Producing Lactic Acid Bacteria (Lab) Isolated from Gastrointestine of Clarias Macrocephalus (Broadhead Catfish)

CHARACTERIZATION OF BACTERIOCIN- PRODUCING LACTIC ACID BACTERIA (LAB) ISOLATED FROM GASTROINTESTINE OF CLARIAS MACROCEPHALUS (BROADHEAD CATFISH)

NUR SHAZANA BT AZHAR

A thesis submitted in fulfilment of the requirement for the degree of Master of Science in Biotechnology

Kulliyyah of Science International Islamic University Malaysia

MAY 2015

ABSTRACT

The increase in demand of aquaculture products has led to intensive aquaculture; unfortunately, aquatic animals are often exposed to stressful conditions. Problems related to diseases and deterioration of environmental conditions often occurs, resulting in serious economic losses. In this study, the LAB strains from gastrointestinal of Broadhead catfish were identified and characterized by Gram staining and by other biochemical tests such as catalase, lactose utilization and API 50CHL (BioMerieux). Out of 50 isolates, 15 isolates from sample 1 and 11 isolates from sample 2 were found to be positive towards lactose utilization test and they were also identified as Gram positive cocci. Only 22 isolates showed negative reactions towards catalase test. Out of 22 isolates, only isolate A5 demonstrated a clear bactericidal effect against Bacillus cereus, Staphylococcus aureus and Salmonella thyphimurium, thus, this isolate was chosen for further characterization. PCR amplification of 16S rRNA sequence (given NCBI accession number, KP 064393) positively identified isolate A5 as a member of Lactococcus lactis with 100% DNA homology. Cell free supernatant fluid collected from Lactococcus lactis strain A5, when evaluated against Gram positive and Gram negative pathogens, showed inhibitory action against indicator microorganisms by using disc diffusion method. Bacteriocin was extracted using chloroform extraction method and complete inactivation of antimicrobial activity after treatment with Proteinase K confirmed the proteinaceous nature of bacteriocin extracted. Stability of the bacteriocin in the presence of catalase enzyme ruled out the possibility of antagonistic activity due to either hydrogen peroxide or organic acids. The bacteriocin extract was heat stable and has a molecular weight of 3.4 kDa based on SDS-PAGE analysis. The antibacterial activity was significantly increased by the addition of SDS, Triton X-100, Tween 20 and Tween 80. This probiotic strain, exclusively isolated from Malaysian catfish, produced an antimicrobial protein or bacteriocin which can be used as probiotic to control and compete with pathogenic bacteria as well as to promote the growth of the cultured organisms in aquaculture feeding.

Keywords: LAB, probiotic, bacteriocin, SDS-PAGE, Lactococcus lactis

خالصة البحث ABSTRACT IN ARABIC

قد أدى ازدياد الطلب على منتجات االستزراع املائي إىل االستزراع املكثف. و لألسف بسبب ذلك غالبا ما تتعرض احليوانات املائية لظروف مثرية للتوتر. املشاكل املتعلقة باألمراض وبتدهور الظروف البيئية غالبا ما حتدث ، مما تؤدي إىل خسائر اقتصادية فقيمة. يف هذا البحث ، مت التعرف على على سالالت الالكتوباسيلوس من اجلهاز اهلضمي لسمك السلور ذي الرأس العريض و متييزها بالصبغ بطريقة غرام و بطرق بيوكيميائية كاستخدام الكاتاالز و استعمال الالكتوز و نظام BioMerieux( API 50CHL(. من اخلمسني معزولة ، مخس عشرة معزولة من العينة رقم واحد و إحدى عشر معزولة من العينة رقم اثنني قد أظهروا نتائج إجيابية يف اختبار استعمال الالكتوز و قد صنفت أيضا على أهنا من القرمزيات اإلجيابية الغرام. اثنني و عشرون فقط من املعزوالت قد أظهرت نتائج سلبية يف اختبار الكاتاالز. من االثنني و العشرين معزولة ، املعزولة A5 وحدها أظهرت خصائص واضحة ضد بكترييا العصوية الشمعية ، و العنقودية الذهبية ،و السلمونيلة التيفية ، و بالتايل فقد أختريت هذه املعزولة ملزيد من التوصيف. احلمض النووي الريبوزي الريبوسومي 16S rRNA احمللل بتفاعل البوليمرياز املتسلسل قد حددها إجيابيا كفرد من الالكتوكوكوس الكتيس بتماثل نووي محضي بنسبة 100 % ، و قد أظهرت ساللة A5 من الالكتوكوكوس الكتيس نشاطا مثبطا ضد املكروبات االختبارية باستعمال طريقة االنتشار القرصي عندما اخترب السائل الطاف اخلايل من اخلاليا املخخوذ منها ضد املمرضات املوببة الغرام و السالبة الغرام. املبيد اجلرثومي قد استخلص باالستعمال الكلوروفورم ، و التثبيط التام للنشاط الضد بكتريي بعد املعاجلة بالربوتيناز K قد أكد الطبيعة الربوتينية للمبيد اجلرثومي املستخلص. استقرارية املبيد اجلرثومية يف وبود انزمي الكاتاالز قد استبعد احتمال النشاط املناهض بسبب بريوكسيد اهليدروبني أو احلموض النووية. مستخلص املبيد اجلرثومي كان مستقرا حتت احلرارة و بوزن بزيئي حوايل 3.4 كيلو دالتون حبسب اختبار SDS-PAGE دوديسيل كربيتات الصوديوم-الرحالن الكهريب هلالم البويل اكريالميد. النشاط الضد بكتريي قد قمع بإضافة محض اخلليك الرباعي ثنائي امني االيثيلني ، و لكن قد ارتفع بشكل مالحظ بإضافة SDS دوديسيل كربيتات الصوديوم ، و Triton X-100 ، و Tween 20 و Tween 80. هذه الساللة الربوبيوتيكية املستخلصة حصريا من مسك السلور املاليزي قد أنتجت بروتينا ضد بكتريي أو مبيدا برثوميا من املمكن استعماله كربوبيويت للتحكم و للمنافسة مع البكترييا املمرضة و أيضا للحث على منو األحياء املستزرعة يف تغذية املستزرعات املائية. الكلمات الرئيسية: الكتوباسيلوس ، بروبيوتيك ، مبيد برثومي ، SDS-PAGE ، الالكتوكوكوس الكتيس.

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APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master of Science in Biotechnology.

...... Tengku Haziyamin Tengku Abdul Hamid Supervisor

...... Noor Hasniza bt Md Zin Co-Supervisor

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master of Science in Biotechnology.

...... Raha Ahmad Raus Internal Examiner

...... Zaharah Ibrahim External Examiner

This thesis was submitted to the Department of Biotechnology Sciences and is accepted as a fulfilment of the requirement for the degree of Master of Science in Biotechnology.

...... Suhaila Mohd. Omar Head, Department of Biotechnology Sciences

This thesis was submitted to the Kulliyyah of Science and is accepted as a fulfilment of the requirement for the degree of Master of Science in Biotechnology.

...... Kamaruzzaman Yunus Dean, Kulliyyah of Science

DECLARATION

I hereby declare that this thesis is the result of my own investigations, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degrees at IIUM or other institutions

Nur Shazana bt Azhar

Signature…………………………. Date ..……………………

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

CHARACTERIZATION OF BACTERIOCIN-PRODUCING LACTIC ACID BACTERIA (LAB) ISOLATED FROM GASTROINTESTINE OF CLARIAS MACROCEPHALUS (BROADHEAD CATFISH)

No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise without the prior written permission of the copyright holder except as provided below.

1. Any material contained in or derived from this unpublished research may only be used by others in their writing with due acknowledgement.

2. IIUM or its library will have the right to make and transmit copies (print or electronic) for institutional and academic purposes.

3. The IIUM library will have the right to make, store in a retrieval system and supply copies of this unpublished research if requested by other universities and research libraries.

Affirmed by Nur Shazana bt Azhar

……………...... ……………...... Signature Date

ACKNOWLEDGEMENTS

First and foremost, I would like to express my utmost gratitude to Allah S.W.T for opening doors of opportunity to me throughout my life and for giving me the strength and health to achieve what I have achieved so far. I would like to convey my heartfelt thanks to my supervisor, Assistant Prof. Dr. Tengku Haziyamin Tengku Abdul Hamid for his invaluable guidance and coaching to do this research. Thank you for giving me the chance to prove to everyone and mostly to myself that I can do it. I would also like to thank my co-supervisor, Dr. Noor Hasniza for her guidance, suggestions and openhearted support throughout my study. I would also like to convey my special thanks to Dr Akbar John for his knowledge sharing on how to construct a phylogenetic tree.

I would also like to convey my highest appreciation and special thanks to Br. Ahmad Muzammil bin Zuberdi, Br. Wadi, Sr. Ain and other lab assistants for the help rendered. I am grateful to all co-operative staffs in IIUM laboratory. We are so privileged at the Department of Biotechnology in Kulliyah of Science to have someone like them helping all the students.

Thank you very much indeed to my beloved husband, Ezzad Ashraff, for his invaluable patience, support and encouragement throughout my two years journey in completing this research project. I would like to extend my warm thanks to my parents, Azhar Mustafa and Putri Hasliza Megat Abu Hassan, with all the support that both of you had given to me. Last but not least to my beloved daughter, Zarra Saafia, thanks for being a great companion to me.

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TABLE OF CONTENTS

Abstract ...... ii Abstract in Arabic ...... iii Approval Page ...... iv Declaration ...... v Copyright Page ...... vi Acknowledgements ...... vii List of Tables ...... xi List of Figures ...... xii List of Abbreviations ...... xiv List of Symbols ...... xv

CHAPTER ONE: INTRODUCTION ...... 1 1.1 Background Study ...... 1 1.2 Research Objectives...... 3 1.3 Significance of the Study ...... 3

CHAPTER TWO: LITERATURE REVIEW ...... 5 2.1 Lactic Acid Bacteria (LAB)...... 5 2.2 Sources of LAB ...... 6 2.3 Catfish ...... 8 2.4 Antibiotic Application in Aquaculture ...... 8 2.5 Potential Use of Probiotic LAB in Aquaculture ...... 10 2.6 Probiotics ...... 11 2.7 Probiotic Characteristics ...... 12 2.8 Benefits of Probiotics LAB ...... 13 2.9 Bacteriocins ...... 15 2.10 Bacteriocins Versus Antibiotics ...... 16 2.11 Classification of Bacteriocins ...... 17 2.12 Bacteriocins as Food Preservatives ...... 18

CHAPTER THREE: MATERIALS AND METHOD ...... 20 3.1 Materials ...... 20 3.1.1 Catfish and Indicator Microorganisms ...... 20 3.1.2 Gram Reaction’s Solutions ...... 20 3.1.3 Culture Media ...... 20 3.1.4 Chemicals and Reagents ...... 21 3.1.5 Components (Stock Solutions and Buffers) for Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS- PAGE) ...... 21 3.2 Methods ...... 21 3.2.1 Overall Methodology ...... 21 3.3 Screening for Lactic Acid Bacteria...... 23 3.3.1 Catfish Gastrointestinal Tract (GIT) Sample Preparation ...... 23 3.4 Phenotypic Characterization of Lactic Acid Bacteria ...... 23 3.4.1 Lactose Utilization Test ...... 23

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3.4.2 Gram Staining ...... 23 3.4.3 Catalase Test ...... 24 3.4.4 Evaluation of Antimicrobial Activity of Isolated LAB by Disc Diffusion Assay ...... 24 3.4.5 Measuring Zone Sizes ...... 25 3.4.6 Identification of Isolates by API 50 CHL ...... 25 3.5 Genotypic Characterization of Lactic Acid Bacteria ...... 26 3.5.1 Genomic DNA Isolation ...... 26 3.5.2 Pre-treatment for Gram Positive Bacteria ...... 26 3.5.3 Purification of Total DNA from Animal Tissues (Spin- Column Protocol) ...... 27 3.5.4 Polymerase Chain Reaction (PCR) Amplification of 16S Ribosomal RNA ...... 27 3.5.5 Agarose Gel Electrophoresis ...... 28 3.5.6 Automated DNA Sequencing ...... 29 3.5.7 DNA Sequence Analysis ...... 29 3.6 Batch Cultivation of Lactococcus Lactis Strain A5 ...... 30 3.7 Characterization of Bacteriocin Produced by Lactococcus Lactis Strain A5 ...... 30 3.7.1 Production of Crude Bacteriocin ...... 30 3.7.2 Extraction of Bacteriocin Using Chloroform Extraction Method ...... 31 3.7.3 Antibacterial Assay by Disc Diffusion Method ...... 31 3.7.4 Assessment of Crude Bacteriocin and Extracted Bacteriocin Production for Antibacterial Activity ...... 32 3.7.5 Antibacterial Activity of Extracted Bacteriocin in Two-Fold Serial Dilution...... 32 3.7.6 Effect of Enzymes, Detergents and Temperature on Antimicrobial Activity ...... 33 3.7.7 Protein Quantitation ...... 33 3.7.8 Molecular Weight Determination by SDS-PAGE ...... 34

CHAPTER FOUR: RESULTS AND DISCUSSION ...... 37 4.1 Screening and Isolation for Lactic acid Bacteria ...... 37 4.2 Phenotypic Characterization of Lactic Acid Bacteria ...... 40 4.2.1 Lactose Utilization Test ...... 40 4.2.2 Catalase Test ...... 41 4.2.3 Antagonistic Test ...... 41 4.2.4 Identification of LAB Isolate Using API 50 CHL System ...... 45 4.3 Genotypic Characterization of Isolate A5 ...... 46 4.3.1 Genomic DNA Isolation ...... 47 4.3.2 PCR Amplification of 16S rRNA Gene Fragment...... 48 4.3.3 DNA Sequence Analysis ...... 49 4.4 Batch Cultivation of Lactococcus Lactis Strain A5 ...... 52 4.5 Characterization of Bacteriocin Produced by Lactococcus Lactis Strain A5 ...... 54 4.5.1 Comparison of Antibacterial Activitty from Crude Bacteriocin and Bacteriocin Extracted Using Chloroform ...... 54 4.5.2 Purification of Bacteriocin Using Chloroform Extraction ...... 57

4.5.3 Antibacterial Activity of Chloroform Extracted Bacteriocin in Two-Fold Dilution Series ...... 59 4.5.4 Effect of Enzymes, Detergents and Temperature on Antimicrobial Activity ...... 61 4.5.5 Molecular Weight Determination by SDS-PAGE ...... 64

CHAPTER FIVE: CONCLUSION AND FUTURE DIRECTION ...... 67

BIBLIOGRAPHY ...... 69

APPENDIX A: MAJOR CHEMICALS AND REAGENTS FOR THE PROJECT ...... 79 APPENDIX B: THE COMPOSITIONS AND PREPARATIONS OF STOCK SOLUTIONS AND BUFFERS FOR SODIUM DODECYL SULPHATE POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) ...... 80 APPENDIX C: THE COMPOSITIONS FOR COOMASSIE STAIN, DESTAIN 1 AND DESTAIN 2 SOLUTIONS...... 82 APPENDIX D: API 50CHL SYSTEM ...... 83 APPENDIX E: BRADFORD METHOD ...... 84 APPENDIX F: EFFECT OF TEMPERATURE, ENZYMES AND DETERGENTS ON ANTIMICROBIAL ACTIVITY ...... 86

LIST OF TABLES

Table No. Page No.

3.1‎ The sequence of 16S rRNA primers 27

3.2‎ PCR mixture (Vivantis 2x Taq Master Mix) 28

3.3‎ PCR condition 28

3.4‎ Volume of reagents for resolving and stacking gel monomer solution 35

4.1‎ Morphological and biochemical characterization of isolates 38

4.2‎ Inhibitory spectrum of LAB strains against gram positive and gram negative pathogens 42

4.3‎ Inhibitory spectrum of isolate A5 against gram-positive and gram- negative bacteria 44

4.4‎ Biochemical profile of isolate A5 46

4.5‎ The main hit list for partial rDNA sequence from similarity search result obtained from NCBI database by using BLASTN search tool 50

4.6‎ Data of pH and optical density (OD) of Lactococcus lactis strain A5 during the batch cultivation 52

4.7‎ Inhibitory spectrum of bacteriocin produced by Lactococccus lactis strain A5 on gram positive and gram negative bacteria correspond to Figure 4.8 to Figure 4.10 57

4.8‎ Titration of bacteriocin extract activity in terms of activity units (AU/mL) 60

4.9‎ Sensitivity of bacteriocin extract from Lactococcus lactis strain A5 to temperature, enzymes and detergents 62

LIST OF FIGURES

Figure No. Page No.

3.1‎ Main steps involved in the study 22

4.1‎ Well-isolated discrete colonies 37

4.2‎ Colonies producing lactic acid changed the pH and caused the plate to turn from purple to yellow 40

4.3‎ Positive antagonistic effect of isolate A5 against indicator microorganisms: 43

4.4‎ Genomic DNA of sample A5. Lane M: Gene Ruler™ DNA Ladder Mix (Fermentas); Lane 1-3: Replicates genomic of A5 47

4.5‎ Agarose gel electrophoresis of the PCR amplified product using universal rDNA primers producing 1.5 kb rDNA gene fragment from isolate A5. Lane M: Gene Ruler™ DNA Ladder Mix (Fermentas); Lane 1-4: Replicates of PCR amplified product from genomic DNA of A5 48

4.6‎ Partial DNA sequence of 16S rRNA of isolate A5 amplified by forward primer; 27F 16S rRNA 640bp 49

4.7‎ Phylogenetic tree obtained by the neighbor joining method based on the alignment of partial 16S rRNA sequences of Lactococcus lactis strain A5 from BLAST analyses 51

4.8‎ Antibacterial activity of crude bacteriocin (plate A) and extracted bacteriocin using chloroform extraction method (plate B) towards Bacillus cereus ATCC 11778. Control for plate A was 0.85% NaCl while control for plate B was a mixture of MRS broth and chloroform. R1, R2 and R3 represents for replicates 55

4.9‎ Antibacterial activity of crude bacteriocin (plate A) and extracted bacteriocin using chloroform extraction method (plate B) towards Staphylococcus aureus ATCC 33592. Control for plate A was 0.85% NaCl while control for plate B was a mixture of MRS broth and chloroform. R1, R2 and R3 represents for replicates 55

4.10‎ Antibacterial activity of crude bacteriocin (plate A) and extracted bacteriocin using chloroform extraction method (plate B) towards Salmonella thyphimurium S1000. Control for plate A was 0.85% NaCl while control for plate B was a mixture of MRS broth and chloroform. R1, R2 and R3 represents for replicates 56

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4.11‎ Antibacterial activity of extracted bacteriocin with two-fold dilution (2n) against: 61

4.12‎ SDS-PAGE (14% gel) of the bacteriocin extract. M: Protein marker (PageRuler Low Range Unstained Protein Marker 3.4-100kDa), A5: triplicates of bacteriocin extract with 3.4 kDa molecular weight 65

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LIST OF ABBREVIATIONS

APS Ammonium Persulphate ATCC American Type Culture Collection BSA Bovine Serum Albumin CFS Cell Free Supernatant CVI Crystal Violet-Iodine complex dH2O Distilled water DNA Deoxyribonucleic Acid dNTPs Deoxynucleotide Triphosphate EDTA Ethylene Diamine Tetraacetic Acid EtBr Ethidium Bromide GIT Gastrointestinal Tract GRAS Generally Regarded As Safe H2O2 Hydrogen Peroxide HCl Hydrochloric Acid LAB Lactic Acid Bacteria MgCl2 Magnesium Chloride MHA Muller Hinton Agar MHB Muller Hinton Broth MRS Man Rogosa and Sharpe NaCl Sodium Chloride NaOH Sodium Hydroxide O2 Oxygen PBS Phosphate Buffer Saline PCR Polymerase Chain Reaction rRNA Ribosomal RNA SDS Sodium Dodecyl Sulphate SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis spp. subspecies TAE Tris-Acetate EDTA Taq Thermus aquaticus TEMED Tetramethylethylenediamine Tris-HCl Trisaminomethane Hydrochloride UV/Vis Ultraviolet/Visible

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LIST OF SYMBOLS

% Percentage g Gram mg Milligram µg Microgram L Liter mL Milliliter µL Microliter cm Centimeter mm Millimeter nm Nanometer w/v Weight per Volume v/v Volume per Volume µg/mL Microgram per Milliliter mg/mL Milligram per Milliliter kDa Kilo Dalton ºC Degree Celsius CO2 Carbon Dioxide rpm Revolutions per Minute mA Milliampere V Volt pH Power of Hydrogen A Absorbance OD Optical Density CFU Colony Forming Unit g Earth’s Gravitational Acceleration AU/ml Arbitrary Unit Per Milliliter M Molarity

CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND STUDY

Aquaculture, a fast growing industry, has become a vital economic activity in many countries including Malaysia due to consumers’ increasing demands for animal protein, nutrition and food security. Catfish (Clarias spp.) is one of the most popular aquaculture fish produced by farmers in Malaysia (Ibrahim et al., 2010). Fishes in aquaculture are vulnerable to conditions that favour diseases, such as overcrowding and deterioration of water quality, due to intensive fish farming and large scale production of fishes. Ultimately, mortality in hatcheries caused by pathogens, costly treatment, loss of productivity and reduced marketability from the lack of appeal of fish appearances have resulted in severe financial losses. Aeromonas hydrophila is an example of fish pathogen that is commonly implicated with ulcer diseases in catfish

(Noga, 2010).

Antibiotics have always been the treatment of choice in the aquaculture industry. It has improved the growth and feed conversion in poultry and some animals

(Shin et al., 2008). Although antibiotics are helpful in treating infected animals, if overused or misused, it can potentially lead to harmful effects such as the emergence of antibiotic resistant bacteria. Also, the presence of residual antibiotics from farming is a concern to consumers as it could cause chronic health effects such as cancer, nerve problems and immunological problems and can accumulate in fish which subsequently may affect food safety for human consumption (Ibrahim et al., 2010).

Lactic acid bacteria (LAB) are a heterologous group of bacteria widespread in nature and are commonly found in milk, dairy products, plant material and intestinal tracts of humans and animals (Utkarsha and Milind, 2013). The LABs are described as

Gram-positive, usually non-motile, non-sporulating bacteria that produce lactic acid as a major product of carbohydrate fermentation (Khalid, 2011). Lactic acid bacteria

(LAB) are regarded as a major group of probiotic bacteria (Patel and Goyal, 2012).

Probiotics are live microbial feed supplements which improve the intestinal balance of the host animal’s microbiota and thereby, improves its health (Fuller, 1989).

Probiotics are disease-resistant and have the potential to improve the health of larval and immature fish, growth performance and body composition (Lara-Flores and

Olvera-Novoa, 2013). Ideally, microorganisms used in feed additives should originate from the microbiota of the targeted animals (Kosin and Rakshit, 2006).

There is high consumer demand for catfish, particularly in South East Asia.

The delicious taste and nutritional value are what make catfish a popular Malaysian dish. Unfortunately, improper disposal of fish gut has led to environmental pollution

(Jini et al., 2011). Hence, to better control fish waste disposal and to avoid environmental problems, lactic acid bacteria are taken from fish guts. Fish guts are ideal for use they high potential to recover new bacteriocins as an alternative treatment and prevent bacterial diseases in the aquaculture industry. Fish gut has shown to be a natural habitat for LAB as it is consistently found in the digestive tracts of many different fish species from different environments (Nirunya et al., 2008).

LAB could inhibit pathogenic organisms by producing antimicrobial substances such as hydrogen peroxide, organic acids, diacetyl, acetoin, reuterin, reutericyclin, antifungal peptides and bacteriocins (Rajaram et al., 2010). Bacteriocins are ribosomally synthesized proteinaceous antimicrobial

substances produced by bacteria that contain antimicrobial activity against bacteria closely associated to the producer strain (Trivedi et al., 2013).

Several reports have suggested that probiotic supplementation can reduce disease outbreaks by enhancing the immune system of fish (Mohideen et al., 2010) and decreasing the feed efficiency of fish (Mohapatra et al., 2012; Tuan et al., 2013).

In addition, the application of probiotics can lead to an improvement in water quality, as better feed efficiency may result in fish producing less waste (Nimrat et al., 2012).

The application of probiotics in aquaculture has been used as a means of controlling disease, enhancing immune response, providing nutritional and enzymatic contributions to the digestion of the host and improving water quality (Qi et al., 2009).

Since catfish is the most extensively cultured freshwater fish in Malaysia, the objective of this study was to isolate LAB strains from Clarias macrocephalus or locally known as Keli Bunga, and investigate the antimicrobial potential of the bacteriocin they produce against pathogenic microorganisms for future use in catfish aquaculture.

1.2 RESEARCH OBJECTIVES

1. To isolate, identify and extract bacteriocin-producing LAB from the

intestine of local catfish (Clarias macrocephalus).

2. To characterize the bacteriocin produced by the selected LAB isolate

(Lactococcus lactis strain A5).

1.3 SIGNIFICANCE OF THE STUDY

It is a common practice in the fish industry, particularly in developing countries to use large amounts of antibiotics to prevent infection. The antibiotics used are often non-

biodegradable and remain in the aquaculture environment for a long period of time.

The use of antibiotics in aquaculture industry is undoubtedly another contributing factor to increasing drug resistance. The addition of low levels of antibiotics to fish feeds raises the efficiency and rate of weight gain to the fishes. However, this also increases the number of drug resistant bacteria in fish intestinal tracts.

Hence, with the aim to discover new probiotic strains and their bacteriocins, it is essential to substitute commercial antibiotics used in the aquaculture industry with this bacteriocin isolated as an alternative treatment to prevent bacterial diseases for controlling fish diseases. Moreover, it can also help to reduce the development of drug- resistant bacteria that can be transferred to human and animal pathogens, leading to increased infectious disease in fish, animals and humans alike. Finally, bacteriocins produced by LAB are suitable for food preservation. In recent years, the increased consumption of foods containing additives such as chemical preservatives and the increase in consumer concerns have created a high demand for natural and minimal processed food. Therefore, the use of bacteriocins in food preservation can also be considered to solve consumer’s great fear of chemical food preservatives in their food products that they consume because the bacteriocin have antagonistic effect towards other bacteria and do not produce adverse effects.

CHAPTER TWO

LITERATURE REVIEW

2.1 LACTIC ACID BACTERIA (LAB)

Nobel Prize winning Russian scientist, Elie Metchnikoff, first proposed a hypothesis that evolved the concept of probiotics at the inception of the 20th century. Elie

Metchnikoff suggested that the poor Bulgarian people’s long and healthy life was due to their significant consumption of fermented milk products. He believed that the fermenting lactobacilli from fermented dairy products improved the microflora of the colon towards a healthy balance, subsequently decreasing toxic microbial activities, when consumed.

LABs are a heterologous group of bacteria widespread in nature and are commonly found in milk, dairy products, plant material, intestinal tracts of humans and animals (Utkarsha and Milind, 2013). The LABs are described as Gram-positive, usually non-motile, non-sporulating bacteria that produce lactic acid as a major product of carbohydrate fermentation (Khalid, 2011). They are either cocci or coccobacilli in shape and generally have a DNA base composition of less than 50% mole Guanine (G) + Cytosine (C). These organisms are fastidious and require carbohydrates, amino acids, peptides, nucleic acids derivatives and vitamins for growth (Djeghri-Hocine et al., 2010). Since most LABs can obtain energy only from the metabolism of sugars, they are usually restricted to habitats in which sugars are present. De Man Rogosa Sharp (MRS) medium is the standard growth medium that is extensively used to cultivate LABs and to produce bacteriocin. MRS medium is highly selective for LAB due to its sodium acetate content which inhibits non-LAB.

According to Arimah et al. (2014), LAB generally needs a fermentable carbohydrate in their sugar fermentation process and they can be categorized in two groups which are known as homofermenter and heterofermenter. The homofermenter

LAB converts hexoses to produce almost exclusively into lactic acid while the heterofementer LAB converts sugars to ethanol, carbon dioxide as well as lactic acid.

LAB lack cytochromes and poryphyrins (components of respiratory chains) and therefore they are catalase negative and oxidative negative. Most LABs are facultative anaerobic microorganisms and grow well under anaerobic condition but they are also capable of surviving in the presence of oxygen (Khalid, 2011). Under the US Food and Administration (FDA) guidelines, LAB is generally regarded as safe

(GRAS) status implying that they have been proven to be safe for human consumption through scientific procedures or through experience based on common use in food

(Patrick, 2012). As LAB grows, it causes the decrease in pH and increase the acidity of its environment. This trait has historically linked LAB with food fermentations and preservation as acidification process inhibits the growth of pathogens and food spoiling organisms, thus prolonging the foods’ shelf life.

2.2 SOURCES OF LAB

Based on Sheeladevi and Ramanathan (2011), LAB genera include Lactobacillus,

Leuconostoc, Pediococcus, Streptococcus, Aerococcus, Carnobacterium,

Enterococcus, Lactococcus, Vagococcus, Oenococcus and Weissella. LAB were first isolated from milk (Carr et al., 2002) and commonly found in many foods and fermented products such as meat, milk products, vegetables, drinks such as tea, milk or wine, and bakery products (Liu, 2003; O’ Sullivan et al., 2002).

Most of the LAB that have been isolated from Boza, a drink usually produced in the past in Bulgaria and prepared from a combination of different cereals, belong to the genera Lactobacillus spp., Lactococcus spp., and Leuconostoc spp. (Todorov and

Dicks, 2007). Furthermore, LAB has been detected in soil, water, manure and sewage

(Holzapfel et al., 2001). LABs occur naturally in fermented food (Caplice and

Fitzgerald, 1999). Lactobacillus plantarum strains have been isolated from fermented maize dough (Felis and Dellaglio, 2007). Some LAB is also associated with animal oral cavities which can cause dental caries (Sbordone and Bortolaia, 2003). Hekkila and Saris (2003) isolated LAB from human milk. Bifidobacterium gives a health- encouraging effect specifically to a child breast-fed by his or her mother (Markino et al., 2011).

Ringo and Gatesoupe (1998) isolated and identified LAB in the gastrointestinal tracts and feces of fishes. LAB were not a dominant population in fishes, but it has been well documented in several investigations that lactic acid bacteria are a part of the native microbiota of aquatic animals from temperate regions

(Ringo, 2004). In addition to the numerous investigations demonstrating the presence of LAB in the digestive tracts of several different fish species, several studies have reported on the isolation of LAB (Carnobacteria, Lactobacilli, Lactococci,

Leuconostocs and Pediococci) from cold-smoked and fermented fish (Nirunya et al.,

2008). Studies have shown the fish gut to be a natural habitat for LAB as it is consistently found in the digestive tracts of fishes from different environment

(Nirunya et al., 2008).

2.3 CATFISH

Catfish (Clarias macrocephalus) is locally known as Ikan keli bunga (Ambak et al.,

2010). It is widely distributed in Perlis, Kedah and Perak (Ambak et al., 2010).

According to Chong et al. (2000), catfish in Malaysia is gray or black in color.

Catfish (Clarias spp.) is one of the most popular aquaculture fish produced by farmers in Malaysia (Ibrahim et al., 2010). Catfish, a delectable dish, is very popular amongst the Malaysians. In addition, it is discovered that catfish is superior in terms of protein and amino acid content. It seems that consumers in Malaysia have begun to accept aquaculture fish as an alternative to sea fish since the production of sea fish has depleted (Ibrahim et al., 2010). It is also an attractive freshwater fish species in

Malaysia and other places due to its wide tolerance to environmental conditions, resistance to diseases and its ability to grow at a fast growth rate in length and weight

(Marimuthu et al., 2010). Hence, broadhead catfish, Clarias macrocephalus, is definitely an ideal aquaculture species in many regions of the world.

2.4 ANTIBIOTIC APPLICATION IN AQUACULTURE

Several countries including Malaysia rely on aquaculture, a thriving industry, as an important source of economy. According to Ibrahim and co-workers (2010), farmers in Malaysia have cultured freshwater fish in earthen ponds, floating net cages in rivers and ex-mining pools for many years.

Intensive fish farming and large scale production have made fishes vulnerable to conditions favouring diseases, such as overcrowding of fishes and declining of water quality. Such problems ultimately result to economic losses due to high mortality rate of fishes, costly treatment, loss of productivity and reduced marketability because of unappealing fish appearances. Fish pathogens such as

Aeromonas spp., Vibrio spp. and Streptococcus spp. are normally associated with mortalities (Kesarcodi-Watson et al., 2008).

Antibiotics have always been the popular choice of treatment in the aquaculture industry. The use of antibiotics in fish farming are common practices to avoid the overgrowth of herbal plants and fish diseases besides promoting the fast growth rate of the fish (Ibrahim et al., 2010). Although antibiotics are helpful in treating infected animals, if overused or misused, they can yield antibiotic resistant bacteria. Human consumption of animal products may have exposed consumers to resistant bacteria (Marshall and Levy, 2011). The identification of some antibiotic resistance in bacteria as well as in humans provides direct evidence for the transfer of antibiotic resistance genes via food handling or consumption.

Based on Marshall and Levy (2011), other researchers found that glycopeptide-resistant Enterococcus faecium of animal origin consumed through chicken or pork can reside in human fecal for up to 14 days after consumption of the meat products. Moreover, concern over the presence of residual antibiotics used at farm level can accumulate in fishes and cause chronic health effects to consumers such as cancer, nerve problems and immunological problems (Ibrahim et al., 2010).

The European Union and USA implemented bans on the use of antibiotics to curb this source of resistance as well as the fatal effect of residual antibiotics of aquaculture products on human health (Kesarcodi-Watson et al., 2008).

The use of antibiotics as animal growth promoters became a political issue due to the emergence of antibiotic-resistant bacteria and was finally banned to counteract the problem (Ohashi and Ushida, 2009). The ban of antibiotics has shown to produce positive results. Resistance to drugs declined dramatically among bacteria isolated