VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE PHAM THI DIU ANTIMICROBIAL ACTIVITY AND PRELIMINARY CHARACTERIZATION OF PEPTIDES PRODUCED BY LACTIC ACID BACTERIA ISOLATED FROM SOME VIETNAMESE FERMENTED FOODS Program: Code: Supervisor: Food technology Master Dr Nguyen Hoang Anh AGRICULTURAL UNIVERSITY PRESS - 2016 COMMITMENTS I assure that the data and the research results in this thesis are true They have not been used And, I assure that all the helps in this thesis have been acknowledged and information used in the thesis has been cited the sources Ha Noi, day month Master candidate Pham Thi Diu i year 2016 ACKNOWLEDGEMENTS During this thesis, I learned many useful experiences in laboratory I would like to express my deepest appreciation to all those who help me to complete thesis First of all, I would like to express the deep gratitude to Dr Nguyen Hoang Anh who helped and supported me to complete my thesis I also thank Dr Nguyen Thi Thanh Thuy and my colleagues from faculty of Food Science and Technology who provided insight and expertise that greatly assisted the research Finally, I thank very much to my friends, my family who supported me in this time Ha Noi, day month Master candidate Pham Thi Diu ii year 2016 TABLE OF CONTENTS Commitments i Acknowledgements ii Table of contents iii List of tables v List of figures vi List of diagram vi Abbreviation & acronyms vii Chapter i Introduction 1.1 Introduction 1.2 Objective of research Chapter ii Literature review 2.1 Lactic acid bacteria 2.1.1 Lactic acid bacteria 2.1.2 Bacteriocins of lactic acid bacteria 2.1.3 Application of bacteriocin 2.2 Popular pathogenic bacteria contaminated in food 2.2.1 Listeria monocytogenes 2.2.2 Bacillus cereus 10 2.2.3 Salmonella spp 11 2.2.4 Escherichia coli 12 2.3 Some vietnamese traditional fermented foods 13 2.3.1 Fermented chili sauce 13 2.3.2 Vietnamese traditional fermented meat (nem chua) 14 2.3.3 Fermented meat 14 2.3.4 Cassava leaf and bamboo pickled 15 Chapter iii Materials and research methodology 16 3.1 Material 16 3.2 Research methodogy 17 iii 3.2.1 Collection of samples 17 3.2.2 Isolation of lactic acid bacteria 17 3.2.3 Storage of isolated bacteria 17 3.2.4 Screening of Lactic acid bacteria 17 3.2.5 Screening of antimicrobial activity of isolated lab strains 19 3.2.6 Effect of cultivation time on peptides production 20 3.2.7 Characterization of crude bacteriocin 20 Chapter iv Results and discussion 22 4.1 Isolation and identification of lactic acid bacteria 22 4.2 Screening of antimicrobial activity of lactic acid bacteria 23 4.3 Effect of cultivation time on production peptides 24 4.4 Characterization of concentrated cell free supernatant 26 4.4.1 Effect of Enzymes 26 4.4.2 Characterization of concentrated cell free supernatant 26 Chapter v Conclusions and recommendation 29 5.1 Conclusions 29 5.2 Recomendations 29 References 31 Appendix 40 iv LIST OF TABLES Table 2.1 Popular bacteriocins produced by Lactobacilli Table 3.1 Materials, tools, chemical and equipment required for the whole experiment 16 Table 3.2 Morphological and Biochemical characteristics of selected potential strains producing Lactic acid 18 Table 4.1 Characteristics of isolated LAB strains 23 Table 4.2 Vietnamese fermented foods that were collected to isolate LAB 24 Table 4.3 Antimicrobial activity of concentrated cell free supernatant of FME1.7 and CS3.7 24 v LIST OF FIGURES Figure 2.1 Structure of Nisin Figure 2.2 Structure of Sakacin P Figure 2.3 Morphology of L monocytogenes cell 10 Figure 2.4 Morphology of B cereus cell 11 Figure 2.5 Morphology of Salmonella spp cell 12 Figure 2.6 Morphology of Escherichia coli cell 13 Figure 2.7 Rice chili and Muong Khuong chili sauce 13 Figure 2.8 Thanh Hoa pork roll 14 Figure 2.9 Thit chua Thanh Son 15 Figure 2.10 Cassava leaf and bamboo pickled 15 Table 3.1 Materials, tools, chemical and equipment required for the whole experiment 16 Table 3.2 Morphological and Biochemical characteristics of selected potential strains producing Lactic acid 18 Figure 4.1 Culture characteristics of selected strain on MRS medium added 1% CaCO3 22 Figure 4.2 Anti- Bacillus and Salmonella activity of concentrated cell free supernatant of FME1.7 (a) and CS3.7 (b) 23 Figure 4.3 Effect of cultivation time on antimicrobial activity of FME1.7 and CS3.7 26 Figure 4.4 Effect of papain enzyme 26 Figure 4.5 Antimicrobial activity of concentrated crude bacteriocin of FME1.7 and CS3.7 to pathogenic bacteria with pH range of 2-9, (Original : Crude peptides) 28 LIST OF DIAGRAM Diagram 3.1 Procedure of Screening of isolates for antimicrobial activity 19 vi ABBREVIATION & ACRONYMS Abbreviation Meaning LAB : Lactic Acid Bacteria MRS : DE MAN, ROGOSA and SHARPE LB : Luria Broth OD : Optical Density vii CHAPTER I INTRODUCTION 1.1 INTRODUCTION Food is essential for human being’s to live, and as a result, food safety has received increased attention Consumption of food contaminated with pathogens may cause certain disease events when it is contaminated with a very low infective dose In addition, foods contaminated with antibiotic resistant bacteria could be a major threat to public health as the antibiotic resistance determinants can be transferred to other pathogenic bacteria that later on cause compromises in the treatment of severe infections Recently, food safety has not only been an intractable problem in developing countries like Vietnam, but also in many countries around the world The risk of pathogenic microorganism contamination is increasing in agricultural products and food processing products Undoubtedly, the major threat to food safety is the emergence of pathogens such as Escherichia coli, Salmonella spp., Campylobacter spp., Listeria monocytogenes, Clostridium botulinum, Clostridium perfringens, or Bacillus cereus, which have been considered to be foodborne microorganisms (Castellano et al., 2008) There are several methods used to prevent foods from pathogenic contamination, such as freezing and thawing or using chemical substances However, food quality is decreased in terms of both nutrition and food safety when using those methods (Parada et al., 2007) So, new approaches to controlling foodborne pathogens in food processing and food preservation have been prompted For the past two decades, many studies have focused on the natural compounds produced by lactic acid bacteria (LAB) to apply in food preservation as LAB have been, so far, considered a food grade organism (Fricourt et al., 1994; Ogunbanwo et al., 2003; Parada et al., 2007) Moreover, LAB produce antimicrobial substances, such as acids, peptides, and hydrogen peroxide, among others, during their growth and development, of which, peptides have been proven to be the main group to have antimicrobial activity and to be safely applied in food preservation (Deegan et al., 2006; Settanni and Corsetti, 2008) A great deal of evidence has been reported that peptides produced by LAB have broad range capabilities against pathogenic bacteria activity (Nomoto, 2005) In addition, peptides are safe and stable in food processing and preservation, and are not deleterious to food Therefore, up to date, many studies on antimicrobial peptides from isolated lactic acid bacteria with expectations for food preservation have been published However, peptides from these studies have narrow range antimicrobial activity, and almost all of them against only gram-positive bacteria (Ivanova et al., 1998) Meanwhile, many bacteria contaminating food are gram-negative bacteria, such as E.coli and Salmonella spp That is why this study aims to isolate lactic acid bacteria from a selection of Vietnamese fermented foods, including fermented vegetables, fermented milks, and fermented meats, to explore new peptides with high ranges of antimicrobial activity and characterize the peptides for further applications 1.2 OBJECTIVE OF RESEARCH  General objective of research: Antimicrobial activity and characterization of peptides produced by lactic acid bacteria from some Vietnamese fermented foods  Specific objectives of this study are:  Isolation and identification of lactic acid bacteria from some Vietnamese fermented foods  Antimicrobial activity screening of isolates  Effect of cultivation time on peptides production  Characterization of bacteriocin: effect of enzymes (proteolytic enzymes, amylase), heat stability, pH sensitivity 34 Farhana S Diba, Khondoker M Hossain, M A Azim and Md Moinul Hoque, 2013 Isolation, Characterization and Determination of Antimicrobial Properties of Lactic Acid Bacteria from Human Milk Jordan Journal of Biological Sciences, pp 111- 116 35 Ennahar, S., Sonomoto, K., and Ishizaki, A (1999) Class IIa bacteriocins from lactic acid bacteria: antibacterial activity and food preservation J Biosci Bioeng 87 pp 705–716 36 Ercolini, D., Moschetti, G., Blaiotta, G., and Coppola, S (2001) The potential of a polyphasic PCRDGGE approach in evaluating microbial diversity of natural whey cultures 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ground beef by lactocin 705 Int J Food Microbiol 29 pp 397-402 96 Vilain S, Luo Y, Hildreth M, Brözel V 2006 Analysis of the life cycle of the soil saprophyte Bacillus cereus in liquid soil extract and in soil Applied and Environmental Microbiology, 72 pp 4970–4977 97 Vinod Kumar Joshi, Somesh Sharma, Neerja S Rana 2006 Production, Purification, Stability and Efficacy of Bacteriocin from Isolates of Natural Lactic Acid Fermentation of Vegetables Bacteriocin from Lactic Acid Fermented Vegetables, Food Technol Biotechnol 44 pp 435–439 Vietnamese Reference 98 Nguyễn Lân Dũng , Nguyễn Đình Quyến , Phạm Văn Ty 1978 Một số phương pháp nghiên cứu vi sinh vật học, 3th, Science and Technology publisher, Hanoi 39 APPENDIX Isolated Lactic acid Bacteria Catalase Gram stain No Sample Morphological colony CS 1.1 Small, circular, glossy, convex, opaque white - + CS 1.2 Small, circular, glossy, convex, creamy white - + CS 1.3 Small, circular, glossy, convex, yellow - + CS 1.4 Large, circular, glossy, opaque white - + CS 1.5 Small, circular, glossy, creamy white - + CS 1.6 Small, circular, glossy, yellow - + CS 2.1 Large, circular, viscous, yellow - + CS 2.2 Small, circular, viscous, yellow - + CS 2.3 Small, circular, convex, creamy white - + 10 CS 2.4 Large, circular, convex, opaque white - + 11 CS 3.1 Small, circular, glossy, opaque white - + 12 CS 3.2 Small, circular, convex, glossy, yellow - + 13 CS 3.3 Small, circular, convex, opaque white - + 14 CS 3.4 Large, circular, convex, yellow - + 15 CS 3.6 Large, circular, glossy, convex, yellow - + 16 CS 3.7 Small, circular, convex, opaque white - + 17 CS 4.1 Small, circular, convex, glossy, opaque white - + 18 CS 4.4 Large, convex, glossy, flat, , opaque white - + 19 CS 4.5 Small, circular, convex, glossy, opaque white - + 20 CS 4.6 Small, circular, convex, glossy, opaque white - + 21 CS 4.7 Large, circular, flat, slightly wave, yellow - + 22 CS 4.8 Small, circular, convex, glossy, yellow - + 23 CS 5.1 Small, circular, glossy, opaque white - + 40 24 CS 5.2 Small, circular, glossy, convex, opaque white - + 25 CS 5.4 Small, circular, glossy, convex, yellow - + 26 CS 6.1 Small, circular, glossy, convex, yellow - + 27 CS 6.10 Large, circular, glossy, convex, yellow - + 28 CS 6.2 Small, circular, convex, opaque white - + 29 CS 6.3 Small, circular, glossy, convex, opaque white - + 30 CS 6.4 Small, circular, glossy, convex, yellow - + 31 CS 6.5 Large, circular, flat, opaque white - + 32 CS 6.7 Small, circular, glossy, convex, opaque white - + 33 CS 6.9 Large, circular, flat, opaque white - + 34 CS3.5 Large, circular, glossy, convex, opaque white - + 35 CS3.8 Small, circular, convex, yellow - + 36 CS4.2 Small, circular, convex, glossy, yellow - + 37 CS4.3 Large, convex, glossy, yellow - + 38 CS5.3 Small, circular, glossy, convex, opaque white - + 39 CS6.8 Small, circular, glossy, flat, yellow - + 40 FBS 1.1 Large, circular, glossy, convex, opaque white - + 41 FBS 1.2 Small, circular, glossy, flat, yellow - + 42 FBS 1.3 Small, glossy, flat, yellow - + 43 FBS 2.1 Small, circular, glossy, convex, opaque white - + 44 FBS 2.2 Small, circular, glossy, convex, creamy white - + 45 FBS 2.3 Large, circular, glossy, opaque white - + 46 FC L1.1 Small, circular, glossy, convex, creamy white - + 47 FCL 1.2 Large, circular, glossy, convex, opaque white - + 48 FCL 1.3 Small, circular, glossy, creamy white - + 49 FCL 1.4 Small, circular, glossy, opaque white - + 50 FCL 1.5 Small, circular, glossy, convex creamy white - + 41 51 FCL 2.1 Small, circular, glossy, convex, opaque white - + 52 FCL 2.10 Small, circular, convex, creamy white - + 53 FCL 2.2 Lager, circular, glossy, convex, creamy white - + 54 FCL 2.3 Small, circular, glossy, flat, opaque white - + 55 FCL 2.4 Small, circular, convex, creamy white - + 56 FCL 2.5 Small, circular, glossy, creamy white - + 57 FCL 2.6 Small, circular, convex, glossy, opaque white - + 58 FCL 2.7 Large, circular, glossy, creamy white - + 59 FCL 2.8 Large, circular, glossy, creamy white - + 60 FCL 2.9 Small, circular, glossy, convex, creamy white - + 61 FE 1.43 Small, circular, glossy, yellow - + 62 FE 1.01 Small, circular, convex, creamy white - + 63 FE 1.02 Large, circular, convex, opaque white - + 64 FE 1.03 Small, circular, convex, opaque white - + 65 FE 1.04 Small, slightly wavy surface, convex, opaque white - + 66 FE 1.05 Small, circular, opaque white, glossy, convex - + 67 FE 1.06 Large, circular, glossy, slightly viscous, opaque - + 68 FE 1.1 Small, circular, glossy, convex, opaque white - + 69 FE 1.2 Small, circular, glossy, convex, creamy white - + 70 FE 1.3 Large, circular, glossy, convex, opaque white - + 71 FE 1.38 Small, circular, glossy, flat, creamy white - + 72 FE 1.39 Small, circular, glossy, flat, creamy white - + 73 FE 1.4 Large, circular, glossy, convex, creamy white - + 74 FE 1.40 Small, circular, glossy, flat, creamy white - + 75 FE 1.41 Small, circular, glossy, convex, creamy white - + 76 FE 1.42 Small, circular, glossy, convex, creamy white - + 42 77 FE 1.44 Small, circular, glossy, sharp, yellow - + 78 FE 1.45 Small, circular, glossy, convex, opaque white - + 79 FE 1.46 Large, circular, glossy, convex, yellow - + 80 FE 1.47 Large, circular, glossy, convex, creamy white - + 81 FE 1.48 Small, glossy, opaque white - + 82 FE 1.49 Small, circular, glossy, convex, yellow - + 83 FE 1.5 Small, circular, glossy, convex, creamy white - + 84 FE 1.50 Large, circular, glossy, sharp, creamy white - + 85 FE 1.51 Large, circular, glossy, convex, creamy white - + 86 FE 1.52 Small, circular, glossy, yellow - + 87 FE 1.6 Large, circular, glossy, convex, creamy white - + 88 FE 1.7 Small, circular, convex, creamy white - + 89 FE 2.07 Large, circular, glossy, convex, opaque white - + 90 FE 2.08 Small, circular, opaque white - + 91 FE 2.09 Large, circular, opaque white - + 92 FE 2.1 Large, circular, glossy, convex, creamy white - + 93 FE 2.10 Large, circular, glossy, convex, opaque white - + 94 FE 2.12 Small, circular, flat, opaque white - + 95 FE 2.15 Small, circular, convex, creamy white - + 96 FE 2.17 Large, circular, convex, creamy white - + 97 FE 2.2 Small, circular, glossy, convex, yellow - + 98 FE 2.20 Small, circular, glossy, convex, yellow - + 99 FE 2.21 Small, circular, glossy, convex, creamy white - + 100 FE 2.23 Small, circular, glossy, flat, yellow - + 101 FE 2.25 Large, circular, glossy, convex, creamy white - + 102 FE 2.26 Small, circular, glossy, convex, creamy white - + 103 FE 2.28 Small, circular, glossy, flat, yellow - + 43 104 FE 2.29 Large, circular, glossy, flat, yellow - + 105 FE 2.3 Small, circular, glossy, convex, opaque white - + 106 FE 2.30 Large, circular, glossy, flat, creamy white - + 107 FE 2.31 Large, circular, glossy, convex, creamy white - + 108 FE 2.32 Small, circular, glossy, opaque white - + 109 FE 2.33 Small, circular, glossy, sharp, creamy white - + 110 FE 2.34 Small, circular, glossy, convex, yellow - + 111 FE 2.35 Large, circular, glossy, sharp, opaque white - + 112 FE 2.36 Large, circular, glossy, sharp, creamy white - + 113 FE 2.37 Small, circular, glossy, convex, yellow - + 114 FE 2.4 Small, circular, creamy white - + 115 FE 2.5 Small, circular, glossy, convex, yellow - + 116 FE 2.6 Large, circular, convex, yellow - + 117 FE 2.8 Small, circular, flat, yellow - + 118 FE 2.9 Small, circular, glossy, flat, yellow - + 119 FE 3.1 Small, circular, glossy, convex, creamy white - + 120 FE 3.10 Small, circular, glossy, convex, opaque white - + 121 FE 3.11 Large, circular, glossy, convex, sharp, creamy white - + 122 FE 3.12 Large, circular, glossy, flat, yellow - + 123 FE 3.13 Large, circular, glossy, convex, yellow - + 124 FE 3.15 Small, circular, glossy, flat, creamy white - + 125 FE 3.16 Small, circular, glossy, convex, creamy white - + 126 FE 3.19 Large, circular, glossy, flat, opaque white - + 127 FE 3.2 Large, circular, glossy, convex, opaque white - + 128 FE 3.3 Small, circular, glossy, convex, yellow - + 129 FE 3.4 Large, circular, glossy, convex, creamy white - + 44 130 FE 3.5 Small, circular, glossy, flat, yellow - + 131 FE 3.6 Small, circular, glossy, convex, creamy white - + 132 FE 3.7 Large, circular, glossy, flat, opaque white - + 133 FE 3.8 Large, circular, glossy, convex, creamy white - + 134 FE 3.9 Small, circular, glossy, convex, creamy white - + 135 FMI 1.01 Small, convex, opaque white - + 136 FMI 1.01 Small, sharp, circular, yellow - + 137 FMI 1.02 Small, convex, creamy white - + 138 FMI 1.02 Large, sharp, convex, opaque white - + 139 FMI 1.03 Very small, convex, opaque white - + 140 FMI 1.03 Small, circular, glossy, opaque white - + 141 FMI 1.04 Large, circular, convex, glossy, opaque white - + 142 FMI 1.04 Small, circular, opaque white - + 143 FMI 1.05 Small, circular, glossy, convex, opaque white - + 144 FMI 1.05 Small, circular, glossy, opaque white - + 145 FMI 1.06 Small, circular, glossy, convex, creamy white - + 146 FMI 1.06 Large, circular, convex, opaque white - + 147 FMI 1.07 Small, circular, slightly wave, convex, opaque white - + 148 FMI 1.07 Large, circular, glossy, opaque white - + 149 FMI 1.08 Small, circular, convex, opaque white - + 150 FMI 1.08 Small, circular, convex, creamy white - + 151 FMI 1.09 Small, glossy, circular, convex, opaque white - + 152 FMI 1.10 Small, glossy, circular, convex, opaque white - + 153 FMI 1.11 Large, circular, convex, creamy white - + 154 FMI 2.09 Small, sharp, convex, creamy white - + 155 FMI 2.10 Small, sharp, creamy white - + 45 156 FMI 2.11 Small, sharp, circular, yellow - + 157 FMI 2.12 Small, sharp, circular, convex, yellow - + 158 FMI 2.12 Small, circular, flat, yellow - + 159 FMI 2.13 Small, circular, glossy, creamy white - + 160 FMI 2.13 Small, circular, creamy white - + 161 FMI 2.14 Large, circular, glossy, convex, yellow - + 162 FMI 2.15 Small, circular, glossy, convex, opaque white - + 163 FMI 2.15 Small, circular, convex, opaque white - + 164 FMI 2.16 Small, circular, glossy, creamy white - + 165 FMI 2.17 Small, circular, glossy, convex, opaque white - + 166 FMI 2.17 Small, circular, glossy, convex, creamy white - + 167 FMI 2.18 Small, sharp, circular, convex, yellow - + 168 FMI 2.19 Large, circular, glossy, slightly viscous, opaque - + 169 FMI 2.6 Small, circular, opaque white - + 170 FMI 2.14 Small, circular, glossy, convex, creamy white - + 46 LAB have antimicrobial activity Antimicrobial activity No Sample 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 CS1.7 CS2.7 CS3.7 FBS1.4 FCL1.1 FCL1.2 FCL1.4 FCL1.4 FCL2 FCL2.1 FCL2.7 FCL2.8 FE 234 FE 235 FE1.38 FE1.40 FE1.44 FE1.45 FE1.46 FE1.47 FE1.51 FE1.52 FE2.1 FE2.20 FE2.21 FE2.23 FE2.26 FE2.28 FE2.3 FE2.30 FE2.31 FE2.32 FE2.33 Salmonella spp E.coli ++ + + + ++ ++ + + + ++ ++ ++ ++ ++ ++ + ++ ++ ++ ++ - 47 L.monocytegenes B.cereus L plantarum + + + + ++ ++ + - + + + +++ +++ +++ +++ ++ + + ++ ++ + + ++ ++ ++ - +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 FE2.34 FE2.35 FE2.36 FE2.37 FE221 FE3.12 FE3.15 FE3.19 FE3.4 FE3.5 FE3.7 FM2.14 FME1.7 FME2.3 FMI1.04 FMI1.10 FMI2.15 FMI2.18 FMI35 ++ ++ + + ++ + ++ + - + ++ + ++ + - + + ++ ++ + + + + ++ + +++ + + + ++ ++ + +: < 2mm; ++: 2-5mm: +++: > 5mm; -: 0mm 48 +++ +++ + + + + ++ ++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ... by lactic acid bacteria from some Vietnamese fermented foods  Specific objectives of this study are:  Isolation and identification of lactic acid bacteria from some Vietnamese fermented foods. .. Isolation of lactic acid bacteria 17 3.2.3 Storage of isolated bacteria 17 3.2.4 Screening of Lactic acid bacteria 17 3.2.5 Screening of antimicrobial activity of isolated. .. isolate lactic acid bacteria from a selection of Vietnamese fermented foods, including fermented vegetables, fermented milks, and fermented meats, to explore new peptides with high ranges of antimicrobial