Effects of culture conditions on the ability to produce bacteriocins of enterococcus faecium strain et02

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY  GRADUATION THESIS TITLE: “EFFECTS OF CULTURE CONDITIONS ON THE ABILITY TO PRODUCE BACTERIOCINS OF ENTEROCOCCUS FAECIUM STRAIN ET02” Hanoi - 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY  GRADUATION THESIS TITLE: “EFFECTS OF CULTURE CONDITIONS ON THE ABILITY TO PRODUCE BACTERIOCINS OF ENTEROCOCCUS FAECIUM STRAIN ET02” Student name : TRAN HUONG LY Class : K63CNSHE Student’s code : 637418 Supervisor : PHAM HONG HIEN, Ph.D NGUYEN XUAN CANH, Assoc.Prof Department : MICROBIAL TECHNOLOGY Hanoi - 2022 COMMITMENT I hereby declare that the data and results stated in the thesis are honest and have never been published by anyone in other studies The graduations with references to documents and action information are indicated in the references section I take full responsibility for the data in this thesis Hanoi, December 5th, 2022 Sincerely Tran Huong Ly i ACKNOWLEDGEMENTS Firstly, I would like to thank the Academy's leadership and the Department of Biotechnology teachers for helping and creating conditions for me to complete the thesis Secondly, I would like to express my most sincere thanks to Assoc Prof Nguyen Xuan Canh was interested, was guided, and created all favorable conditions to help me in my research and completed my thesis Thirdly, I would also like to thank the teachers of the Department of Microbiology, Assoc.Prof Nguyen Van Giang, Msc Tran Thi Hong Hanh, Msc Nguyen Thanh Huyen, Msc Tran Thi Dao and fellow researchers Duong Van Hoan, Nguyen Thi Thu helped me during the course of my thesis My completion of this project could not have been accomplished without their support of them At last but not least, I am thankful to my friends who conducted the thesis in the Department of Microbiology and my family for their encouragement, encouragement, and help so that I could complete my graduation thesis I sincerely thank you! Hanoi, December 5th, 2022 Sincerely Tran Huong Ly ii CONTENTS COMMITMENT i ACKNOWLEDGEMENTS ii CONTENTS iii LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS viii ABSTRACT ix INTRODUCTION PART I LITERATURE REVIEW 1.1 Overview of Enterococcus spp 1.1.1 Concept of Enterococcus faecium 1.1.2 Role of Enterococcus faecium 1.2 Overview of bacteriocins 1.3 Classification of bacteriocins 1.3.1 Class I bacteriocins 1.3.1.1 Class Ia 1.3.1.2 Class Ib 1.3.2 Class II bacteriocins 1.3.2.1 Class IIa 1.3.2.2 Class IIb 1.3.2.3 Class IIc 1.3.3 Class III bacteriocins 1.3.4 Class IV bacteriocins 1.4 The mechanism of action of bacteriocins 1.5 Biochemical properties of bacteriocins 11 1.5.1 Heat resistance 11 1.5.2 pH stability 11 1.5.3 Enzyme stability 11 iii 1.6 The application of bacteriocins 13 1.6.1 Application in food preservation 13 1.6.2 Applications in health care 14 1.6.3 Commercial bacteriocins 14 1.6.4 Probiotics 15 1.7 Advantages and disadvantages of bacteriocins 16 1.7.1 Advantages of bacteriocins 16 1.7.2 Disadvantages of bacteriocins 16 1.8 Effect of medium composition 16 1.9 Effect of cultivation condition 18 1.9.1 Temperature 18 1.9.2 pH 19 1.10 Bacteriocins is produced Enterococcus faecium 20 1.11 Research status of bacteriocins from Enterococcus faecium 22 1.11.1 Domestic studies 22 1.11.2 Studies in the world 22 PART II MATERIALS AND METHODS 24 2.1 Research subjects and materials 24 2.1.1 Location and time of the study 24 2.1.2 Materials 24 2.1.3 Equipment and chemicals 24 2.1.4 Medium 24 2.2 Methods 25 2.2.1 Identification and analysis by 16s rRNA gene sequencing 25 2.2.2 Investigation of antibacterial ability 25 2.2.3 Effect of the medium ingredients on bacteriocin production 26 2.2.3.1 Effects of cacbon sources on bacteriocin production 26 2.2.3.2 Effect of nitrogen sources on bacteriocin production 26 2.2.3.3 Effect of NaCl concentration on bacteriocin production 27 2.2.4 Effect of pH on bacteriocin production 27 2.2.5 Effect of temperature on bacteriocin production 27 iv 2.2.6 Effect of time on bacteriocin production 27 PART III RESULTS AND DISCUSSION 29 3.1 Enterococcus faecium strain ET02 29 3.2 Effect of the medium ingredients on bacteriocin production 30 3.2.1 Effects of cacbon sources on bacteriocin production 30 3.2.2 Effect of nitrogen sources on bacteriocin production 32 3.2.3 Effect of NaCl concentration on bacteriocin production 33 3.3 Effect of pH on bacteriocin production 33 3.4 Effect of temperature on bacteriocin production 35 3.5 Effect of time on bacteriocin production 36 CONCLUSION AND PROPOSAL 37 REFERENCES 38 APPENDIX 47 v LIST OF TABLES Table 1.1 Heat, pH, and enzymatic hydrolytic stability of some bacteriocins biosynthesized by lactic acid bacteria 12 Appendix Effect of culture medium composition on bacteriocin .50 Appendix Effect of cultivation condition 52 vi LIST OF FIGURES Figure 3.1 A: Colony of the Enterococcus faecium strain ET02; B: Phylogenetic tree of Enterococcus faecium strain ET02 29 Figure 3.2 The effect of C-source on the antibacterial ability of Enterococcus faecium strain ET02 30 Figure 3.3 The effect of different glucose concentrations on the antibacterial ability of Enterococcus faecium strain ET02 31 Figure 3.4 The effect of diferent peptone concentrations on the antibacterial ability of Enterococcus faecium strain ET02 32 Figure 3.5 The effect of adding NaCl 1%, 2%, 3%, 4% on the antibacterial ability of Enterococcus faecium strain ET02 33 Figure 3.6 The effect of pH on the antibacterial ability of Enterococcus faecium strain ET02 34 Figure 3.7 The effect of temperature on the antibacterial ability of Enterococcus faecium strain ET02 35 Figure 3.8 The effect of time on the antibacterial ability of Enterococcus faecium strain ET02 36 Appendix Effect of culture medium composition on bacteriocin .47 Appendix Effect of cultivation condition 48 vii LIST OF ABBREVIATIONS Abbreviation Full word LAB Lactic acid bacteria Spp Several species E faecium Enterococcus faecium E faecalis Enterococcus faecalis Sp specie LB Luria Bertani NB Nutrient Broth MRS De Man, Rogosa and Sharpe µl Microliter S aureus Staphylococcus aureus A hydrophila Aeromonas hydrophila kDa Kilodalton viii REFERENCES Vietnamese Lê Thanh Bình, Phan Thị Khánh Hoa, Phạm Thị Ngọc Lan, Trần Thị Thúy (2000) Hoạt tính sinh tổng hợp bacteriocin khuẩn lactic Các cơng trình nghiên cứu ứng dụng công nghệ sinh học công nghiệp thực phẩm giai đoạn 1996-2000, Bộ Công Nghiệp, Viện công nghiệp thực phẩm Nxb Khoa học kỹ thuật Lê Quang Huấn, Đinh Duy Kháng, Trương Nam Hải, Đồng Văn Quyền, Lê Trần Bình (2000) Sử dụng kỹ thuật di truyền việc thu nhận peptit tái tổ hợp có hoạt tính kháng khuẩn Báo cáo khoa học Hội nghị Sinh học Quốc gia 89-93 Phạm Thùy Linh (2011) Nghiên cứu khả taọ chất diệt khuẩn enterocin P tái tổ hơp ṇ hằm ứng dụng bảo quản thực phẩm Luận án Tiến sĩ, Trường Đại học Khoa học Tự nhiên, Hà Nội English And H C & Hoover D (2003) Bacteriocins and their food applications Comprehensive reviews in food science and food safety 2(3): 82-100 Arias C A & Murray B E (2012) The rise of the Enterococcus: beyond vancomycin resistance Nature Reviews Microbiology 10(4): 266-278 Baquero F (2004) From pieces to patterns: evolutionary engineering in bacterial pathogens Nature Reviews Microbiology 2(6): 510-518 Basanta A., Sánchez J., Gómez-Sala B., Herranz C., Hernández P E & Cintas L M (2008) Antimicrobial activity of Enterococcus faecium L50, a strain producing enterocins L50 (L50A and L50B), P and Q, against beer-spoilage lactic acid bacteria in broth, wort (hopped and unhopped), and alcoholic and non-alcoholic lager beers International journal of food microbiology 125(3): 293-307 BE M (1990) The life and times of the Enterococcus Clin Microbiol Rev 3: 46-65 38 Bhardwaj A., Gupta H., Kapila S., Kaur G., Vij S & Malik R K (2010) Safety assessment and evaluation of probiotic potential of bacteriocinogenic Enterococcus faecium KH 24 strain under in vitro and in vivo conditions International journal of food microbiology 141(3): 156-164 10.Bhardwaj A., Kapila S., Mani J & Malik R K (2009) Comparison of susceptibility to opsonic killing by in vitro human immune response of Enterococcus strains isolated from dairy products, clinical samples and probiotic preparation International journal of food microbiology 128(3): 513515 11.Björkroth J., Ristiniemi M., Vandamme P & Korkeala H (2005) Enterococcus species dominating in fresh modified-atmosphere-packaged, marinated broiler legs are overgrown by Carnobacterium and Lactobacillus species during storage at degree C International journal of food microbiology 97(3): 267-276 12.Bonten M J., Willems R & Weinstein R A (2001) Vancomycin-resistant enterococci: why are they here, and where they come from? 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Staphylococcus aureus 47 ❖ NaCl 1%, 2%, 3%, 4% N1 N2 N1 control control N3 N3 N4 Aeromonas hydrophila N2 N4 Staphylococcus aureus Appendix Effect of cultivation condition ❖ pH Aeromonas hydrophila Staphylococcus aureus Aeromonas hydrophila Staphylococcus aureus ❖ Time 48 ❖ Temperature Aeromonas hydrophila Staphylococcus aureus 49 Appendix Effect of culture medium composition on bacteriocin ❖ Different carbon sources 2% Composition of culture medium Antibacterial ring diameter (mm) A Hydrophila S Aureus Glucose 12.00 ± 1.00 11.00 ± 1.00 Fructose 10.67 ± 1.15 7.67 ± 2.30 Saccarose 9.33 ± 0.58 6.67 ± 2.89 Lactose 9.67 ± 1.15 6.33 ± 2.31 ❖ Glucose 1%, 2%, 3%, 4% Composition of culture medium Antibacterial ring diameter (mm) A Hydrophila S Aureus Glucose 1% 0 Glucose 2% 10.00 ± 1.00 11.67 ± 0.58 Glucose 3% 13.33 ± 0.58 14.67 ± 0.58 Glucose 4% 12.00 ± 0.00 13.67 ± 0.58 ❖ Peptone 1%, 2%, 3%, 4% Composition of culture medium Antibacterial ring diameter (mm) A Hydrophila S Aureus Peptone 1% 11.67 ± 0.58 14.33 ± 0.58 Peptone 2% 12.00 ± 0.00 14.33 ± 0.58 Peptone 3% 15.33 ± 0.58 18.33 ± 0.58 Peptone 4% 13.00 ± 0.00 15.67 ± 0.58 50 ❖ NaCl 1%, 2%, 3%, 4% Composition of culture medium Control Antibacterial ring diameter (mm) A Hydrophila S Aureus 11.67 ± 0.58 13.67 ± 0.58 NaCl 1% 8.33 ± 0.58 9.67 ± 0.58 NaCl 2% 8.33 ± 0.58 9.00 ± 0.00 NaCl 3% 9.00 ± 0.00 9.33 ± 0.58 NaCl 4% 8.67 ± 0.58 9.67 ± 0.58 51 Appendix Effect of cultivation condition ❖ pH Antibacterial ring diameter (mm) pH A Hydrophila S Aureus 4.5 15.33 ± 0.58 14.67 ± 0.58 5.5 14.33 ± 0.58 15.33 ± 0.58 6.5 17.67 ± 0.58 17.33 ± 0.58 7.5 14.67 ± 0.58 13.33 ± 0.58 8.5 13.00 ± 1.00 10.33 ± 0.58 9.5 0 ❖ Time Time Antibacterial ring diameter (mm) A Hydrophila S Aureus 2days 10.83 ± 0.29 16.83 ± 0.29 4days 12.17 ± 0.29 18.00 ± 0.00 6days 10.83 ± 0.29 17.17 ± 0.29 8days 11.17 ± 0.29 16.67 ± 0.29 ❖ Temperature Antibacterial ring diameter (mm) Temperature A Hydrophila S Aureus 25°C 14.67 ± 0.58 14.5 ± 0.58 30°C 16.67 ± 0.58 16.5 ± 0.58 35°C 14.33 ± 0.58 14.5 ± 0.58 52