop modern technologies for crop improvement and ensure food security. We should not hesitate to use new technologies to develop alternative options that should coexist with the established techniques. What are the other alternatives? Certainly, GM crops are not the only option to increase crop yield – they are an efficient and complementary tool to the established tools at our disposal. It is recognized that efficient crop management will improve yield to some extent. These include the use of right amount of chemical fertilizers and pesticides, because excess use of such agricultural chemicals leads to yield loss as well as air and water pollution. Ecological engineering is another concept being tested with positive effect. For example, the use of beneficial insects for pest control by growing flowering plants around the fields is one such ecological modification tool. This method helped to reduce pesticide use by 5


Global Food Security and Sustainability ~21% in Vietnam according to a study coordinated by IRRI. Alternate Wetting and Drying (AWD) method is another crop management tool. AWD can lead to ~35% less water requirement for rice growing compared to the traditional farming method, with ~10% yield increase and attendant reduction in greenhouse gas (methane) emission from the fields. The reduction in methane emission is due to the reduction in methanogenic bacteria growing in the root zones of rice plants when cultivated under permanent submergence (leading to anaerobic soil favoring the methanogenic bacteria). The AWD management practice leads to reduced growth of such bacteria along with reduced water needs. Marker-assisted breeding, genome-wideassociation study (GWAS) leading to Genomic Selection (GS) are established as non-GM alternatives for crop improvement in the past decade or so (Meuwissen et al., 2001, Desta and Ortiz, 2014). Genomic Selection is based on the powerful tools of genome-wide prediction in plant improvement. GWAS helps to generate statistical models to predict how a plant will perform before conducting a genetic cross and field-testing of actual crops. This involves the combined use of genomic information, statistics, bioinformatics tools, and prediction-based breeding for crop improvement. Of course, this requires that the scientists establish robust association between DNA seqence (SNPs) and specific traits for a large number of breeding lines (1000s of individual plants). This will then be used to estimate Genomic Breeding Values (GEBV). New plants to be used for breeding will then be sequenced; for each new breeding line a DNA-profile will be determined and using such profiles from each line scientists will estimate GEBV for every trait of interest. Based on the genetic potential for the different traits the breeder ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Kumar can design a new variety on the computer which combines plant lines with highest scores (using the predictive models built). One of the more recent GWAS studies with impressive results is on oil palm (Teh et al., 2016). Also, several improved crops generated using these techniques are being released and more will be coming in the near future. The gene that facilitates deep rooted phenotype beneficial to develop drought tolerant crops, e.g., DEEP ROOTED1 gene or DRO1 of rice was identified by GWAS approaches while as mentioned above, the SUB1 rice was developed by marker assisted breeding. Genetically modified (GM) crops: to use or not? Based on ample evidence from the use of GM crops during the past couple of decades it is safe to conclude that GM food can save millions of kids from dying due to malnutrition. The use of GM crops can also help to reduce pesticide usage and environmental pollution. In this connection it is worth remembering that technological advances are continually occurring. Accordingly, new genome editing methods may be seen as good alternative tools to generate the new generation, advanced crop plants. Besides GM, several non-GM strategies are being developed and some of the novel strategies for crop breeding by genome editing include: (a) TALENs (b) Zinc Finger Nucleases (ZFN) (Petolino and Kumar, 2016) and (c) CRISPR-Cas9 system (Chen and Bailey, 2016; Kleinstiver et al., 2016). TALEN stands for Transcription Activatorlike Effector Nucleases; while CRISPR/Cas9 stands for Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated(Cas) system. CRISPR/Cas9 was first named as Short Regularly Spaced Repeats (SRSR) in 2000. 6


Global Food Security and Sustainability It was renamed as CRISPR in 2002. CRISPR/Cas9 induces double-strand break (DSB), generates deletion and/or insertion of short sequences at the break leading to altered gene of interest. In fact, the common feature of all these genome editing tools is that they induce DSBs in the genomic DNA at specific locations (gene of interest) – with or without short insertions. The DSB will then be repaired by one of two repair processes, namely, Non-Homologous End Joining (NHEJ), or Homology Dependent Repair (HDR). This results in gene editing. FUTURE PERSPECTIVES Gene-edited CRISPR mushroom has been generated recently and it was exempted from having to go through US Regulatory process; because, such organisms do not harbor foreign genes and are similar to induced mutants. The white button mushrooms generated have their polyphenol oxidase gene edited, which helps to prevent browning (Waltz, 2016). Scientific American (14 April 2016) also indicated that this is one of almost 30 genome edited organisms to avoid regulatory requirement currently. These represent the first few improved crops and more such crops will be released by various companies in the years to come (Wolt et al., 2016).

Kumar direct beneficiaries until now. But, when we carefully examine it there are already plenty of indirect benefits for all. GM papayas (ring-spot virus resistant) saved the papaya industry in Hawaii, GM crops will play an important role in protecting soil and water resources, minimizing crop diseases and attendant loss of produce, and responding to the pressures of climate change. Cultivation of Bt-cotton has reduced the amount of neurotoxic insecticide use by millions of kilograms every year in many countries where it is grown. Hence, one way of looking at it is that the concept of organic crops and GM crops coexist! GM crops do not pose any conflict with traditional or organic farming. A book published by Oxford University Press in April 2008 by authors Pamela Ronald and Raoul Adamchak (UC, Davis) advocates a food system that is organic and genetically engineered! They argue that a judicious blend of GM and organic farming representing two important strands of agriculture is key to helping feed the world's growing population in an ecologically balanced manner.

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

Even newer GM crops that will directly benefit the consumers are coming, for example, the purple tomatoes produced by Prof Cathie Martin’s lab, John Innes Centre, UK. These GM tomatoes have high amounts of plant nutrients (e.g., anthocyanins found in some berries), which are super foods that will confer tremendous health benefits to the consumers. The purple tomatoes were grown in controlled greenhouses in Canada and juice from these GM tomatoes is undergoing tests and evaluations. Clearly, this represents a ‘super food’ that could not be otherwise made available to us in plentiful quantities. They have already shown that inclusion of these high-anthocyanin GM tomatoes in the diet of cancer-prone mice can extend their healthy life-span by 30% - a most welcome

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Global Food Security and Sustainability prospect indeed! It is a firm belief of the author that people will line up for this type of GM superfoods and demand that more be supplied! From the foregoing discussion it is clear that the future global food supply can benefit greatly by the use of new initiatives in research. We should be open to employ a multidisciplinary approach involving molecular genetics, cell and developmental biology, systems biology, as well as ecology. GM crops have been grown and consumed for over 20 years now and the underlying technology is continually being improved. Hence, the emerging novel tools such as genome editing will help us alleviate food scarcity in the future decades. We should be mindful to develop ‘safe technology’ and develop suitable alternative strategies such as GM, molecular marker assisted breeding and genome editing. Collectively, these will serve us well in ensuring future food security. REFERENCES ADB

(2009). Operational plan for sustainable food security in Asia and the Pacific. Document downloadable from ADB web site. http://www.adb.org/publications

Brookes G, Barfoot P (2009). Global impact of biotech crops: Income and production effects, 1996-2007. AgBioForum 12(2), 184-208. Chen H, Bailey S (2016). Cas9, poised for DNA cleavage. Structural rearrangements explain how Cas9 cuts DNA. Science 351, 811-812. Desta ZA, Ortiz R (2014). Genomic selection: genome-wide prediction in plant improvement. Trends in Plant Science 19, 592-601. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Kumar FAO paper (2009). How to Feed the World in 2050? Document available at the FAO web site. http://www.fao.org/fileadmin/template s/wsfs/docs/expert_paper/How_to_Fee d_the_World_in_2050.pdf James, C. (2013). Global status of commercialised biotech/GM crops: 2013, ISAAA Brief No. 46. International Service for the Acquisition of Agri-Biotech Applications, Ithaca, NY. ISBN 9781-892456-55-9. www. isaaa. org/resources/publications/briefs/46, 2014. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK (2016). High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490-495. Meuwissen THE, Hayes BJ, Goddard ME (2001). Prediction of total genetic value using genome-wide dense marker maps. Genetics 157, 1819– 1829. Pamela C. R. and Raoul, W. A. (2008). Tomorrow's Table: Organic Farming, Genetics, and the Future of Food, Oxford University press (OUP). Petolino JF, Kumar S (2016). Transgenic trait deployment using designed nucleases. Plant Biotechnology Journal 14, 503–509. Teh CK, Ong AL, Kwong QB, Apparow S, Chew FT, Mayes S, MohamedM, Appleton D, Kulaveerasingam H (2016). Genome-wide association study identifies three key loci for high mesocarp oil content in perennial crop

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Global Food Security and Sustainability oil palm. Scientific Reports 6, 19075 | DOI: 10.1038/srep19075 Waltz E (2016). Gene-edited CRISPR mushroom escapes US regulation. A fungus engineered using CRISPR– Cas9 can be cultivated and sold without oversight. Nature 532, 293.

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Kumar Wolt JD, Wang K, Yang B (2016). The regulatory status of genome-edited crops. Plant Biotechnology Journal 14, 510–518.

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Res. Highl. 4Bs (2016), P10-19

Nano- and Bio-technological Advancement to assist in the Determination of Halal Products Quamrul Hasan1, 2, * 1

College of Business, Universiti Utara Malaysia (UUM), 06010 Sintok, Kedah, Malaysia; 2Japan Halal Research Institute for Products and Services (JAHARI), Kobe, Japan; *corresponding author, e-mail: [email protected] / [email protected]

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

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Determination of Halal Products INTRODUCTION As processed and prepared ready-to-eat food products with animal origins have become increasingly available to consumers, due to technological advances, the possibility of fraudulent adulteration and substitution of the expected species (source) with other sources has also increased. The same goes to confectionery and functional cosmetic products especially containing gelatin or collagen peptides. Such practices pose a substantial concern to consumers in terms of economic loss, allergies, religious observance, loss of traceability, and food safety. In many cases, porcine derivatives are used due to cheaper price and readily available. For Muslim consumers, the major authenticity concerns are in meat and meat products include porcine gelatin, collagen, fat, and so on. The analytical methods used for Halal authentication of meat and meat products include: Polymerase Chain Reaction (PCR); Enzyme Linked Immunosorbent Assays (ELISA); Mass spectrometry; Chromatography, Electronic nose and Spectroscopy. An overview of the currently available analytical methods or techniques is given in Table 1. The analytical methods currently in use to detect the presence of porcine materials are mainly protein and DNA-based. These are described below. DNA-based detection PCR is capable of amplifying very few copies of DNA and its detection limit is much lower than what is observed with protein based assays. PCR amplification is based on hybridization of specific oligonucleotides to a target DNA and synthesis of million copies flanked by these primers. The simplest PCR strategy applied to evaluate presence of any species in a meat product is the amplification of DNA fragments, followed by agarose gel electrophoresis for fragment size verification. To successfully ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Hasan detect a species with PCR, adequate genetic markers are chosen to develop the assay. Either nuclear or mitochondrial genes can be targeted (Fajardo et al., 2008). However, the use of mitochondrial DNA (Mt DNA) offers a series of advantages over cell nucleus DNA. Mitochondrial DNA facilitates PCR amplification, even in cases where the availability of DNA template after its extraction is insufficient for detection (Murugaiah et al., 2009). This is attributed to the fact that Mt DNA is several fold more abundant than that of nuclear genome; each mitochondrion is estimated to contain 2 to 10 Mt DNA (Murugaiah et al., 2009). Furthermore, Mt DNA evolves much faster than nuclear DNA and henceforth contains more sequence diversity facilitating the identification of phylogenetically related species (Fajardo et al., 2010; Girish et al., 2005; Murugaiah et al., 2009). Among the mitochondrial genes, cytochrome b (cyt b) (Aida et al., 2005; Murugaiah et al., 2009) and 12S rRNA (Chen et al., 2010; Girish et al., 2005) are the most commonly used markers in the development of DNA methods for meat species authentication. Protein-based detection Porcine protein, due to it is being cheap and readily available, might fraudulently be used to substitute other animal proteins. ELISA is the most commonly used method to detect animal proteins and a number of commercial immunoassays are available. Chen and Hsieh (2000) were the first ones to develop the immunoassay (ELISA) using a monoclonal antibody to a porcine thermostable muscle protein for detection of pork in cooked meat products. They observed no cross-reactivity with common food proteins. By employing this technology, the first pork detection kit was developed in Japan by a Japanese company (Okamoto, 2016). This kit is immuno-chromatographic employing nano-sized colloidal gold 11


Determination of Halal Products particles to detect presence of pork in food samples. It can detect pork in both raw and cooked food. It allows rapid detection of pork in food samples at low cost without

Hasan using any special equipment or requiring skill. The basic principle of immunechromatographic kit is shown in Figure 1.

Table 1: Summary of analytical techniques applicable in the halal authentication of meat products♦. Authenticity issue Pork adulteration Species identification

Analytical Techniques PCR-RFLP Real time PCR

Species-specific PCR

Pork protein

Pork fat (lard)

RAPD PCR sequencing ELISA Chromatography Peptide examination Isoelectric focusing FTIR spectroscopy

DSC Electronic nose

Blood plasma

Isoelectronic focusing ELISA Immunodiffusion LC – MS/MS

References Murugaiah et al. (2009), Aida, Che Man, Raha, and Son (2007), and Aida et al. (2005) Martín et al. (2009), Kesmen, Gulluce, Sahin, and Yetim (2009), Tanabe et al. (2007), Fumière, Dubois, Baeten, von Holst, and Berben (2006), and López-Andreo, GarridoPertierra, and Puyet (2006) Soares, Amaral, Mafra, and Oliveira (2010), Alaraidh (2008), Che Man et al. (2007) and Montiel-Sosa et al. (2000) Martinez and Malmheden Yman (1998) Karlsson and Holmlund (2007) Chen and Hsieh (2000); Chen and Hsieh (2000) Chou et al. (2007) Aristoy and Toldra (2004) Hofmann (1985) Rohman, Sismindari, Erwanto, and Che Man (2011a, 2011b), Che Man, Abidin, & Rohman, 2010, Rohman and Che Man (2011a, 2011b), Rohman and Che Man (2009), Che Man, Gan, NorAini, Nazimah, and Tan (2005), Che Man, Syahariza, Mirghani, Jinap, and Bakar (2005) and Che Man and Mirghani (2001) Marikkar, Ghazali, Man, and Lai (2003) and Marikkar, Lai, Ghazali, and Che Man (2001) Nurjuliana, Che Man, and Mat Hashim (2011a), Nurjuliana, Che Man, Mat Hashim, and Mohamed (2011b), Che Man, Gan, et al. (2005), and Che Man, Syahariza, et al. (2005) Bauer and Stachelberger (1984) Church and Hart (1995) Price, Hart, and Church (1992) Grundy et al. (2007) and Grundy et al. (2008)

♦

Source: Nakyinsige et al. (2012).

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Determination of Halal Products

Hasan

Figure 1 The basic principle of the immuno-chromatographic kit.

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

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

Table 2 Sequence of Porcine and Bovine specific primers. Target Primer Sequence Porcine Forward GAC CTC CCA GCT CCA TCA AAC ATC TCA TCT TGA TGA AA Reverse GCT GAT AGT AGA TTT GTG ATG ACC GTA Bovine Forward GAC CTC CCA GCT CCA TCA AAC ATC TCA TCT TGA TGA AA Reverse CTA GAA AAG TGT AAG ACC CGT AAT ATA AG 13


Determination of Halal Products Table 3 Condition of PCR Temperature Step (°C) Initial 94 denaturation Denaturation 94 Annealing 60 Extension 72 Final 72 Extension Hold 4

Hasan B. FAMIC method: Time

Cycle

1 min

1

30 sec 30 sec 30 sec

30

7 min

1

Primer:FAMIC Porcine mitochondrial DNA DNA: 20 ng PCR steps: Step



Comparative study on the PCR-based detection of the Porcine and Bovine genes using different primers

Initial denaturation Denaturation Annealing Extension Final Extension Hold

primer-

Temperature (°C)

Time

Cycle

95

9 min

1

92 60 72

30 sec 1 min 1 min

45

72

5 min

1

4



A. Neogen kit method: Primer: Neogen genomic DNA DNA: 20 ng PCR steps:

Porcine

primer-

Step

Temperature Time Cycle (°C) Initial 94 10 1 denaturation min Denaturation 94 15 sec Annealing 64 15 30 sec Extension 72 15 sec Final 72 3 1 Extension min Hold 4 

Primer:FAMIC Bovine mitochondrial DNA DNA: 20 ng PCR steps: Step Initial denaturation Denaturation Annealing Extension Final Extension Hold

Temperature (°C)

primer-

Time

Cycle

95

9 min

1

92 55 72

30 sec 30 sec 30 sec

45

72

5 min

1

4



RESULTS Detection of Porcine and Bovine DNAs from different products containing gelatin/collagen from different sources

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Determination of Halal Products Table 4: Summarized results of the detection of porcine, bovine and vertebrate DNA Porcine Bovine Vertebrate Sample 1 Not Not Detected* detected detected Sample 2 Detected* Not Not detected detected Sample 3 Detected* Not Not detected detected Sample 4 Detected* Not Detected* detected *When one of the test samples from duplicate was found positive, it was taken as the positive (as shown in the following three figures).

Hasan 3. Two products (one marshmallow, one functional cosmetic) showed positive result for vertebrate material. Only one of the two marshmallow products might contain fish material (gelatin) since it was found porcine negative.

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

Figure 4 Result of PCR using the primer that common to a vertebrate; Lane 1: Marker (25bp ladder), 2: Negative control, 3: sample 1 (n=1), 4: sample 1 (n=2), 5: sample 2 (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]. 15



Hasan

Comparative study on the PCR-based detection of the Porcine and Bovine genes using different primers Result-1 (DNA concentration) The values are shown in the following table DNA extraction kit: Neogen (Speciation) Samples: No Sample ID DNA 260/280 (ng/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, no porcine DNA was detected in any of the two samples tested in duplicates. B. FAMIC method: As shown in the Figure 6, Porcine DNA was detected for the two samples though in single from duplicate (MB and T-A). No bovine DNA was detected from the two samples tested.

Figure 5 Porcine DNA; PCR product size: 380-bp (housekeeping fragment), 314-bp (porcine fragment); from left: marker, M-A, M-B, T-A, T-B, positive control (380-bp, 314-bp).

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

Figure 6 Porcine DNA; PCR product size: 126-bp; from left: M-A, M-B, T-A, T-B, positive control. 16



Hasan tested. PCR-based detection was performed by using PK mastermix/control POD from Neogen Europe Ltd., UK, and porcine and bovine specific primers from Food and Agricultural Materials Inspection Center (FAMIC), Japan.

Figure 7 Bovine DNA; PCR product size: 126-bp; from left: M-A, M-B, T-A, T-B, positive control and jelly containing gelatin) and functional cosmetic product containing collagen peptides by employing conventional PCR and two different primers (nuclear and mitochondrial genes). Earlier, two other groups also reported about the successful application of the conventional PCR in meat species detections including from the processed foods (Tanabe et. al., 2007; Matsunaga et. al., 1999). These conventional PCR methods are simple and useful. That is why we have employed these. The choice of the target gene and the design of the primers have a great impact on the sensitivity and specificity of a detection system. It is wellknown that very sensitive PCR assays can be established when the primer target is a multicopy gene, such as a mitochondrial gene (Holzhauser et. al., 2006). In this study, we chose both mitochondrial and nuclear (genomic) genes as the target to detect porcine and bovine materials for comparing the efficiency of these two primers while using the processed products. DNA extraction was performed by using the commercial kit from Neogen Europe Ltd., UK and following the manufacturer’s instructions. The DNA extraction was carried out in duplicate for each sample ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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


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

Hasan Meat species the loop amplification DNA sensor. 605.

identification based on mediated isothermal and electrochemical Food Control 21, 599-

Aida, A. A., Che Man, Y. B., Wong, C. M. V. L., Raha, A. R. and Son, R. (2005). Analysis of raw meats and fats of pigs using polymerase chain reaction for halal authentication. Meat Science 69(1), 47–52. Chen, F. C., and Hsieh, Y. H. P. (2000). Detection of pork in heat-processed meat products by monoclonal antibody-based ELISA. Journal of AOAC International 83(1), 79–85. Chen, S. Y., Liu, Y. P. and Yao, Y. G. (2010). Species authentication of commercial beef jerky based on PCR–RFLP analysis of the mitochondrial 12S rRNA gene. Journal of Genetics and Genomics 37(11), 763–769. Fajardo, V., González, I., Martín, I., Rojas, M., Hernández, P. E., García, T. et al. (2008). Differentiation of European wild boar (Susscrofa scrofa) and domestic swine (Susscrofa domestica) meats by PCR analysis targeting the mitochondrial D-loop and the nuclear melanocortin receptor 1 (MC1R) genes. Meat Science 78(3), 314–322. Fajardo, V., González, I., Rojas, M., García, T. and Martín, R. (2010). A review of current PCR-based methodologies for the authentication of meats from game animal species. Trends in Food Science & Technology 21(8), 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

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Determination of Halal Products Girish, P. S., Anjaneyulu, A. S. R., Viswas, K. N., Shivakumar, B. M., Anand, M., Patel, M. et al. (2005). Meat species identification by polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) of mitochondrial 12S rRNA gene. Meat Science 70(1), 107–112. Holzhauser, T., Stephen O., and Vieths, S. (2006). Polymerase chain reaction (PCR) methods for the detection of allergenic foods. In “Detecting Allergens in Food,” S.J. Koppelman and S.L. Hefle, edc. CRC Press, Boca Raton, pp. 125-143. Matsunaga, T., Chikuni, K., Tanabe, R., Muroya, S., Shibata, K., Yamada, J., & Shinmura, Y. (1999). A quick and simple method for the identification of meat species and meat products by PCR assay. Meat Science 1, 143-148.

Hasan Radu, S. (2009). Meat species identification and halal authentication analysis using mitochondrial DNA. Meat Science 83(1), 57–61. Nakyinsige, K., Che Man, Y.B., & Sazili, A.Q. (2012). Halal authenticity issues in mean and meat products. Meat Science 91, 207-214. Okamoto, K. (2016). Development of Porcine Immunochromato. Bioindustry, April 2016, Vol. 33(4), 26-32, CMC Books, Tokyo. (in Japanese). Tanabe, S., Miyauchi, E., Muneshige, A., Mio, K., Sato, C., and Sato, M. (2007). PCR method of detecting pork in foods for verifying allergen labeling and for identifying hidden pork ingredients in processed foods. Bioscience, Biotechnology, and Biochemistry 71(7), 1663–1667.

Murugaiah, C., Noor, Z. M., Mastakim, M., Bilung, L. M., Selamat, J., &

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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: [email protected]

ABSTRACT This article is an overview to depict the potential of bacteriophages as biocontrol agents in controlling the foodborne pathogens for enhancing the food safety. Pathogenic strains of Salmonella spp., Campylobacter spp., E. coli, Listeria spp., Vibrio spp. and many other foodborne bacteria are a significant cause of foodborne illnesses in humans worldwide and especially in the developing world. Antibiotic resistance is increasing in many foodborne pathogens and the development pathway for new antibiotics is time consuming. Thus very few new antibiotics are discovered in recent years. Hence there is an urgent need for alternatives to antibiotics and bacteriophage biocontrol is one of the viable alternatives to antibiotics especially for the multiple drug resistant pathogens. The concept of using bacteriophages as food safety tool is emerging rapidly and they are becoming the logical agents for targeted control of pathogenic foodborne bacteria to overcome the food safety concerns regarding the entry of such pathogens in food chain and thereby affecting the public health. Thus, the bacteriophage biology, their structure, morphology, classification, mode of action, their usage as biocontrol agents to control foodborne bacteria and their advantages are discussed in this paper. Keywords: Antibiotic resistance; Bacteriophages; Biocontrol; Food safety; Foodborne bacterial pathogens.

INTRODUCTION Antimicrobial or antibiotic resistance (AMR/ABR) is an increasing concern world over. The World Health Organization in its Global Surveillance Report on ABR states that “The problem is so serious that it threatens the achievements of modern medicine. A post-antibiotic era - in which common infections and minor injuries can kill – is a very real possibility for the 21st century.” The US President (Whitehouse, 2014) and UK Prime Minister (O’Neill, 2014) have already asked their Governments to prepare a roadmap to tackle this issue. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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


Phage Biocontrol of Foodborne Pathogens Salmonella to the antibiotics has been reported (Fluit, 2005). The multiple drug resistance in zoonotic non typhoidal Salmonella from food animals and food sources is of great concern all over the world due to their entry into human food chain (O’Mahony et al., 2005). Similar antibiotic resistance is noticed in Campylobacter species (Bester and Essack, 2008), E. coli (Diarrassouba et al., 2007 and Saenz et al., 2001) and Listeria species (Li et al., 2007). Drug resistance emerges through excessive usage of antimicrobials in humans and in food animals, which imposes a selection pressure for bacteria that are resistant to antibiotics (Huges and Heritage, 2006). Particularly, in fish farming antibiotics are given to the whole population with inaccurate doses and thus healthy fishes along with sick ones are exposed to antibiotics and thus development of antibiotic resistance is inevitable (Sorum, 2008). In USA about 40% of total antibiotics produced are used on livestock and about 80% of that is used as growth promoters (Hileman, 1999). Thus, considering the threat to public health the European Commission has imposed an EU wide ban on the use of antibiotics as growth promoters in animal feed from January 1, 2006 (European Commission, 2006). The development pathway for new antibiotics is time consuming and very few new antibiotics are discovered in recent years. Hence, there is an urgent need for alternatives to antibiotics and bacteriophage biocontrol is one of the viable alternatives to antibiotics especially for the multiple drug resistant pathogens (Barrow, 2001; Matsuzaki et al., 2014). Lytic phages are responsible for limiting bacterial numbers in an aquatic environment with up to 80 % of mortality evidenced in the bacterial population (Weinbauer, 2004). Phages are strain specific and thus attempting a biocontrol of single strain of bacteria at a ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Bhandare time is advisable and if successful another strain can be tackled. Eventually, a cocktail of phages will give a broad-spectrum activity. Recently, the scientific community has witnessed the sudden surge of interest in bacteriophage research. Bacteriophage therapy is a promising and natural way of reducing Salmonella (Atterbury et al., 2007), Campylobacter (Atterbury et al., 2005) and E. coli (Raya et al., 2006) as a pre harvest treatment. While, post harvest application of phages to reduce Listeria monocytogenes (Leverentz et al., 2003) contamination in later stages of food production is logical. Phages can help to reduce specific bacterial load in food animals through proactive ante mortem interventions rather than reactive end product testing or treatment of patients. They can be applied directly to foods to extend the shelf life and also as hygiene indicators in assessing food quality (Hudson et al., 2005). The isolation and characterization of bacteriophages is uncomplicated and is possible on large scale (Toro et al., 2005). Hankin in 1896 first noticed the bactericidal activity of phages from the Ganges and Jumna river waters in India, which when filtered had antibacterial properties against Vibrio Cholerae. Phage therapy was pioneered by Felix D’Herelle, in early 19th century and his major contributions came from his work in India and he could discover the phages in Ganges, the holy river in India (Summers, 2001). The former Soviet Union has been using phage therapy since 1920s (Chanishvili et al., 2001) but after the discovery of antibiotics in early nineteenth century scientists stopped working with phages until recently. The reappraisal of phage therapy in the Western world was done by H. Williams Smith and his colleagues for oral E. coli infection control in neonatal animals and for systemic 21


Phage Biocontrol of Foodborne Pathogens infection control of E. coli in mice (Smith and Huggins, 1982; Smith and Huggins, 1983). The concept of using bacteriophages as food safety tool is emerging rapidly (Hagens and Offerhaus, 2008) and they are becoming the logical agents for targeted control of pathogenic foodborne bacteria to overcome the food safety concerns regarding the entry of such pathogens in food chain and thereby affecting the public health. There are regulatory concerns over the human phage therapy but their application for food safety has relatively less regulatory concern owing to the presence of phages already in the food environment.

Bhandare Phage structure and morphology They consist of a nucleic acid genome surrounded by a protein coat called as capsid. Many phages contain additional structures such as collar, tails, basal plate and spikes or fibers (Figure 1). Structure of phages may be icosahedrons, spherical shapes consisting of triangular faces, or filamentous or complex structures consisting of icosahedral heads with helical tails. Their genomes can consist of either DNA or RNA, single (ss) or double (ds) stranded, circular or linear (Nicklin et al., 1999).

BACTERIOPHAGE BIOLOGY Bacteriophages (in Greek language “phagein” means ‘to eat’ or ‘to devour’, thus they are bacteria eaters), normally abbreviated to phage are a family of naturally occurring viruses that can be isolated from all those habitats where bacteria can thrive. They are the most abundant in the environment and almost ten bacteriophages are supposed to be there for each bacterial cell (Skrunik and Strauch, 2006). Phages can persist outside the host cells under a great variety of conditions, and usually persist much better than their bacterial hosts under adverse conditions. Most phages are far more resistant to heat, freezing, radiation, chemical disinfection and natural inactivation than their host bacteria (Jofre and Muniesa, 2000). They can infect and kill specific bacteria and do not affect other bacteria or cells, meaning the dysbiosis (imbalance of commensal gut flora) often resulting from the use of broad spectrum antibiotics can be avoided. They are obligate intracellular parasites that are capable of existing as phage particles outside the bacterial cell but can only reproduce inside the cell. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Figure 1: Schematic representation of bacteriophage.

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

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Phage Biocontrol of Foodborne Pathogens Table 1: Classification of bacteriophages as per ICTV (Source: Bhandare, 2015). Order

Caudovirales Myoviridae

dsDNA linear

Genome size (Kb) 31-317

Caudovirales Podoviridae

dsDNA linear

Caudovirales Siphoviridae

dsDNA linear

Unassigned Unassigned Unassigned Unassigned

Family

Genome

Corticoviridae dsDNA circular supercoiled Plasmaviridae dsDNA circular supercoiled Tectiviridae dsDNA linear Inoviridae ssDNA (+) circular

Unassigned

Microviridae

Unassigned

Cystoviridae

Unassigned

Leviviridae

ssDNA (+) circular dsRNA three linear segments ssRNA (+)

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Bhandare

Envelope Morphology

Virion size

No

Icosahedral head with tail

Icosahedral heads: 60-45 nm; Elongated heads: 80-110 nm; Tail: 16-20 × 80-455 nm Icosahedral heads: 60-70 nm; Tail: 10-20 nm Icosahedral heads: 40-80 nm; Tail: 5-10 × 100-210 nm 60 nm

16-78

No

Icosahedral head with short tail

21-134

No

Icosahedral head with long tail

10

No

Icosahedral

12

Yes

15 Inoviruses: 5.812.4 Plectroviruses: 4.58.2 4.4-6.1

No No

Quasi-spherical, pleomorphic Icosahedral Inoviruses: filamentous Plectroviruses: rod shaped

No

Icosahedral

66 nm Inoviruses: 7 × 700-3500 nm; Plectroviruses: 15 × 200400 nm 25-27 nm

6.4-7.1; 3.6-4.7; 2.6-3.2 3.5-4.3

Yes

Spherical

85 nm

No

Icosahedral 23

26 nm

50-125 nm


Phage Biocontrol of Foodborne Pathogens

Bhandare

Figure 2: Schematic representation of major phage groups. (Source: Ackermann, 2006) Phages have binary (consisting of two parts), cubic, helical and pleomorphic symmetries. Most phages have double stranded (ds) DNA, but there are few phages with single stranded (ss) DNA, ssRNA or ds RNA. Few phages contain lipid containing envelope surrounding the capsid (the protein coat covering viruses). The majority (96%) of the phages are tailed phages (binary symmetry), which are classified into the order Caudovirales and three very large phylogenetically related families, while remaining 4% are other types. Taxonomic names of orders, families, and genera are typically constructed from Latin or Greek roots and end in -virales, -viridae and – virus, respectively (Ackermann, 2006). Phage mode of action

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

Figure 3: Replication cycle of Bacteriophage.

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Phage Biocontrol of Foodborne Pathogens cell wall can become unstable and rupture, without replication of the viruses inside, this phenomenon is called ‘passive inundation’ (Atterbury, 2006). The stages in the life cycle of the phages are recognized when virus particles infect cells in culture and are illustrated in Figure 4, which exhibits a one-step growth curve. This graph displays the results of a single round of viral multiplication in a population of cells. Following adsorption, the virus particles undergo uncoating and viral nucleic acid is injected into bacterial cell, thus there is no growth observed and the phenomenon is called eclipse. During the latent period, replication of viral nucleic acid and proteins occurs. The maturation period follows, when virus nucleic acid and protein are assembled in to mature virus particles. At this time, if the cells are prematurely lysed (by the use of chloroform for example), mature virus particles can be detected. Finally, release occurs, either with cell lysis (e.g. lytic phage) or without cell lysis (e.g. temperate phage). The timing of the one-step growth cycle varies with the Eclipse

Bhandare viruses as different viruses have different latent periods and burst sizes. With many bacterial viruses, the whole cycle may be complete in 30-60 min (Madigan et al., 2003). The burst size is the average yield of phage particles per cell, which is very important for determination of inoculum size in phage therapy. Many times small doses are not sufficient and even at large doses some times there is no active replication and therapeutic benefits are obtained by using very large or repeated doses of phage (Payne and Jansen, 2001). Large dosage may cause lysis from without so that bacterial cells are destroyed without any phage replication if the phages are applied at high (> 100) MOI i.e. Multiplicity of Infection, which means number of phage per bacterium. The latent period is the minimum length of time from the adsorption of phage to its host, until the release of newly formed phage particles, which is crucial for phage therapy because the inoculations given in right time taking latent period in to account would be more effective. Rise

Plaque forming units

Latent period

Extracellular phage Burst size

Intracellular phage

Time

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

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Phage Biocontrol of Foodborne Pathogens BACTERIOPHAGES APPLICATION AS BIOCONTROL AGENTS TO CONTROL FOODBORNE BACTERIA There are several reports on successful usage of bacteriophages in controlling the foodborne pathogens and some of the important reports are discussed in this section. Phages can help to reduce Salmonella in poultry through proactive antemortem interventions rather than reactive end product testing or treatment of sick birds. Berchieri, et al. (1991) successfully used bacteriphages to control Salmonella colonization in the poultry gastrointestinal tract. The mortality was reduced from 60% to 3% although large numbers of phages were needed. Their study revealed that the production of large numbers of phages is practicable on farms. It was also noticed that phages spread readily between bacterially infected birds. Similarly, E. coli infection in the poultry can be controlled by using phage therapy. Toro, et al. (2005) stated that there is a beneficial effect of the phage treatment on weight gain performance in chickens along with reduction of Salmonella colonization. Huff, et al. (2006) used aerosol spray of phages to bring down the mortality of birds due to E. coli infection. The mortality was brought down to 7% in phage treated birds while it was 48% in the untreated ones. Campylobacter spp., the celebrity bug in poultry as referred by Butzler (2004) is also being targeted by the phage therapy. Wagenaar et al. (2005); Loc et al. (2005) and Atterbury et al. (2005) have carried out successful phage therapy trials against Campylobacter spp. in poultry. Amongst the phage therapy reports in large food animals Smith and Huggins (1983) showed an effectiveness of phages in treating experimental E. coli diarrhea in calves, piglets and lambs. Infections were

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Bhandare caused by three different E. coli strains in calves, piglets and lambs and a mixture of two phages were given orally to protect them. E. coli infection was reduced to cure the diarrhea in all three species. The phage resistant mutants were emerged in the calves and piglets but they were less virulent than their parent strains. A cocktail of phages significantly reduced the numbers of E. coli O157:H7 in the intestinal tract of sheep (Callway and colleagues, 2008). Even in the fisheries the successful phage therapy is carried out by Karunasagar et al. (2007). They could achieve biocontrol of pathogens in shrimp hatcheries by using bacteriophages (Douglas, 1974). Also, the Japanese researchers (Park et al., 2000; Park and Nakai, 2003) studied the potential use of phages to control the fish infections caused by Lactococcus and Pseudomonas by giving phages orally as phage impregnated feed. Largely it is a win-win situation for the mankind if phage therapy is thoroughly scrutinized for applications in the food animals and then used because it will help to reduce the antibiotic usage to avoid multiple drug resistance along with the reduction in foodborne pathogen load. Rather than applying the phages in live animals before their slaughter the application of phages after slaughter to the carcasses and to the food products during their processing prior to the consumption is one more option to get rid of foodborne pathogens (Thorns, 2000). The significant reductions in the pathogenic bacteria can be achieved by their bacteriophage biocontrol in food processing. In number of studies the poultry products are used for their surface decontamination by phages. Atterbury et al. (2006) achieved a reduction in S. enteritidis and S. typhimurium below the detectable levels by applying phages to the chicken skin. Similarly, Goode and colleagues (2003) could reduce the Campylobacter

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Phage Biocontrol of Foodborne Pathogens jejuni by 1-1.3 log10 CFU within 24 hours by application of phages to the surface of chicken skin. The phage biocontrol of Salmonella upon whole carcasses of broiler chickens and turkeys was achieved by Higgins et al. (2005). Phages have also been used to reduce the numbers of Salmonella from chicken sausages (Whichard et al., 2003). In case of beef the bacteriophage biocontrol has been attempted to extend shelf life by reducing spoilage organisms (Greer and Dilts, 2002). The numbers of E. coli O157:H7 from the beef surfaces were brought down to below the detectable levels by O’Flynn et al. (2004). A 3 log10 CFU reduction in the Listeria counts on salmon was achieved by Hagens and Loessner (2007) after applying high titres of phage. L. monocytogenes, is more likely to occur during food processing, and thus at this point of time phage biocontrol of this pathogen can be done successfully. AntiListeria phages as food additives have been approved by the US FDA by according it a Generally Recognized As Safe (GRAS) status and such products are available in the market for use in food processing. For control of biofilms in the food processing environment the phage application is being envisaged. Roy et al. (1993) and Hibma and colleagues (1997) have showed that the formation of Listeria biofilms in the food processing environments can be reduced by application of phages. A great deal of research is being carried out for non-thermal ways of controlling foodborne pathogens during processing for the want of maintaining nutritive values of food and phage-based non-thermal intervention is an ideal way in such situations.

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Bhandare ADVANTAGES OF USING PHAGES FOR FOOD SAFETY ♦ Phages are ubiquitous in the environment. Every known bacterium in nature is supposed to have its complementary phage and thus it is possible to use phage biocontrol against any type of bacteria. ♦ Bacteriophages are strain specific to an individual bacteria and they don’t harm the other beneficial bacterial flora of the gut as is the case with antibiotics which harm the commensal gut flora. They can also be given in cocktails for broad spectrum effect. ♦ Bacteriophages are self replicating and thus the given dosage can self amplify in due course of the treatment so that they effectively tackle the target bacteria. Also, they will only replicate till the target bacterium is present and thus they are naturally self limiting. ♦ As there is resistance development in bacteria for antibiotics, the phage resistance may also develop but it has been reported (Loc Carrillo et al., 2005; Atterbury et al., 2007 and Smith and Huggins, 1983) that such bacteria would have low virulence or less survival rate owing to the ‘fitness penalty’. Also, the resistance to phage does not affect their sensitivity to antibiotics and the relevant phages naturally evolve alongside as bacteria evolve resistance. ♦ There are no reports of allergic reactions to phage as in antibiotics and no serious side effects have been described so far either in animals or humans. This may be due to the abundance of phages in our environment and regular exposure of animals and humans to them.

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Phage Biocontrol of Foodborne Pathogens ♦ It is relatively cheaper and easier to produce phage preparations especially as topical applications for surface decontamination of foods. ♦ As the human phage therapy is fraught with the limitations of lack of public confidence and more regulatory concerns, the application of phages as biocontrol agents in food animals and in food processing is an ideal way of harnessing the potential of this nature’s weapon for food safety. Though not totally eliminated, the foodborne pathogens can be reduced to the acceptable levels by application of bacteriophages as pre-harvest and post-harvest interventions. Phages can be effectively used along with other food safety tools to protect public health. REFERENCES Ackermann, H.W. (2006). ‘Classification of bacteriophages’, In: Calendar, R. (Ed). The Bacteriophages. Oxford University Press, New York. pp. 816. Atterbury, R. J., Van Bergen, M.A., Ortiz, F., Lovell, M.A., Harris, J.A., De Boer, A., Wagenaar, J.A., Allen, V.M., and Barrow, P.A. (2007). ‘Bacteriophage therapy to reduce Salmonella colonization of broiler chickens’, Applied and Environmental Microbiology 73, 4543. Atterbury, R.J. (2006). The age of phage? Poultry International 6, 18-22. Atterbury, R.J., Dillon, E., Swift, C., Connerton, P.L., Frost, J.A., Dodd, C.E., Rees, C.E. and Connerton,

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Bhandare I.F. (2005). ‘Correlation of Campylobacter bacteriophage with reduced presence of hosts in broiler chicken ceca’, Applied and Environmental Microbiology 71, 4885-7. Atterbury, R.J., Van Bergen, M.A., Ortiz, F., Lovell, M., Harris, J.A. and De Boer, A. (2006). ‘Control of Salmonella in poultry using bacteriophage’, In Proceedings of the 13th International Symposium Salmonella Salmonellosis. Colin, P., and Clement, G. (eds). Saint Malo, France: 10–12 May, pp. 579–580. Barrow, P. A. (2001). "The use of bacteriophages for treatment and prevention of bacterial disease in animals and animal models of human infection." Journal of Chemical Technology and Biotechnology 76(7), 677-682. Berchieri, A. Jr, Lovell, M.A. and Barrow, P.A. (1991). ‘The activity in the chicken alimentary tract of bacteriophages lytic for Salmonella typhimurium’, Research in Microbiology 142, 541-549. Bester, L.A. and Essack, S.Y. (2008). ‘Prevalence of antibiotic resistance in Campylobacter isolates from commercial poultry suppliers in KwaZulu-Natal, South Africa’, Journal of Antimicrobial Chemotherapy 62(6), 1298-1300. Bhandare, S. G. (2015). Biocontrol of V. cholerae using bacteriophage. PhD, Thesis. University of Nottingham, UK.

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Phage Biocontrol of Foodborne Pathogens Butzler, J.P. (2004). ‘Campylobacter, from obscurity to celebrity’, Clinical Microbiology & Infection 10, 868. Callaway, T.R., Edrington, T.S., Brabban, A.D., Anderson, R.C., Rossman, M.L. and Engler, M.J. (2008). ‘Bacteriophage isolated from feedlot cattle can reduce Escherichia coli O157:H7 populations in ruminant gastrointestinal tracts’, Foodborne Pathogens and Disease 5, 183–191. Chanishvili, N., Chanishvili, T., Tediashvili, M. and Barrow, P.A. (2001). ‘Phages and their application against drug-resistant bacteria’, Journal of Chemical Technology and Biotechnology 76, 689–699. Diarrassouba, F., Diarra, M.S., Bach, S., Delaquis, P., Pritchard, J., Topp, E. and Skura, B.J. (2007). ‘Antibiotic resistance and virulence genes in commensal Escherichia coli and Salmonella isolates from commercial broiler chicken farms’, Journal of Food Protection 70(6), 1316-27. Douglas, J. (1974). Bacteriophages. Chapman and Hall, London. pp. 2148. European Commission (2006). Ban on antibiotics as growth promoters in animal feed enters into effect. Press release of 22/12/2005. http://europa.eu/rapid/pressrelease_IP-05-1687_en.htm (accessed on 10/5/2016). Fluit, A.C. (2005). Towards more virulent and antibiotic-resistant Salmonella?

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Bhandare FEMS Immunology & Microbiology 43, 1-11.

Medical

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Res. Highl. 4Bs (2016), P33-41

Cloning and Expression of the Urease Operon from Helicobacter pylori J99 Mohamad CWSR.1, 2, *, Abdul-Manaf U.2 and Mat-Arip Y.2 1

Biomedical Electronic Engineering, School of Mechatronic Engineering, Pauh Putra Main Campus, Universiti Malaysia Perlis, Arau, 02600 Perlis, Malaysia; 2 School of Biology Science, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia; *corresponding author, e-mail: [email protected]

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 Helicobacter pylori is one of the common bacterial infections in human and recognized as the etiologic agent for majority of upper gastro duodenal diseases. H. pylori has been

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established as the causative agent for acute or chronic gastritis (Mitchell et al., 1999) and could be further developed into peptic ulcer disease, gastric carcinoma and others upper gastro duodenal diseases (Kiesslich et al., 2005; Ardekani et al., 2013). According

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Cloning and Expression of the Urease to the World Health Organization (WHO) statistic, H. pylori infection is on the rise and proportional to the progress of a country. Almost 50% of the world's population is infected by H. pylori (Sasidharan et al., 2008). In Malaysia, H. pylori infection is on the raise as well. From the year 2000 until 2007, patients infected by H. pylori were 30.4% of the gastro duodenal cases reported (Sasidharan et al., 2008). Urease is one of the pathogenic factors that help H. pylori colonizes the epithelium in the acidic environment of the stomach (Ardekani et al., 2013). H. pylori urease displays enzyme-independent effects in mammalian models, mostly through lipoxygenases-mediated pathway (Uberti et al., 2013). The urease would induce edema, neutrophil chemotaxis and shows apoptosis inhibition reverted in the presence of the lipoxygenase inhibitors esculetin (Uberti et al., 2013). In addition to its involvement in the pathogenesis process of H. pylori infection, urease is also a target for vaccination development (Voland et al., 2006) besides being a suitable marker to use as a target protein for detecting presence of H. pylori infection among suspected gastrointestinal patient. In this study, urease operon were isolate and examined by colony PCR, restriction enzyme digestion and nested PCR with specific primer to confirm orientation of urease operon was done before continue to protein expression. The sodium dodecyl sulphate polyacrylamide (SDS-PAGE) gel was performed to examine the immune functioning assay of recombinant urease operon of Helicobacter pylori J99 and protein sequencing to make sure this protein was functioning.

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Mohamad et al. MATERIALS AND METHODS Escherichia coli growth and maintenance The E. coli strains (Merck Inc.) used in this study and their genotypes are shown in Table 1. Escherichia coli strains were grown on LB broth at 37°C for 16 hours in a shaker incubator (Shel Lab, UK). For storage purposes, E. coli strains were stored in LB broth containing 50% glycerol (50%), then, kept at -80oC for long term storage. Table 1: Genotypes of E. coli strains. Strain Genotype Nova Blue endA1 hsdR17(rK12– mK12+) supE44 thi-1 recA1gyrA96 relA1 lac [F’ proA+B+ lacIqZΔ M15 ::Tn10] BL21 (DE3) F– ompT hsdSB (rB– mB–) gal dcm (DE3) Helicobacter pylori J99 growth and maintenance Helicobacter pylori J99 (ATCC 700824) was grown on Eugon agar with 10% human expired blood at 37°C, 10% CO2 and 100% humidity in an incubator (Shel Lab, UK). Subcultured was performed every four to seven days to maintain fresh bacterium. For storage purposes, H. pylori J99 strain was stored in TSB containing 20% glycerol (20%), then, kept at -20oC for short term storage and -80oC for long term storage. Plasmid cloning vector The plasmid cloning vector used in this study was pET-32-Ek/LIC (Merck, Germany). This vector contains 109aa Trx•TagTM which encodes for the 109 amino acid of thioredoxin protein. The pET-32Ek/LIC vector was specially designed for cloning and high-level expression of target protein. Thioredoxin protein and histidine tag would be fused to the target protein to enhance purification of the expressed proteins. 34


Cloning and Expression of the Urease Genomic extraction of H. pylori J99 Genomic of H. pylori was extracted by employing GENEAll ExGene Cell SV kit (Intron, Korea). The extracted genomic DNA was then stored at -20oC for further analysis. This genomic extraction of H. pylori J99 was used as a template for amplification of urease gene. Primer pair (pET32UOHP-F & pET32UOHP-R) would amplify the urease operon. The specific PCR primers for urease operon with pET-32 Ek/LIC complimentary overhangs are shown in Table 2. Table 2: Specific PCR primers sequence with vector-compatible overhang. Primer Sequences pET32UOHP-F 5’ GAC GAC GAC AAG ATG AAA CTC ACC CCA AAA GAG 3’ pET32UOHP-R 5’ GA GGA GAA GCC CGG TTC AAA CCT TTT GCG TGG TGG 3’ Amplification of urease gene The total volume for PCR reaction was 25 µl [1x PCR buffer, 0.1 mM dNTPs (NHK Inc), 0.4 pmol of each pET32UOHP-F, pET32UOHP-R primers, 1 unit of i-Taq DNA polymerase (NHK Inc) and 50 to 100 ng of H. pylori J99 DNA template]. Gradient temperatures for amplification of urease operon were from 64oC to 74oC by using gradient PCR thermal cycler (Biometra, USA). PCR cycler was programmed for 30 cycles with 95oC for 1 minute, 57oC for 2 minutes for full length urease operon and 72oC for 1 minute. The final elongation was set at 72oC for 10 minutes. Negative control was included consisting of all mixture except the DNA template. After PCR ended, 1 µl of ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Mohamad et al. the PCR product was analyzed on 1% agarose gel then stained with ethidium bromide (EtBr) and 1Kb DNA ladder (Promega, USA) was used as a marker. Preparations of pET32 Ek/LIC insert The purification PCR product of H. pylori urease operon by employing PCR DNA Extraction System (Intron, Korea). After that, the annealing procedure between purified PCR products (Ek/LIC insert) with pET32 Ek/LIC vector was follow according to manufacturing protocol (Merck Inc.). Transform recombinant into cloning host Three microliter (3 µl) of amplification product was transferred into a new 1.5 ml Eppendorf tube which had been cooled on ice and the remaining annealing product was stored at -20°C. An uncut plasmid (0.1 ng) was used as a control for transformation efficiency of the competent cells. Escherichia coli NovaBlue competent cells were thaw on ice before transformation procedure. Transformation involved mixing 50 µl of the competent cell with 2 µl of annealing product and mixed gently. After that, the mixture was incubated on ice for 5 minutes, then, heat-shocked for 30 seconds at 42°C. Immediately after heat-shock, the tube was placed on ice for 2 minutes. Next, 80 µl of LB was added and the mixture was incubated at 37°C, shaker at 250 rpm for 60 minutes. The mixture was spread onto LB agar plate containing ampicillin (100µg/mL). The plates were incubated overnight (16 - 20 hours) at 37°C. Screening of recombinant urease operon Colonies that formed had the possibilities of carrying the insert. Thus, the colonies were screened for inserts by performing colony PCR using specific primers as in Table 3. The potential clones were also subjected to restriction enzyme digestion to confirm the 35


Cloning and Expression of the Urease presence of the inserts. The used of restriction enzymes either as single (Sal1) or double digestions (EcoR1/ Pst1 and EcoRV/Bgl II) followed the manufacturer protocol (Promega, Inc.). The orientation of the urease gene was determined so that it was in the correct reading frame by nested PCR. The specific primers cws1, cws2 and cws3 were used to confirm the target insert as shown in Table 3. Finally, the clones were verified using DNA sequencing which was carried out by a service provider, NHK Bioscience Solution Sdn Bhd. The selected recombinant plasmid was named as pET32UOA6 for urease operon recombinant for expression urease UreA/UreB. Table 3: Specific nested PCR primers used for insert confirmation. Gene Sequences cws-1-F 5-CGT TAT GTC CTT AAG GAA AAA AC-3’ cws-1-R 5-CCC ATG AGC GAT CGC TGG GTT AAT GG-3’ cws-2-F 5’- CCA TTA ACC CAG CGA TCG CTC ATG GG -3’ cws-2-R 5’- CTA TGG GGC ATG CTT ACG GTT AAG -3’ cws-3-F 5-CAA AGC TGA ATT CCA ACG ATC GCT TAA CCG TAA GCA TGC C-3’ cws-3-R 5-GGT TAA AAA GAC TCG AGG GTT TTT TAA TC-3’ Transformation of pET32UOA6 into expression host The recombinant plasmids were sub-cloned into an expression host, E. coli BL21 (DE3) following the manufacturer protocol (Promega Inc.) Expression of urease operon A single colony was inoculated into a universal bottle containing 5 ml of LB broth ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Mohamad et al. with ampicillin (100µg/mL) and incubated at 250 rpm, 37oC for 3 to 4 hours until OD600 reached 0.4 to 1.0. Next, 2 ml of this culture was aliquot into Mc Courtney bottles and kept at 4oC overnight. The next day, 2 ml pre-culture from 4oC was centrifuged at 13 000xg for 15 seconds, the supernatant was discarded and the cell pellet was suspended with 2 ml LB broth with ampicillin (100µg/mL). The suspended cell with fresh media then inoculated into a 250 ml flask containing 50 ml LB broth with ampicillin (100µg/mL). The cells were grown at 37oC, 250 rpm until OD600 reached approximately 0.6 (~3 to 4 hours). One ml of this culture was removed as un-induced sample for SDS-PAGE analysis. Cells were induced with 0.4mM IPTG at 37oC, 250 rpm for 3 hours. Subsequently, the culture was incubated on ice for 10 minutes and harvested by centrifugation at 5000 x g for 20 minutes at 4oC. The cell pellet was washed with 0.25 culture volume of cold 20 mM Tris-HCl pH 8.0, then centrifuged at 5000 x g for 20 minutes at 4oC. The supernatant were discarded and the cells pellet was kept at 80oC for further analysis. The expression of urease protein was detected by Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDSPAGE). The preparation and the assembly of SDS-PAGE electrophoresis was carried out according to the manufacturer protocol (Bio-Rad, USA). Briefly, the resolving gel was at 12.5% with standard 4% stacking gel. Cell pellets were suspended with PBS before 5X SDS-PAGE sample buffers at the ratio 3:1 was added. Next, the sample was heated at 95oC for 5 minutes. After heated, this sample became viscous and 29cc gouge needle was used to reduce the viscosities of the sample. Twenty microliter of sample 36


Cloning and Expression of the Urease was loaded per lane on the SDS-PAGE gels. The SDS-PAGE running buffer was filled into the bottom and upper chambers and all the bubble were removed from the wells of the gels using a syringe. The gel was run at 80V constant current for 35 minutes and 100V for 2 hours until the stacking dye had reached the end of the gel. After that, the gel was carefully removed from the glass plates and stained with Coomassie Blue stain for 1 hour and destained with destining solution overnight (~16 hours). The expression of constructed recombinant urease operon was important to measure the urease expression level and to know the biological active of the recombinant urease. Immune functioning assay The immune functioning assay was used to verify the recombinant protein of pET32UOA6. The electrophoresis transfer of proteins to 0.45 µm Polyvinylidene fluoride (PVDF) membrane (Millipore Inc.) was accomplished by a modification of the method described by Towbin et al. (1979). Protein was electro blotted from the gel onto PVDF membrane by electrophoresis transfer using a Mini Trans-bolt® Electrophoretic Transfer Cell (Bio-Rad, USA). Lastly, the membrane was put in a plastic wrap and the image was captured within 1 minute using Gene Genius Bio Imaging System. Protein sequencing Recombinant urease clones were grown, induced with 0.4 mM IPTG, followed by SDS-PAGE as described in Section 3.6.9. After SDS-PAGE, the band which represented the recombinant UreA and UreB was purified and sent to 1st BASE, Inc. for protein sequencing. Finally, the amino acid sequence resulted from protein sequencing was aligned using Protein Basic Local Alignment Search Tool (NCBI BLASTp) of National Center for Biotechnology Information (Stephen et al., 1997) and this ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Mohamad et al. protein sequencing was carried out to ensure the expressed recombinant urease was identical to H. pylori J99 urease protein. RESULTS AND DISCUSSION Determine of recombinant urease operon The urease operon was determined by their size based on urease gene sequence information, accessed from NCBI database (Accession No. NC_000921.1). Complete urease operon that encode for urease enzymes and accessory proteins. As shown in Figure 1, the presence of the urease operon PCR amplified was detected with the expected size of 5974 bp.

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


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

5865 & 6026

Sal I

11891

Figure 2: Restriction enzyme analysis of potential recombinant clones. The restriction enzyme digestion of urease operon fragment. Lane 1: 1 Kb DNA ladder; Lane 2 and 6: undigested plasmids; Lane 3 and 7: EcoRI & Pst I digestion; Lane 4 and 8: EcoRV & Bgl II digestion; Lane 5 and 9: Sal I digestion and Lane 10: λ Hind III DNA ladder. DNA Sequencing ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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


Cloning and Expression of the Urease the presence of additional amino acids fused to protein of interest could change the protein conformation. Therefore, it was essential to determine whether the expressed recombinant UreA and UreB still maintained their immune properties.

Figure 3: SDS-PAGE analysis for expression of recombinant urease protein. The expression profiles for (A) pET32UOA6 (B) pET32ureA3 and (C) pET32ureB2. Lane 1: DGelTM Marker; Lane 2: Pre-culture urease gene; Lane 3 and 6: uninduced culture; Lane 4: Induced culture with 0.4 mM IPTG at 2 hours; Lane7: Induced culture with 0.4 mM IPTG at 3 hours; Lane 5: Induced culture with 1.0 mM IPTG at 2 hours and Lane 8: Induced culture with 1.0 mM IPTG at 3 hours. Figure 4 shows immune functioning of UreA and UreB from cell crude of recombinant urease clones compared with other bacterial crude cells, as negative controls and H. pylori J99 crude cell as the positive control. Helicobacter pylori ureaseα and urease-β antibodies (Santa Cruz, Inc.) were very specific and sensitive to UreA and UreB of H. pylori J99 crude cell (lanes 8 and 18). Similar specificity and sensitivity were observed for recombinant UreA and UreB, expressed in full operon unit (lanes 2-3 and 12-13). As suggested by the manufacturer, crude cells from Salmonella (lanes 4 & 14), Pseudomonas (lanes 7 & 17) and E. coli (lanes 9 & 19) gave negative results that indicate H. pylori urease-α and urease-β antibodies were very specific and sensitive to the native, as well as, the recombinant ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Mohamad et al. ureases. These results confirmed the recombinant UreA and UreB maintained their immune properties.

Figure 4: Western blot analysis for determination of recombinant ureases immune functioning in of UreA (A) and UreB crude cell (B). Lane 1, 10, 11 and 20: Kaleidoscope Prestained protein ladder; Lane 2-3 and 12-13: pET32UOA6 cell crude; Lane 5-6: pET32ureA6 cell crude; Lane 15-16: pET32ureB2 cell crude; Lane 4 and 14: Salmonella cell crude; Lane 7 and 17: Pseudomonas cell crude; Lane 8 and 18: H. pylori J99 cell crude; Lane 9 and 19: E. coli cell crude. Immune functioning assay verified the recombinant UreA and recombinant UreB still maintained their antigenicity, equivalent to native enzyme regardless of the presence of additional amino acids fused to them. These were supported by immune functioning assay through Western blotting and previous work by Hu et al. (1992). Purification of both recombinant ureases did not affect the antigenicity, as evidence by Western blotting (Figure 4). Regardless of this condition, both recombinant ureases still maintained their antigenicity in immune functioning assay. Purification process failed to separate UreA from UreB since H. pylori urease-α and urease-β antibodies (Santa Cruz, Inc, USA) still detecting both 39


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Mohamad et al.

of them in the immune functioning assay. Thus, clone pET32UO expressed and assembled the urease apoenzyme and perhaps together with the accessory proteins to form the holoenzyme.

This study was supported by the Universiti Malaysia Perlis for Academic Training Scheme Funding (SLAI).

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

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

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

Figure 5: The percentage identity of recombinant ureases against H. pylori J99 ureases, pET32UOA6. CONCLUSION In this study, the availability of a functional replicon carries urease operon has potential benefit related to H. pylori pathogenesis. A through and better understanding of H. pylori pathogenesis process could contribute to the improvement of diagnostic methods. ACKNOWLEDGEMENT

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REFERENCES

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

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Cloning and Expression of the Urease Medicine and Hygiene 102, 12261232. Stephen, F., Altschul, Thomas, L., Madden, Alejandro, A., Schäffer, Jinghui Zhang, Zheng Zhang, Webb, M. and David, J. L. (1997), Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389-3402. Towbin, H., Staehelin, T. and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences 76(9), 4350-4354.

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Mohamad et al. Uberti, A. F., Olivera-Severo, D., Wassermann, G. E., ScopelGuerra, A., Moraes, J. A., Barcellos-de-Souza, P. and Carlini, C. R. (2013). Pro-inflammatory properties and neutrophil activation by Helicobacter pylori urease. Toxicon 69, 240-249. Voland, P., Zeitner, M., Hafsi, N. and Prinz, C. (2006). Human immune response towards recombinant Helicobacter pylori urease and cellular fractions. Vaccine 24(18), 3832-3839. Zheng, Z., Scott, S., Lukas, W. and Webb, M. (2000). A greedy algorithm for aligning DNA sequences. Journal of Computer Biology 7(1-2), 203-214.

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Res. Highl. 4Bs (2016), P42-56

Production of Butter Flavour Concentrate from Butter fat with Lactic Acid Bacteria by Solid Substrate Fermentation Nadaraj Sivan1, Thambirajah, J. J.2 and Guruswamy Prabhakaran*1 1

Department of Biotechnology, AIMST University, Bedong 08100, Kedah Darul Aman, Malaysia; 2Faculty of Business Management, AIMST University, Bedong 08100, Kedah Darul Aman, Malaysia; *corresponding author, e-mail: [email protected]

ABSTRACT Aim: The aim of this study was to investigate the fermentation of butter fat with different lactic acid bacterial strains by solid substrate fermentation (SSF) for the production of butter flavour concentrates. Methodology and results: Lactic acid bacteria (LAB) were isolated from dairy products and environmental samples using Mann Rogosa and Sharpe (MRS) agar. These, together with two reference ATCC lactic acid bacterial strains were evaluated for the production of flavor components which were determined by GC-MS. The SSF was found to be effective in producing sweet and buttery notes within 24 hours of fermentation. Scale up studies with 120 g of butter fat supplemented with 10% galactose enhanced butter flavour production. Butter oil recovered from the fermented samples was subjected to sensory evaluation by 120 volunteers. The butter flavour compounds in butter oil samples were quantitatively analyzed in GC-MS. The untreated butter fat recorded the lowest concentration of diacetyl (211.5 ppm) and acetoin (161.7 ppm) whereas, butter fat supplemented with galactose and fermented showed a significant increase in concentration of acetoin (1321.2 ppm) and diacetyl (511.4 ppm). The formulation of butter powder with maltodextrin was investigated. Conclusion, significance and impact of study: The results obtained from this study will pave the future investigations for development of a microbial process for production of butter flavour concentrate from butter fat. Keywords: Acetoin; Butter fat; Diacetyl; Butter flavor; GC-MS; Lactic acid bacteria; Solid substrate fermentation.

INTRODUCTION Flavour is a combination of taste and aroma. It results from the perception of odor-active volatile compounds. Food flavours are mixtures of natural and/or artificial aromatic compounds. They are designed to impart, modify, or even mask an undesirable flavour. Flavours along with fragrances are highly prized in the global market. Currently there are three known methods of acquiring flavour compounds (Bicas et al., 2010). These include, extraction from pre-existing

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

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Microbial Production of Butter Flavour Currently, diacetyl and acetoin, the flavour compounds are chemically synthesised at commercial scale. The inhalation of intense synthetic butter flavour at the source of manufacturing and application results in bronchiolitis obliterans, commonly termed as ‘popcorn lung’ disease (Morris and Hubbs, 2009). The increasing demand for natural butter flavour has lead to the development of biotechnological processes. However, the fermentation processes which involve lactic acid bacteria in obtaining high yields of desirable enantiomeric butter flavour compounds from butter fat are yet to be established. The development of solid state fermentation techniques for the bioconversion of butter fat and the recovery of butter flavour compounds are important challenges for researchers in this field. Hence, this study was undertaken to optimize the fermentation conditions for the production of butter flavour concentrate from butter fat by lactic acid bacteria in solid substrate fermentation.

Nadaraj et al. MATERIALS AND METHODS The process flowchart in Figure 1 highlights the major stages in the production of butter flavour concentrate. Isolation of Lactic Acid Bacteria (LAB) from various samples Raw milk was purchased from a local market. Soured milk was prepared by allowing the raw milk to sour for a day. Pasteurized milk (Marigold, Dutch Lady), cheese (Emborg, Kraft), unsalted butter (Devondale, Tatura), salted butter (Devondale, Anchor), cultured drink (Solivite, Nutrigen), yoghurt (Dutch Lady) were purchased from TESCO supermarket in Alor Setar, Kedah Darul Aman, Malaysia. Soil, grass and water samples were collected from a nearby cattle farm. Lactic Acid Bacteria (LAB) were isolated from these samples as per the method (Bettache et al., 2012). The isolates were stored in agar slants at 4°C. The individual isolates were tested for their ability to ferment butter to produce flavour concentrates.

Figure 1: Flowchart for the production of butter flavour concentrate. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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

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

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

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

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

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


Microbial Production of Butter Flavour selected lactic acid bacteria and incubated at 37°C. One petri dish was designated as control. Sensory evaluation of samples was performed at fixed time intervals (24th, 48th and 72th hour) as reported previously. Scale up studies of Solid Substrate Fermentation (SSF) Scales up studies were performed with increased quantities of butter fat. In order to increase the surface area for solid substrate fermentation, experiments were carried out with various amounts of butter fat taken in conical flasks (30 g in 250 ml conical flask, 60 g in 500 ml conical flask and 160 g in 1000 ml conical flask). Overnight cultures were prepared individually and the volume of overnight inoculum used was 10 ml for 30 g, 20 ml for 60 g and 40 ml for 160 g substrates, respectively. These cultures were incubated at 37ºC under anoxic conditions. At specified time intervals of 24th, 48th and 72th hour, samples were acquired for sensory evaluation by 10 random volunteers. Scale up studies of Solid Substrate Fermentation (SSF) with 10% galactose Scale up studies was carried out with 30 g, 60 g and 160 g of butter fat, supplemented with 10% of galactose individually. The flasks were sterilized and inoculated with overnight inoculum respectively. They were incubated and samples were drawn and tested as previously. Sensory evaluation of butter oil samples from different fermented butter samples Sensory evaluation of butter oil samples that were obtained from the fermented butter samples were performed as per the methods by Anvoh et al. (2009). A 5-point hedonic scale was utilized where 1 represents dislike the most, 3 represents neither like nor dislike and 5 represents like the most. Sensory scores were evaluated based on preferences of odour by 120 students of Biotechnology from the Faculty of Applied Sciences, AIMST ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Nadaraj et al. University. Prior to sampling, volunteers were first requested of their health status and free of any respiratory illnesses. During inhalation of aroma, volunteers were required to inhale the samples and suspend breathing for 2-3 seconds and mark their preferences in the flavour survey form provided. Coffee powder was provided as a neutralizer to eliminate traces of previous aromas. Samples indicating high preferences were selected for further analysis by GC-MS (Gas chromatography-mass spectrometry). GC-MS analysis of butter oil for acetoin, diacetyl and fatty acid Volatile compounds such as acetoin and diacetyl are generally analysed by GC-MS (Gokce et al., 2014). Extraction of diacetyl and acetoin from the treated butter oil was performed. Chromatographic analyses of the treated and control butter samples for aroma compounds and fatty acid components were performed using a split less injector system gas chromatograph coupled with a mass spectrometer (SHIMADZU GCMS-QP2010). The carrier gas used was ultra-pure helium with a flow rate of 1.0 mL/min. The injection port was worked at 250°C in split less mode coupled with 1 minute split less time. A 1 µl injection volume was applied for each sample analysis and the syringe was washed with hexane upon completion of injection. Separation was performed using a DB-WAX (60 m x 0.25 mm x 0.15 µm) capillary column with a 0.15 µm stationary film. The oven temperature programme was set as follows: initial temperature 40°C, increased by 7°C min-1 to 200°C and held for 1 minute. Mass spectrometric parameters were set as follows: electron impact ionization with 70 eV energy and 250°C ion source. The aromatic compounds and fatty acid profiles were detected and quantified based on their retention time and peak areas on the chromatogram respectively.

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Microbial Production of Butter Flavour Butter powder formulation with maltodextrin Maltodextrin was used as a carrier material, as it is edible, slightly sweet, and low in cost (Wandrey et al., 2010). For the production of butter oil powder, the extracted butter oil was mixed with maltodextrin with different anti-caking agents and the entire mixture was then treated with liquid nitrogen and mixed evenly using a mixer. The addition of liquid nitrogen was to imitate freeze drying conditions. Formulation of butter powder from carrier material (maltodextrin) and active material (butter oil) was performed individually by using 50 g of maltodextrin powder and varying amounts of butter oil (5 g, 10 g, 15 g, 20 g, 25 g and 30 g). The maltodextrin powder was added into a plastic container, and butter oil of the concerned weight was poured into the container. Simultaneously, the mixture was added with liquid nitrogen (approximately 100 ml) and manually stirred. The prepared powder was then stored in air-tight glass containers and kept at room temperature away from any light source. Anti-caking agents are required in the formulation of powdered food or drug preparations to either stop or postpone occurrences of caking (Lipasek et al., 2011). For this, 50 g of the prepared butter powder was placed in a plastic container and supplemented individually with 2%, 4% and 6% (w/w) anti-caking agent that comprised sodium chloride, sodium silicate, sodium bicarbonate, bentonite, mannitol and potato starch. The mixture was manually stirred with intermittent shaking to enable even dispersion of the anti-caking agent and the powder. The prepared powder was then stored in a glass container that was sealed and stored in a dry area. RESULTS AND DISCUSSION Over the years lactic acid bacteria (LAB) 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

Nadaraj et al. LAB are lactic acid production which results in the improvement of flavour, aroma, keeping quality and enhancement in nutritional content (Halász, 2009). Butter is an important flavouring additive and creates the distinctive aroma and taste in bakery, dairy and other food products. Diacetyl and acetoin are two important constituents in butter fat that contribute to unique butter flavour. Natural butter flavour is expensive, hence artificial butter flavour compounds are chemically synthesized from petroleum-derived precursors. Synthetic flavours are produced in bulk at low cost and it is a mixture of racemic compounds. The prolonged exposure to synthetic flavour compounds to the line workers was reported harmful by National Institute for Occupational Safety and Health (NIOSH) (Kreiss, 2007). Biotechnological approaches in producing butter flavour compounds are being investigated as a replacement to chemicalbased processes (Longo and Sanromán, 2006). This study was aimed at producing butter flavour concentrate from butterfat by a microbial process. The investigation included isolating LAB strains as well as Lactobacillus acidophilus (ATCC 314) and Lactobacillus casei (ATCC 393) in the fermentation of butter fat by solid substrate fermentation (SSF). After fermentation, removal of solids by centrifugation and recovery of butter oil from the treated butter was performed. The butter oil was then subjected to sensory evaluation and analysed for butter flavour compounds by GC-MS. Isolation of LAB from various samples Raw milk, soured milk, cheese, unsalted butter, salted butter, yoghurt and grass, soil, and water samples from cattle grazing areas were niches of LAB strains. A total of ten colonies were isolated from the different samples.

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Microbial Production of Butter Flavour Pasteurization and sterilization of butter fat Two different methods namely pasteurization and sterilization of butter fat were evaluated. Compared to pasteurization (63ºC±5ºC for 30 minutes), the sterilization of butter fat samples was better. Hence, sterilization was used subsequently. Recovery of butter oil Butter oil was recovered for sensory evaluation, detection and quantification of diacetyl, acetoin and fatty acids in the fermented butter samples which constituted the major butter flavour compounds. Three different methods were adopted for extraction of butter oil from 6 g of butter fat (Figure 2). Percentage recovery of butter oil by different methods On comparative analysis, it was found that the percentage recovery of oil was high (69.67%), when the butter fat was melted at 50 °C for 8 minutes and centrifuged at 3,700 rpm for 12 minutes (Table 2). The oil recovery was recorded low in the other two methods. However, the clarity of butter oil was found to be good in the second method when compared to methods 1 and 3. This may be attributed to high centrifugal forces for efficient separation of denser materials from lighter counterparts (Anlauf, 2007). Solid Substrate Fermentation (SSF) of butter fat in glass petri dishes (Figure 3) and sensory evaluation After 24 hours of incubation, there was a significant improvement in the flavour content in the fermented samples with CFS and CFG strains individually (Table 3). Whereas, with prolonged incubation the off flavour production was recorded. Butter flavour production is based on multi-step reactions which involve lipolytic, proteolytic and glycolytic reactions (Smit et al., 2005). It was ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Nadaraj et al. reported that diacetyl may also contribute to the formation of undesirable, offflavours as observed in spirits manufacture (Krogerus and Gibson, 2013). The fermented butter fat samples were subjected to sensory evaluation by ten volunteers.

Figure 2: Picture showing the recovery of butter oil by using three methods. Table 2: Percentage recovery of butter oil by different methods. Butter oil recovery (g) M* Trial Trial Trial Average Recovery % 1 2 3 1 4.14 4.11 4.12 4.12 68.67 2 4.17 4.19 4.18 4.18 69.67 3 3.79 3.81 3.77 3.79 63.17 Values represent the mean of three replicates; *Method 1: Melting of butter fat at 40 ºC (±5ºC) for 10 minutes and centrifugation at 800 rpm for 3 minutes (Chongcharoenyanon et al., 2012); Method 2: Melting of butter fat at 50 ºC (±5ºC) for 8 minutes and centrifugation at 3,700 rpm for 12 minutes (Krause et al., 2008); Method 3: Melting of butter fat at 70 ºC (±5ºC) for 15 minutes and centrifugation at 3,500 rpm for 5 minutes (Miura et al., 2004). Sensory evaluation of SSF (Solid Substrate Fermentation) samples with various concentrations of butter fat The fermented butter fat samples were recorded with preferred sweet and buttery flavour after 24 hours of incubation with both the isolated strains (CFS and CFG) in Table 4. The increase in butter fat quantity 47



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Figure 3: Solid substrate fermentation of butter fat in glass petri dishes; CFS: bacterial strain isolated from cattle field soil sample.CFG: bacterial strain isolated from cattle field grass sample. (esters, methylketones, lactones, etc.) formation (Smit et al., 2005). Among the 2 Table 3: Sensory evaluation of SSF (Solid strains tested with 160 g butter fat, CFS Substrate Fermentation) fermented butter strain was recorded with increased flavour samples§. production. Solid Sensory evaluation Supplementation of butter fat with substrate CFS CFG various carbohydrate sources fermentation strain strain Lactic acid bacteria are able to ferment (SSF) various hexose sugars, from simple (hour) (galactose) to complex (lactose) sugars but 24 Buttery Sweet is species-dependant (Halász, 2009). 48 Sour, Sour, Galactose moieties present with lactose alcoholic alcoholic molecules in milk when metabolized, may 72 Stale, Stale, lead to intense aroma production odourless odourless § (Hugenholtz et al., 2002). In order to Scores based on the aroma description by determine if addition of lactose, skim milk 10 volunteers; CFS: bacterial strain and galactose would enhance the butter isolated from cattle field soil sample; flavour production, solid substrate CFG: bacterial strain isolated from cattle fermentation was carried with both the field grass sample. strains. The butter fat (6 g) was supplemented with 10 % (w/w) substrate in the medium may have facilitated the individually and fermented with CFS and volunteers in scoring more for sweet and CFG strains. Table 5 summarizes sensory buttery flavours. The increased amounts of evaluation results of the experiment by raw material (fats and milk sugars) are the intermediates for flavour compounds ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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Table 4: Sensory evaluation of SSF samples with varying concentrations of butter fat*. Solid substrate fermentation (SSF) (hour) 24

Butter fat 30 g CFS CFG

Sensory evaluation Butter fat 60 g CFS CFG

Butter fat 160 g CFS CFG

Sweet, Sweet Sweet, Sweet Sweet, Sweet buttery buttery buttery 48 Sour, Sour, Sour, Sour, Sour, Sour, alcoholic alcoholic alcoholic alcoholic alcoholic alcoholic 72 Rancid Rancid Rancid Rancid Rancid Rancid *Scoring based the aroma descriptions by 10 volunteers; CFS: bacterial strain isolated from cattle field soil sample; CFG: bacterial strain isolated from cattle field grass sample. Table 5: Sensory evaluation of SSF samples fermented with CFS and CFG strains supplemented with carbohydrate sources Solid substrate fermentation (SSF) (hour) 24

Sensory evaluation CFS strain CFG strain Galactose Lactose Skim Milk Galactose Lactose Skim Milk Sweet, Sour Milky Sweet Sour Milky buttery 48 Sour Sour Milky Sour Sour Milky 72 Stale Sour Sour Stale Sour Sour * Scoring based the aroma descriptions by 10 volunteers; CFS: bacterial strain isolated from cattle field soil sample; CFG: bacterial strain isolated from cattle field grass sample. undergraduate students (Figure 4). The sensory evaluation of the samples by students is depicted in Table 6. A sweet, butter flavour production was enhanced with both the CFS and CFG strains in 24 hours of incubation. Upon prolonged incubation (48 and 72 hours), stale and sour odour were recorded. The results indicated that butter fat supplemented with 10 % galactose and fermented specifically with the CFS strain was effective in producing butter oil with intense butter flavour. Butter fat supplemented with skim milk recorded a more pronounced milk flavour, compared to butter flavour. Therefore, scale up studies were carried out with increased amounts of butter fat (30 g, 60 g and 160 g) supplemented with 10 % galactose individually.

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Scale up studies of Solid Substrate Fermentation (SSF) with galactose Scale up studies were performed with increased butter fat (30 g, 60 g and 160 g) supplemented with 10% galactose individually. Figure 5, indicates the scale up studies of 160 g butter fat supplemented with 16 g (10%) galactose. After fermentation, the butter oil was extracted and subjected to sensory evaluation. Based on the aroma description (sour, sweet, buttery, milky, stale and rancid). Scoring was done by 10 volunteers. Table 6, indicates the sensory evaluation results of the fermented sample. Sensory evaluation of butter fat samples supplemented with galactose at scale up studies Based on the sensory evaluation, the butter fat samples fermented with strains CFS

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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 and buttery within 24 hours of incubation (Table 6). Extended incubation period (48 and 72 hours) resulted in formation of undesired, foul (stale or rancid or alcoholic) odours. Therefore, the experiment was repeated and after 24 hours of incubation period, the fermentation was arrested by freezing the samples at -20°C. The respective samples were thawed at room temperature. Butter ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

oil was extracted and subjected to sensory evaluation. Sensory evaluation of butter oil samples by 120 students Compared to the control sample (BF), all other samples were preferred (Figure 6). Most individuals (38%) did not approve of sample BF with 53% of the volunteers being unsure of their preference. The least amount (8.3%) of evaluators prefer this 50



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Figure 5: Scale up studies of 160 g butter fat supplemented with 16 g (10%) galactose; 1: BF=Untreated butter fat (160 g) (control); 2: BF+Gal+CFS=Butter fat (160 g) supplemented with galactose (16 g) and fermented with CFS strain; 3: BF+Gal+CFG=Butter fat (160 g) supplemented with galactose (16 g) and fermented with CFG strain. sample on an overall basis. In comparative analysis of unsupplemented butter fat fermented with isolated LAB, sample BF+CFS was more preferred (45%) by students as compared to sample BF+CFG (19.2%). For supplemented butter fat with galactose and fermented with isolated LAB, a large number of evaluators (56.7%) preferred sample BF+Gal+CFS as compared to sample BF+Gal+CFG (45%). Hence, samples BF+CFS and BF+CFS+Gal were selected for further GC-MS analysis of constituents with sample BF serving as the control. Analysis of butter oil volatile constituents It was inferred that the butter fat samples fermented with CFS strain either with or without galactose was most preferred based on the sensory analysis of five different samples by 120 volunteers. Therefore, butter oil samples extracted from BF+CFS (butter fat fermented with strain CFS), BF+Gal+CFS (butter fat ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

supplemented with galactose and fermented with strain CFS) and from BF (untreated butter as control) were analysed for diacetyl and acetoin content by GC-MS method. The results were tabulated. Comparative analysis of diacetyl and acetoin content in the unfermented and fermented butter fat samples with CFS strain Acetoin content was highest (1321.2 ppm) in the butter fat sample fermented with CFS strain (BF+CFS). In contrast, untreated butter fat (BF) reported lowest amount of acetoin (211.5 ppm) amongst all 3 samples (Figure 7). With regards to diacetyl content, butter fat supplemented with galactose and fermented with CFS strain had the highest concentration (511.4 ppm). Lowest content of diacetyl was found to be from untreated butter fat (161.7 ppm). Unlike acetoin, diacetyl strongly contributes to the buttery aroma (Fuquay et al., 2011). Only in cohesion 51



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Figure 6: Sensory evaluation of butter oil samples by 120 participants; BFS: Butter Fat Sample; BF: untreated butter; BF+CFS: butter fermented with strain CFS; BF+CFG: butter fermented with strain CFG; BF+CFS+Gal: butter supplemented with galactose and fermented with strain CFS; BF+CFG+Gal: butter supplemented with galactose and fermented with strain CFG. with diacetyl, does acetoin impart strong, pleasant, mild, overall buttery aroma in addition to toning down diacetyl roughness (Bai et al, 2014). The results of diacetyl and acetoin analysis tallies with the preference of volunteers who preferred supplemented butter fat and fermented with CFS strain the most (56.7%). Untreated butter fat was the least preferred (8.3%) and is reflected by the lowest concentrations of diacetyl and acetoin content. Butter powder formulation Butter powder formulation was made with maltodextrin and to enhance its flow properties anti-caking agents (bentonite, sodium silicate, sodium chloride, potato starch and mannitol) were evaluated (Figure 8). The samples were subjected to sensory evaluation. The recovered butter oil was then formulated to powder form by addition of a carrier material and antiISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

caking agent. This was performed to enhance flow capability and storage property of the butter powder. Standardization of butter oil and maltodextrin ratio for butter concentrate powder formulation It was observed that the use of 25 g butter oil resulted in the formation of minor clumping with maltodextrin (Table 7). Although lower amounts of butter oil did not result in clumping, desired intensity of butter aroma was not imparted. Maltodextrin is a well-known carrier material used for various preparations. It is digestible, lacks artificial colouring or flavour, low in cost and miscible in liquids (Zuidam and Shimoni, 2010). In addition, its good binding and high viscosity capabilities enables spray or freeze drying of active ingredients to be made into powders easily (Akhilesh et al., 2012). From the various anti-caking agents tried, 52



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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. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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Microbial Production of Butter Flavour Table 7: Standardization of butter oil and maltodextrin ratio for butter concentrate powder formulation. Maltodz Butter Quality attributes (g) oil (g) Texture Aroma 50 5 No clumps Faint 50 10 No clumps Faint 50 15 No clumps Decent 50 20 No clumps Decent 50 25 Less clumps Strong 50 30 Grainy clumps Strong zMaltodextrin

Nadaraj et al. method for optimum recovery of butter oil from the butter fat samples was standardized, which resulted in 69.67 % recovery of butter oil. Upon preliminary screening of the LAB isolates, the CFS and CFG isolates were selected based on the sensory evaluation by 10 volunteers.

none are able to enhance the flow rate of the formulated powder at various concentrations (2%, 4% and 6% w/w). Such findings are in contrast to reports by (Ganesan et al., 2008) who stated application of 2% anticaking agent is adequate for enhancing powder flow rates. A possible reason for failing to achieve the desired powder texture might be due to structural collapse of the sample. This may be attributed to decreased product molecular porosity and overall volume, as a result of improper sample drying and storage (Bhadra et al., 2011).

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

CONCLUSION

REFERENCES

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

Akhilesh, D., Faishal, G. and Kamath, J.V. (2012). Comparative study of carriers used in proniosomes. Int J Pharm Chem Sci 3, 6–12. Anlauf, H. (2007). Recent developments in centrifuge technology. Sep. Purif. Technol. 58, 242–246. Anvoh, K.Y.B., Bi, A.Z., Gnakri, D. et al. (2009). Production and characterization of juice from mucilage of cocoa beans and its transformation into marmalade. Pak. J. Nutr. 8, 129–133. Bai, J.A. and Ravishankar, R.V. (2014). Beneficial Microbes in Fermented and Functional Foods. CRC Press.

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Microbial Production of Butter Flavour Bettache, G., Fatma, A., Miloud, H. and Mebrouk, K. (2012). Isolation and identification of lactic acid bacteria from Dhan, a traditional butter and their major technological traits. World Appl. Sci. J. 17, 480–488. Bhadra, R., Rosentrater, K.A. and Muthukumarappan, K. (2011). Effects of varying condensed distillers solubles, drying and cooling temperatures on glass transition temperature of distillers dried grains. Can. Biosyst. Eng. 53, 3–9. Bicas, J.L., Silva, J.C., Dionísio, A.P. and Pastore, G.M. (2010). Biotechnological production of bioflavors and functional sugars. Food Sci. Technol. Camp. 30, 07– 18. Chongcharoenyanon, B., Yamashita, N., Igura, N., Noma, S. and Shimoda, M. (2012). Extraction of volatile flavour compounds from butter oil in a low-density polyethylene membrane pouch. Flavour Fragr. J. 27, 367–371. Fuquay, J.W., Fox, P.F. and McSweeney, P.L. (2011). Encyclopedia of Dairy Sciences 2nd Edition, Four-Volume set. Academic Press. Ganesan, V., Rosentrater, K.A., and Muthukumarappan, K. (2008). Flowability and handling characteristics of bulk solids and powders–a review with implications for DDGS. Biosyst. Eng. 101, 425–435. Gokce, R., Akdogan, A., Divriklib, U. and Elci, L. (2014). Simultatenous determination of diacetyl and acetoin in traditional turkish butter

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Nadaraj et al. stored in sheep’s rumen (Karinyagi). Grasas Aceites 65, 10. Halász, A. (2009). Lactic acid bacteria. Food Qual. Stand. 3, 70–82. Hua, D., Ma, C., Song, L., Lin, S., Zhang, Z., Deng, Z. and Xu, P. (2007). Enhanced vanillin production from ferulic acid using adsorbent resin. Appl. Microbiol. Biotechnol. 74, 783–790. Hugenholtz, J., Sybesma, W., Groot, M.N., Wisselink, W., Ladero, V., Burgess, K., van Sinderen, D., Piard, J.-C., Eggink, G., Smid, E.J. et al. (2002). Metabolic engineering of lactic acid bacteria for the production of nutraceuticals, in: Lactic Acid Bacteria: Genetics, Metabolism and Applications. Springer, pp. 217– 235. Juffs, H. and Deeth, H. (2007). Scientific evaluation of pasteurisation for pathogen reduction in milk and milk products. Food Standards Australia New Zealand. Kreiss,

K. (2007). Flavoring-related bronchiolitis obliterans. Curr. Opin. Allergy Clin. Immunol. 7, 162–167.

Krause, A.J., Miracle, R.E., Sanders, T.H., Dean, L.L. and Drake, M.A. (2008). The effect of refrigerated and frozen storage on butter flavor and texture. J. Dairy Sci. 91, 455–465. Krogerus, K. and Gibson, B.R. (2013). 125th Anniversary Review: Diacetyl and its control during brewery fermentation. J. Inst. Brew. 119, 86–97.

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Microbial Production of Butter Flavour Lipasek, R.A., Taylor, L.S. and Mauer, L.J. (2011). Effects of anticaking agents and relative humidity on the physical and chemical stability of powdered vitamin C. J. Food Sci. 76, C1062–C1074. Longo, M.A. and Sanromán, M.A. (2006). Production of food aroma compounds: microbial and enzymatic methodologies. Food Technol. Biotechnol. 44, 335–353. Miura, S., Tanaka, M., Suzuki, A. and Sato, K. (2004). Application of phospholipids extracted from bovine milk to the reconstitution of cream using butter oil. J. Am. Oil Chem. Soc. 81, 97–100. Morris, J.B. and Hubbs, A.F. (2009). Inhalation dosimetry of diacetyl and butyric acid, two components of butter flavoring vapors. Toxicol. Sci. Off. J. Soc. Toxicol. 108, 173– 183. doi:10.1093/toxsci/kfn222.

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Nadaraj et al. Smit, G., Smit, B.A. and Engels, W.J. (2005). Flavour formation by lactic acid bacteria and biochemical flavour profiling of cheese products. FEMS Microbiol. Rev. 29, 591–610. Wandrey, C., Bartkowiak, A. and Harding, S.E. (2010). Materials for encapsulation, in: Encapsulation Technologies for Active Food Ingredients and Food Processing. Springer, pp. 31–100. Zuidam, N.J. and Shimoni, E. (2010). Overview of microencapsulates for use in food products or processes and methods to make them, in: Encapsulation Technologies for Active Food Ingredients and Food Processing. Springer, pp. 3–29.

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Reconfigurable Filter Bank for Accurate Spectral Decomposition of EEG Signals Biju K. S.1, *, Hareeshkumar M.2, Girishkumar C.3, Jibukumar M. G.1 1

Electronics Engineering Division, School of Engineering, Cochin University of Science & Technology, Kochi, 682022, India. 2Electronics and Communication Engineering Department, Government Engineering College, Bartonhill, Thiruvananthapuram, 695035, India. 3 Faculty of Engineering & Computer Technology, AIMST University, Malaysia; *corresponding author, e-mail: [email protected]

ABSTRACT Aim: Electroencephalography (EEG) signals contain vital information which is extremely helpful for studying the functionalities and disorders of brain. For detailed analysis, the spectral decomposition of EEG signals are split into different EEG rhythms. Since the different EEG rhythms are of non-uniform bandwidth, existing techniques results in inaccurate decomposition and which in turn inaccurate results. The reconfigurable filter bank proposed can replace the existing methods for an efficient and accurate spectral decomposition of EEG signals. Methodology and results: The structure of the reconfigurable filter bank (RFB) includes a uniform filter bank followed by frequency response masking (FRM) filters. The uses of FRM filters provide a sharp transition bandwidth which optimizes the design. The first masking filter for delta band was designed such that it extracts the 0.5-4 Hz band. The second masking filter for theta band was of extracts 4-8 Hz. The masking filter for alpha band extracted about 8-13 Hz. The beta band was extracted using the masking filter applied to the sub filter of fourth stage extract greater than 14Hz. Analysis of RFB magnitude spectrum of both healthy EEG and seizure EEG signal is carried out. Conclusion: The proposed reconfigurable filter bank is based on frequency response masking technique and it provides an accurate extraction of EEG rhythms. The spectra of each band are very much clear that the extracted rhythms have much less components from the adjacent spectra. Keywords: Frequency response masking; Reconfigurable filter banks; Spectral decomposition.

INTRODUCTION Brain is the most complex part in the human body and therefore studying and analysis of the same for understanding the features, functioning and artifacts are difficult as compared to the other organs and or parts in the body. Brain waves are analyzed for the study of functioning and diagnosis of brain disorders. A number of techniques have ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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


Spectral Decomposition of EEG Signals analysis, these EEG rhythms have to be extracted separately. For spectral decomposition and analysis lots of techniques like time-frequency analysis have been introduced earlier (Shayan et al., 2014). The various time-frequency analysis techniques in practice include Short Time Fourier Transform (STFT), Wavelet Transform (WT), Wavelet Packet Transform (WPT), Filter Banks, Moving Average filtering, Auto Regressive analysis, Auto Regressive Moving Average model (Saeid and Chambers, 2007). The STFT, WT, WPT, Filter Banks etc. enhance decomposition of signals into sub-bands and simultaneous analysis of the various (wanted) spectral components. Spectral analysis is performed on the signals to extract various key-information (Bhagwat and Vinod, 2013). For the spectral analysis lots and lots of transform techniques and their variants have been proposed and are being used through decades. Since, in particular, EEG is a non-stationary signal, transforms like DFT, FFT etc. cannot describe it completely, and some transform technique localized in time and frequency as well is needed (Bhagwat and Vinod, 2013). The wavelet transform technique is in wide practice for analysis of EEG signal for decomposition into different bands of equal widths (Bhushan, 2013; Rafiee et al., 2011). Wavelet Packets has been found better in the analysis of biological signals. At higher frequencies WT fails to localize time with required accuracy. Discrimination of frequency is sacrificed at higher frequencies for localization of time. WPT generalizes the time-frequency analysis of WT. Filter Bank is also a candidate of sub-band decomposition of EEG signals (Alfred, 1999; Chen et al., 2014; Darak et al., 2011). In all these cases the sub band bandwidths are fixed. This leads to an inaccurate decomposition and analysis and in turn ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Biju et al. inaccurate results. Use of Reconfigurable Filter Banks helps solving this issue. Various methods have been proposed for achieving the reconfigurability in the cutoff frequency and bandwidths of filters. One approach is to implement a variable cut off digital filter in which the cut off frequency could be controlled through a single parameter which uses the principle of frequency transformation of linear phase FIR filters (Oppenheim et al., 1976). In fractional delay method, each unit in delay operator in fixed coefficient FIR filter is replaced with second order FIR fractional delay structure (Sasikumar et al., 2013). Cut-off frequency is changed by changing the FD value which in turn changes the bandwidth. The frequency response masking (FRM) technique comprises the complete finest transition-band filter using many wide transition-band sub-filters (Lim, 1986; Lim and Boroujeny, 1992). In the modified frequency transformation technique the fixed-coefficient low-pass sub filter in the first stage of a Fast Filter bank (FFB) is replaced by a modified second-order frequency transformation (MFT) based low pass variable digital filter (Filipe et al., 2007). In this paper the design of a reconfigurable filter bank for EEG spectral decomposition is proposed. Here a uniform filter bank is used as a prototype filter bank and FRM Technique is applied for achieving reconfigurability in sub band bandwidths. METHODOLOGY The brain cells transfer the information by means of biochemical reactions across small spaces which are termed as synapses. The nerve cells can be considered as a dipole with its own particular orientation as well as polarity. Such dipoles with identical polarity will receive similar inputs and they add up 58


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

delta () ∶ 0.5 ≤ f < 4𝐻𝑧 theta () ∶ 4 ≤ f < 8𝐻𝑧 alpha (α) ∶ 8 ≤ f ≤ 13Hz beta(β) ∶ 13Hz  f In general, EEG signals do not reveal information about any abnormalities or disorders of brain. Time-frequency analysis is required to extract such information, if any, from EEG signals. Spectral decomposition of EEG signals into EEG rhythms have been always a challenge since the EEG rhythms are of low frequency and accurate decomposition is very difficult (Shayan et al., 2014). The non-uniform band widths of the sub bands also add up to the complexity in spectral decomposition. Lots of methods like Wavelet Transform, Wavelet Packet transform, Filter Banks, etc. have been used for the same spectrum (Alfred, 1999; Shayan et al., 2014). The analysis using WT and WPT decomposes the finite energy signal in successive stages each with different scaling factor, Thus it gives the information in both time and frequency domains. But at higher frequencies discrimination of frequencies is sacrificed for localization of time (Alfred, 1999). Wavelet Packet Transform mitigates this problem. But, the bandwidth of packets is multiples of two; hence, it is suitable only for uniform sub band decomposition. The existing methods have not succeeded in nonuniform sub band decomposition of EEG ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Biju et al. signals. Since each rhythm of EEG represents the activity of brain in different stages of activity accurate spectral decomposition is of great importance (Saeid and Chambers, 2007). So far no work has been done on the design of a method for accurate sub band decomposition. The proposed reconfigurable filter bank is the first attempt of its kind in the scenario of EEG signal processing. In the proposed Reconfigurable Filter Bank, the center frequency and bandwidth of the sub filters can be varied according to the requirements. The design requirements include sub band filters with sharp transition bands and considerably large stop band attenuation. Proposed design The proposed RFB consists mainly of two stages: i) A perfect reconstruction Uniform Filter Bank and ii) Frequency Response Masking based masking filters. In section 3.1, the design and response of Uniform Filter Bank is described. In the following section the principle of Frequency Response Masking and the design steps of FRM masking filters is discussed. Design of perfect reconstruction 16 channel filter bank The filter banks comprise two stages, analysis filter banks and synthesis filter banks filter banks (Vaidyanathan, 1993). Each stage consists of low pass, band pass and high pass filters. In analysis filters the input finite energy signal is applied to M filters which will be decomposed into M uniform width sub-bands. The resulting signals are sub-sampled by a factor N to avoid redundancies. At the synthesis filter bank stage, up sampling is done to recover the sampling rate of input signal. The up sampled signals are applied to synthesis filters which will compose the original signal. If number of filter stages equals the A.

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

(3)

Combining these equations, input-output relation is given by

the

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

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

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

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


Spectral Decomposition of EEG Signals filter bank are given by the following design equations.

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ℎ𝐿𝑃 (𝑖 + 1)= ℎ𝐿𝑃 (𝑖) * 𝑔𝐿𝑃 (𝑖) ̅ℎ𝐿𝑃 (𝑖 + 1) = ℎ𝐿𝑃 (𝑖)* 𝑔̅𝐿𝑃 (𝑖 + 1)

(5) (6)

single hardware structure can be used for mapping these sub-filters. Hence, just one filter structure is needed and the number of adders and multipliers can be reduced. This reduces the complexity in design.

ℎ𝐻𝑃 (𝑖 + 1) = ℎ̅𝐿𝑃 (𝑖) ∗ 𝑔̅𝐿𝑃 (𝑖 + 1)

(7)

RESULTS AND DISCUSSION

̅ℎ𝐻𝑃 (𝑖 + 1) =ℎ̅𝐿𝑃 (𝑖) ∗ 𝑔𝐿𝑃 (𝑖)

(8)

The EEG signals were adopted from CHBMIT scalp EEG database prepared and hosted by Children's Hospital Boston (CHB) and the Massachusetts Institute of Technology (MIT). The signals are in the European data format (.edf), which is a standard file format used for the storage and exchange of medical time series. The sampling frequency is 256 Hz and the file includes signals from 16 channels. Another source of database was that from Department of Epileptology, University of Bonn.

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

The procedure for simulation of the reconfigurable filter bank as follows. The

Figure 1: Magnitude response of 16-channel uniform filter bank. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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

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Figure3: Magnitude spectrum of Uniform Filter Bank response. Filter Bank is shown in the Figure 2 and Figure 3 respectively. From the waveforms it is clear that there is an overlap in the spectra; hence, the obtained result is inaccurate. The adjacent spectral components are present in the desired spectrum which will affect the accurate diagnosis of diseases. Use of Reconfigurable Filter Bank helps to mitigate this problem. The proposed RFB provides accurate extraction of EEG rhythms with high resolution. The waveforms in the Figure 4 and Figure 5 are magnitude spectrum of healthy EEG signal and the abnormal (seizure) EEG signal respectively. From the spectra it is clear that the extracted rhythms have much less components form adjacent spectra. Hence, it will be easier to analyze different bands separately and to determine in which band abnormality occurs. This makes the spectral decomposition analysis for EEG signals and 62



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to detect the seizure activity which is dominating in the sub-bands.

provides considerably narrow transition band width without higher orders. Hence, the circuit complexity is less. Since FRM filters are FIR based, the design retains all the advantages of FIR filters including guaranteed stability, low coefficient sensitivity, free of phase distortion etc.

The main advantage of using FRM filters for reconfiguring bandwidth is that no hardware overhead is required for changing the bandwidth and centre frequency. Only thing required is the change in coefficients. Also it

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Figure 5: RFB Magnitude Spectrum of seizure EEG signal. CONCLUSION In this paper design of a reconfigurable filter bank for efficient and accurate spectral decomposition of EEG signals is proposed. The existing techniques employed for spectral decomposition such as Wavelet Transform, Wavelet Packet transform and DFT Filter bank could only extract bands of uniform bandwidth. The proposed RFB based on FRM technique provides accurate ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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


Spectral Decomposition of EEG Signals sharp transition width narrow band low pass and band pass filters to be used for extracting desired bands. Since the sub filters are identical they can be mapped into a single hardware structure, it reduces the complexity of hardware implementation. Since the reconfigurability is achieved without a change in hardware the technique is found to be optimum for EEG rhythm extraction. REFERENCES Alfred, M. (1999). Signal Analysis: Wavelets, Filter Banks, TimeFrequency Transforms and Applications John Wiley & Sons Ltd. Singapore. pp. 225-245. Bhagwat, S. D. and Vinod, J. (2013). EEG Data Sets Signal Processing Using Wavelet Transforms. International Journal of Innovative Technology and Exploring Engineering 2(6), 108-111. Bhushan, N. P. (2013). A review of ECG monitoring system using Wavelet Transform. International Journal of Innovative Technology and Exploring Engineering 2(4), 334341. Chen, J. Ding, W. and Zhou, J. (2014). Design of Hardware Efficient Modulated Filter Bank for EEG Signals Feature Extraction. In: IEEE Proceeding of 57th International Midwest Symposium on Circuits and Systems. College Station, Texas. pp.793-796. Darak, S. J. Vinod, A. P. and Lai, E. M. K. (2011). A new variable digital filter design based on fractional delay. In: IEEE Proceeding of ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Biju et al. International Conference. Acoustics, Speech and Signal Processing, Prague. pp. 1629-1632. Filipe, C.C.B. D. IuriKothe, S. L. N. and Luiz, W. P. B. (2007). High selectivity Filter Banks for spectral analysis of music signals. Advances in Signal Processing 1-13. Lim, Y. C. and Lian, Y. (1993). The optimum design of One- and TwoDimensional FIR filters using the frequency response masking technique. Circuits and Systems II: Analog and Digital Signal Processing 40(2), 88-95. Lim, Y. C. (1986). Frequency-response masking approach for the synthesis of sharp linear phase digital filters. Circuits and Systems 33, 357-364. Lim, Y. C. and Boroujeny, B. F. (1992). Fast filter bank (FFB). Circuits and Systems II: Analog and Digital Signal Processing 39(5), 316–318. Mahesh, R. and Vinod, A. P. (2011). Reconfigurable Low Area Complexity Filter Bank Architecture Based on Frequency Response Masking for Non-uniform Channelization in Software Radio Receivers. Aerospace and Electronic Systems 47(2), 1241-1255. Oppenheim, A. Mechlenbräuker, W. and Mersereau, R. (1976). Variable cutoff linear phase digital filters. Circuits and Systems 23(4), 199– 203. Rafiee, J. Rafiee, M. A. Prause, N. and Shoan, M. P. (2011). Wavelet basis functions in biomedical signal 65


Spectral Decomposition of EEG Signals processing. Expert Systems with Applications 38(5), 6190-6201. Saeid, S. and Chambers, J. A. (2007). EEG Signal Processing, John Wiley & Sons Ltd. pp.51-75. Sasikumar, G. VudiS. M. and Rittwika, G. (2013). Analysis and simulation of brain signal data by EEG signal processing technique using MATLAB. International Journal of Engineering and Technology 5(3), 2771-76. Shayan, M. Mohamed, M. Martyn, H. Catherine, M. H. and Paul, R. W. (2014). Signal processing techniques applied to human sleep EEG signals—A review. Biomedical Signal Processing and Control 10, 21–33.

Biju et al. reconfigurable channel filter based on decimation, interpolation and frequency response masking. In: IEEE Proceeding of International Conference on Acoustics, Speech and Signal Processing. Vancouver, Canada. pp. 5583-87. Teplan, M. (2002). Fundamentals of EEG Measurement. Measurement Science Review 2 (2), 1-11. Vaidyanathan, P. P. (1990). Multirate Digital Filters, Filter Banks, Polyphase Networks, and Applications: A Tutorial. Proceedings of the IEEE 78(1), 5693. Vaidyanathan, P. P. (1993). Multirate Systems and Filter Banks. Pearson Education, Inc. New Delhi. pp.188234.

Sumedh, D. Smitha, K. G. and Vinod, A. P. (2013). A low complexity

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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: [email protected]

ABSTRACT Aim: Antitubercular therapy leads to the development of acute renal injury (ARI) in some individuals. Though isoniazid (INH) has been evidenced as an ARI inducing drug, mounting evidences from several studies identify rifampicin (RIF) as the most common ARI inducing antitubercular drug. Current study was carried out to evaluate the nephroprotective effect of the oral supplementation of coenzyme Q10 in INH and RIF treated Wistar albino rats. Methodology and results: Rats were administered with INH and RIF (50 mg/kg b.w. each/day) for 28 days. The effect of concomitant treatment with coenzyme Q10 (10 mg/kg b.w./day) on INH and RIF-induced renal injury was evaluated by estimating the serum levels of renal functional markers such as creatinine, urea, uric acid and acid phosphatase. In addition, the antioxidant profile, levels of non-enzymic antioxidants and lipid peroxidation were assessed in renal homogenates of experimental rats. Histological studies were also performed. The standard hepatoprotective drug silymarin (25 mg/kg b.w./day) was used for the purpose of comparison. The tested parameters of the coenzyme Q10 treated INH and RIFinduced rats were compared with that of the normal control rats and silymarin-treated INH and RIF-induced rats. Coenzyme Q10 significantly reduced the elevated levels of serum renal functional markers in INH and RIF-administered rats. Also, the food supplement was able to restore near normal the antioxidant status and noted to prevent renal damage in experimental rats. Conclusion, significance and impact of study: Current study reveals the potential of coenzyme Q10 in minimizing the renal injury due to antitubercular therapy. Hence, CoQ10 supplementation would be useful to patients on antitubercular regimen. Keywords: Acute renal injury; Coenzyme Q10; Isoniazid; Rifampicin.

INTRODUCTION Oxidative stress plays a major role in the induction and progression of renal failure both acute and chronic. Several conditions like hypertension, diabetes, infection, obstruction in the urinary tract, autoimmune disorders such as lupus erythematosus, genetic disorders such as polycystic kidney disease, drugs such as antibiotics, diuretics and anti-inflammatory drugs induce oxidative stress in renal tissues (Schattner et al., 2000; Crispín et ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

al., 2008; Chevalier et al., 2010). Several medications are known inducers of acute interstitial nephritis which includes commonly used non-steroidal antiinflammatory drugs (NSAIDS), interferon and antibiotics such as penicillins, cephalosporins and anti-tubercular drugs such as rifampicin (Rossert, 2001). Studies exploring the sub-cellular mechanisms of renal injury causing AIN have been carried out recently (Mitchell et al., 1977; Servais et al., 2007; Chevalier et al., 2010). It has

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

Udhaya and Sabina MATERIALS AND METHODS Chemicals and drugs Synthetic coenzyme Q10 was purchased from Sigma Aldrich, India. INH and RIF were purchased from Lupin Ltd., Aurangabad, India and the standard hepatoprotective drug silymarin from Quality Pharmaceuticals Ltd., India. INH and RIF were dissolved in normal saline while silymarin was dissolved in sterile distilled water. Coenzyme Q10 was dissolved in 0.2 ml corn oil. All the other chemicals and reagents used were of analytical grade procured from SD Fine Chemicals Pvt. Ltd., Mumbai, India. Animals 30 female Wistar albino rats of body weight 143.26±12.81 g were procured from the Animal House, VIT University, Vellore, Tamilnadu. The rats were divided into 5 groups and treated as follows for 28 days: group I was normal control; group II was treated with INH and RIF (50 mg/kg b.w. each/day); group III was INH and RIF-induced rats co-administered with coenzyme Q10 (10 mg/kg b.w./day); group IV was INH and RIF-induced rats coadministered with silymarin (25 mg/kg b.w./day); group V was treated with coenzyme Q10 (10 mg/kg b.w./day) alone. The animals were sacrificed after the study duration using ether anaesthesia. Blood and kidneys were procured for further analysis. Renal homogenates were prepared using 5 % phosphate buffered saline (PBS). Estimation of serum markers of renal function Serum levels of renal functional markers such as creatinine, urea, uric acid and acid phosphatase were estimated using commercial diagnostic kits obtained from AutoSpan Diagnostics Ltd., India. Assessment of antioxidant profile Renal homogenates were used to assess the activities of superoxide dismutase 68


Coenzyme Q10 Dietary Supplementation (Marklund and Marklund, 1974), catalase (Sinha, 1972), glutathione peroxidase (Rotruck et al., 1973) and Glutathione-Stransferase (Habig et al., 1974); levels of total reduced glutathione (Moron et al., 1979) and lipid peroxidation (Ohkawa et al., 1979). Histopathological analysis A portion of the kidneys were fixed in 10 % formalin after thorough washing with ice-cold 5 % PBS. The tissues were then dehydrated with descending grades of isopropanol. After treating with xylene, the tissues were embedded in molten paraffin wax and tissue sections (5 µm) were cut. The sections were stained with hematoxylin and eosin and examined microscopically for pathological changes.

Udhaya and Sabina Effect of coenzyme Q10 on serum markers of renal function in INH and RIF induced rats INH and RIF induced rats showed significant increase in the levels of urea, creatinine, uric acid and acid phosphatase while concomitant administration of coenzyme Q10 caused significant (P