THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY TRINH THI MY DUYEN Topic title: STUDY ON THE EVALUATION OF PROBIOTICS AS ENVIRONMENTAL CLEANING AGENTS BACHELOR THESIS Study Mode : Full-time Major : Environmental Science and Management Faculty : Advanced Education Program Office Batch : 2015 – 2019 Thai Nguyen, 10 / 2019 Luan van THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY TRINH THI MY DUYEN Topic title: STUDY ON THE EVALUATION OF PROBIOTICS AS ENVIRONMENTAL CLEANING AGENTS BACHELOR THESIS Study Mode : Full-time Major : Environmental Science and Management Faculty : Advanced Education Program Office Batch : 2015 – 2019 Supervisors : Assoc. Tran Van Dien Thai Nguyen, 10 / 2019 Luan van DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Major Environmental Science and Management Student name Trinh Thi My Duyen Student ID DTN155429007 Study on the evaluation of probiotics as environmental cleaning Thesis Title agents 1. Tran Van Dien Abstract: This is an innovative and innovative project developed from a project to use probiotics to enhance the health of animals and plants into projects on environmental protection. This is a study of probiotic bacteria.
About the effectiveness of probiotic bacteria in different biological and abiotic conditions. From there, it is possible to create disinfectant reagents to replace the use of chemicals from the antibacterial activities of each strain of bacteria. Health group bacteria and joint group bacteria have been used in this experiment. Health bacteria contain three strains of biological bacteria.
Its function is to maintain health. Joint bacteria contain three strains of biological bacteria. The function of the general group is to strengthen the health of the joint. By sequencing the 16S rRNA and DNA genes amplified through polymerase chain reaction using 16S-F3R3 primers.
The sequence of bacteria was sequenced after that the result was compared with the gene bank on the NCBI website. Genetic analysis showed that the strains identified were Lactobacillus rhamnosus, Lactobacillus paracasei. Lactobacillus pentosus or Lactobacillus Plantarum subsp, Lactobacillus Plantarum. i Luan van Then test the antibacterial activity of each strain with pathogenic bacteria are staphylococcus aureus and pseudomonas aeruginosa.
This study was created to evaluate the environmental cleanup feature of probiotic bacteria. Keywords Antibacterial, 16S rRNA, 16S-F3R3, NCBI, Number of pages 48 Date of Submission 10 / 2019 ii Luan van ACKNOWLEDGMENT From the bottom of my heart, I would like to express my deepest appreciation to all those who provided me the opportunity to complete this research First and foremost, I would like to express my sincere gratitude and deep regard to my supervisors: Dr. Shyu of National Pingtung University of Science and Technology. Who kindly was very patient with my knowledge gaps and in guiding me wholeheartedly when I implemented this research.
I also want to express my thanks to Dr. Tran Van Dien of Thai Nguyen University of Agriculture and Forestry, the second supervisor, for his supervision, encouragement, advice, and guidance in writing this thesis. In addition, formal thanks should be offered to the dean and Associate Professor of office of international affairs of National Pingtung University of Science and Technology, Dr. Pomin Li for granting my internship acceptance.
I would also like to acknowledge with much appreciation to the Douglas J. Shyu, PhD Associate Professor Functional Genomics Laboratory Department of Biological Science and Technology National Pingtung University of Science and Technology, who gave the permission to use all required equipment and the necessary materials to conduct my research in Laboratory of Department of Biological Science and Technology I wish to thank the technicians who work in the Department of Biological Science and Technology My supervisor Dr. Shyu who provided me probiotic bacteria for free. Without them, this research could not be accomplished on time.
iii Luan van Of course, I would like to thank my Taiwan friends – Yu Wen, Liyu, Wily, Ito, Chau, and others for their invaluable support and encouragement when I stayed in Pingtung. Finally, special thanks to my family, my friends for their love and moral support throughout my studies. Thank you very much and best regards. Thai Nguyen, September 25 Student Trinh Thi My Duyen iv Luan van TABLE OF CONTENT DOCUMENTATION PAGE WITH ABSTRACT.
iii TABLE OF CONTENT. v LIST OF FIGURES. 1 LIST OF TABLES. 3 LIST OF ABBREVIATIONS.
Scope of study. Definition of Probiotics. MATERIAL AND METHOD .1 Equipment and materials .1 Culture of bacteria.7 Cloning of Screened Gene into yT&A-Vector following Transformed into DH5α.8 Antibacterial activity assay. The result of the culture of bacteria.
The result of gram staining. The results of the DNA extraction of six strains in the study. The results of PCR amplification. The results of gene cloning.1 Results of transforming plasmid DNA into variable cells of E.
The results of DNA Plasmid Extraction by Restriction Enzyme digestion. Identify and analyze the nucleotide sequence of the DNA markers.7 The results of the antibacterial activity assay .1 Antibacterial of mix strains of bacterial.2 Antibacterial of each strain. 45 vi Luan van LIST OF FIGURES Figure 2. The powder of bacteria in this study.
MRS broth medium and American Bacteriological Agar. Lysozyme Buffer for DNA extraction. The cell of Escherichia coli (DH5α) in this study. The process of DNA extraction bacteria.
The process of DNA purification from Agarose gel. The process of transformation reaction. The process of Plasmid DNA extraction.9 The image illustrates how to prepare work on the MRS agar plate. The two groups of bacteria isolated growth on media .2: The shape and arrangements of bacteria observed under microscope.
The results of DNA extraction. The results of PCR amplification with primer pair 16S-F3R3. The result of transforming the recombinant vector into competent cells. The results of electrophoresis of enzyme-cut products by EcoRI and HindIII enzyme of the 16S-F3R3 primer.
The software to compare sequences on gene banks on the NCBI website. Phylogenetic tree gene and homologous rate of J2-16S-F3R3 strain 38 Figure 3. Phylogenetic tree gene and homologous rate of J6-16S-F3R3 strain 38 Figure 3. Phylogenetic tree gene and homologous rate of Ht4-16S-F3R3 strain.
Phylogenetic tree gene and homologous rate of Ht6-16S-F3R3 strain .12 The level of anti-Staphylococcus aureus. 40 1 Luan van Figure3.13 The level of anti- Pseudomonas aeruginosa.14 The anti- Staphylococcus aureus level of J2, J6, HT4, and HT6 strain .15 The anti-Pseudomonas aeruginosa level of J2, J6, HT4, and HT6 strain. 42 2 Luan van LIST OF TABLES Table 2. Equipments of studies.
50X TAE buffer composition. 1,2 % Agarose gel composition. The component of PCR reaction amplification gen 16S rRNA. The sequences of primer used for PCR reaction to identify Characterization of rhizobacteria in sesame.
The component of the ligation reaction. The component of restriction enzyme digestion reaction. Identify 6 strains in the study on gene bank. The antibacterial level of mix strains is measured in units of millimeters.
The anti-Staphylococcus aureus level. The anti-Pseudomonas aeruginosa. 42 3 Luan van LIST OF ABBREVIATIONS DNA Deoxyribonucleic acid % Percentage PCR Polymerase chain reaction °C Degree Celsius µg Microgram (s) µg/ml Micrograms per milliliter µl Microlitre NCBI National Center for Biotechnology Information EDTA Ethylenediaminetetraacetic acid LB broth Luria Bertani Broth G Gram H2 O Distilled water g/l Gram per liter Mm Millimeter HCl Acid hydrochloric EtBr Ethidium bromide min. Minute (s) Ml Milliliters (s) ml/l Milliliter per liter TAE Triethanolamine V Volt Rpm Rotation per minute MRS Broth De MAN, ROGOSA, and SHARPE broth 4 Luan van PART I.
Background Multi-drug resistant bacteria are the cause of numerous clinical problems throughout the world. Increased resistance among pathogens causing nosocomial and community-acquired infections is known to be related to the widespread utilization of antibiotics (Pfaller et al. Infectious diseases caused by resistant microorganisms are accountable for increased health costs as well as high morbidity and mortality, especially in developing countries. Pseudomonas aeruginosa is an opportunistic gram-negative bacterium which is a major cause of nosocomial infections, usually occurring in the context of serious underlying diseases and accounting for nearly 10% of all hospital-acquired infections of surgical sites, the respiratory tract and the urinary tract (Jarvis et al.
High genotypic diversity of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis in the Czech Republic. Res in Microbiol. It is also prevalently related to otitis media and nasal infections and represents a leading cause of morbidity due to burn wound infection (Brook, 1994; Lyczak et al. Aeruginosa has an inherent resistance to most available antibiotics, including aminoglycosides, anti-pseudomonal penicillins, newer cephalosporins, imipenem, and fluoroquinolones as treatment options for systemic infections (Burgess, 2005; Paul et al., 2004; Patzer et al.
Similarly, Staphylococcus aureus (S. aureus) belongs to a gram-positive group with a cocci shape. It was found in grape-like clusters. aureus is mostly found in the anterior nares in humans which are the favorable conditions for maintenance and infection.
Especially, 5 Luan van S.aureus has a characteristic of biofilm formation. aureus enters into the circulatory system, it avoids the detection by the immune system, binds to a specific surface including infection area and forms a biofilm to survive in the host (Lowy et al. Moreover, its biofilm can be created on both biotic and abiotic surface; so, S. aureus shows resistance to antibiotics that becomes a problem in treatment (Fedtke et al., 2004; Greenber et al.
Joh et al. Park et al., 1996; Patti et al. Some infectious diseases related to the S .aureus’ biofilm formation were arthritis, endocarditis, and cystic fibrosis (Costa et al. Bacteria could have characteristics to form a biofilm.
The extracellular polymeric matrix is made from the combination of exopolysaccharides, proteins, teichoic acids, enzymes, and extracellular DNA (Melchior et al., 2006; Parra-Ruiz et al. Regarding to previous studies, the matrix’s structure is changed from strains to strains due to the environment and the conditions (Rohde et al. By living in a community, biofilms have various benefits and advantages from their parts and one of them is resistant to the immune system and antibiotics. Recent reports have documented the role of exogenous Lactobacilli in the prevention and treatment of some infections.
Lactobacillus acidophilus is gram-positive bacteria naturally living in the human and animal digestive system. Lactobacillus is also used in dairy products including milk, yogurt… in combination with other microbes. Lactobacillus has the potential to be used as an antibiotic medicine and a drug deliver ( TTV Doan et al. Recent reports have documented the role of exogenous Lactobacilli in the prevention and treatment of some infections.
6 Luan van Lactobacillus strains are commensal in the human body. Oral administration of Lactobacillus strains has been found to be useful in various bacterial infections (Gordon et al., 1957; Vandenbergh, 1993; Carson, Riley, 2003). Its beneficial effect may be associated to its ability to inhibit the growth of pathogens, apparently by the secretion of antibacterial substances including lactic acid, hydrogen peroxide and etc. As the above statement, the aim of the study of Lactobacillus was to know whether Lactobacillus inhibits Staphylococcus aureus and Pseudomonas aeruginosa or not.
Objectives This in vitro experiment was designed to identify strains of bacteria is a base to analysis the antibacterial activity and to evaluate the antibacterial effectiveness of probiotic bacteria in different biological and abiotic conditions. From there, it is as a premise possible to create disinfectant reagents to replace the use of chemicals from the antibacterial activities of each strain of bacteria identified. The main aim of this study is to contribute to protecting the environment. Propose the cleaning agent from probiotic to substitute chemicals cleaning agent, which does not eliminate pathogenic bacteria only but also be an environmentally friendly agent.
Scope of study - The provided bacteria cultured in MRS broth environment. - The bacteria DNA extraction. The amplification genes by PCR reaction using 16S rRNA primers. - Ligation and transformation reactions to joining of two nucleic acid fragments through the action of an enzyme performed using T4 DNA ligase.