(LUẬN văn THẠC sĩ) study on the evaluation of probiotics as environmental cleaning agents

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 download by : skknchat@gmail.com 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 Prof Douglas J H Shyu Assoc Prof Tran Van Dien Thai Nguyen, 10 / 2019 download by : skknchat@gmail.com 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 Assoc Prof Douglas J H Shyu Supervisors Assoc Prof 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 download by : skknchat@gmail.com 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 Number of pages Date of Submission Antibacterial, 16S rRNA, 16S-F3R3, NCBI, 48 10 / 2019 ii download by : skknchat@gmail.com 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 Douglas J H 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 H 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 Douglas J H Shyu who provided me probiotic bacteria for free Without them, this research could not be accomplished on time iii download by : skknchat@gmail.com 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 download by : skknchat@gmail.com TABLE OF CONTENT DOCUMENTATION PAGE WITH ABSTRACT i ACKNOWLEDGMENT iii TABLE OF CONTENT v LIST OF FIGURES LIST OF TABLES LIST OF ABBREVIATIONS PART I INTRODUCTION 1.1 Background 1.2 Objectives 1.3 Scope of study PART II LITERATURE REVIEW 2.1 Definition of Probiotics 2.2 Biofertilizers 10 PART III MATERIAL AND METHOD 12 3.1 Equipment and materials 12 3.1.1 Equipment 12 3.1.2 Materials 13 3.2 Method…… …15 3.2.1 Culture of bacteria 15 3.2.2 Gram staining 17 3.2.3 DNA extraction 18 3.2.4 DNA Electrophoresis 20 3.2.5 PCR amplification 21 3.2.7 Cloning of Screened Gene into yT&A-Vector following Transformed into DH5α 25 v download by : skknchat@gmail.com 3.2.7.1 Ligation 25 3.2.8 Antibacterial activity assay 30 PART IV RESULTS 32 4.1 The result of the culture of bacteria 32 4.2 The result of gram staining 32 4.3 The results of the DNA extraction of six strains in the study 33 4.4 The results of PCR amplification 34 4.5 The results of gene cloning 35 4.5.1 Results of transforming plasmid DNA into variable cells of E coli DH5α 35 4.5.2 The results of DNA Plasmid Extraction by Restriction Enzyme digestion 36 4.6 Identify and analyze the nucleotide sequence of the DNA markers 37 4.7 The results of the antibacterial activity assay 39 4.7.1 Antibacterial of mix strains of bacterial 39 4.7.2 Antibacterial of each strain 41 PART V CONCLUSIONS 43 5.1 Conclusion 43 5.2 Recommendations 44 REFERENCES 45 vi download by : skknchat@gmail.com LIST OF FIGURES Figure 2.1 The powder of bacteria in this study 13 Figure 2.2 MRS broth medium and American Bacteriological Agar 14 Figure 2.3 Lysozyme Buffer for DNA extraction 14 Figure 2.4 The cell of Escherichia coli (DH5α) in this study 15 Figure 2.5 The process of DNA extraction bacteria 18 Figure 2.6 The process of DNA purification from Agarose gel 23 Figure 2.7 The process of transformation reaction 26 Figure 2.8 The process of Plasmid DNA extraction 29 Figure 2.9 The image illustrates how to prepare work on the MRS agar plate 31 Figure 3.1 The two groups of bacteria isolated growth on media 32 Figure 3.2: The shape and arrangements of bacteria observed under microscope 33 Figure 3.3 The results of DNA extraction 33 Figure 3.4 The results of PCR amplification with primer pair 16S-F3R3 34 Figure 3.5 The result of transforming the recombinant vector into competent cells 35 Figure 3.6 The results of electrophoresis of enzyme-cut products by EcoRI and HindIII enzyme of the 16S-F3R3 primer 36 Figure 3.7 The software to compare sequences on gene banks on the NCBI website 37 Figure 3.8 Phylogenetic tree gene and homologous rate of J2-16S-F3R3 strain 38 Figure 3.9 Phylogenetic tree gene and homologous rate of J6-16S-F3R3 strain 38 Figure 3.10 Phylogenetic tree gene and homologous rate of Ht4-16S-F3R3 strain 38 Figure 3.11 Phylogenetic tree gene and homologous rate of Ht6-16S-F3R3 strain 39 Figure3.12 The level of anti-Staphylococcus aureus 40 download by : skknchat@gmail.com Figure3.13 The level of anti- Pseudomonas aeruginosa 40 Figure3.14 The anti- Staphylococcus aureus level of J2, J6, HT4, and HT6 strain 41 Figure3.15 The anti-Pseudomonas aeruginosa level of J2, J6, HT4, and HT6 strain 42 download by : skknchat@gmail.com was relatively successful, there is no DNA was broken, the bands were bright and clear, qualified to make materials for subsequent experiments 4.4 The results of PCR amplification Figure 3.4 The results of PCR amplification with primer pair 16S-F3R3 After optimizing the specific priming temperature, the DNA samples cloned by PCR reaction with a pair of research primers The PCR products after successful amplification follow electrophoresis on 1.2% agarose gel for 30 minutes The figure indicated PCR product was only one bright band, clear and not broken with a size of approximately 1100 bp with primer pair 16S-F3R3 The reaction of PCR products can be carried out into the next splitting vector 34 download by : skknchat@gmail.com 4.5 The results of gene cloning 4.5.1 Results of transforming plasmid DNA into variable cells of E coli DH5α Colony J2 Colony Ht1 Colony J3 Colony J6 Colony Ht4 Colony Ht6 Figure 3.5 The result of transforming the recombinant vector into competent cells Figure 3.5 showed the results of the transformation of the recombinant vector into variable cells had good results On the culture plate, there were white colonies with relative density and quantity and enough for the process of selecting colonies was easy Then colony lines will be reared in ml LB liquid medium with ampicillin supplemented with 1ml/l, shake 200 rounds/minute overnight in order to conduct DNA plasmid extraction 35 download by : skknchat@gmail.com 4.5.2 The results of DNA Plasmid Extraction by Restriction Enzyme digestion The plasmids after extraction should be plasmid-cut with EcoRI and Hind III restriction enzyme to confirm whether the plasmid carries the gene segment of interest Theoretically, after limiting the recombinant enzyme plasmid, there will be parts with DNA: i) the bottom is DNA fragment has been transformed and ii) above is the plasmid remains after the DNA fragment has been lost Joint colony group Healthy colony group Figure 3.6 The results of electrophoresis of enzyme-cut products by EcoRI and HindIII enzyme of the 16S-F3R3 primer The plasmid separation results are divided into two parts, including size DNA bands of 600 bp and 1100 bp respectively that exactly as originally planned 36 download by : skknchat@gmail.com 4.6 Identify and analyze the nucleotide sequence of the DNA markers The DNA segments after successful cloning will be sent to the Kiron Meek Genomics company (the company that takes the lead in sequencing in Taiwan) The sequence is determined on an automatic sequence reader according to Sanger's principle After processing the sequence using Bioedit software and comparing it with the information on GenBank (NCBI) (Figure 3.9), we conducted an analysis of the results with a primer studied as follows Figure 3.7 The software to compare sequences on gene banks on the NCBI website Results of identification and sequence analysis of 16S-F3R3 gene The J2-5; J3-1; J6-7; Ht1-6; Ht4-4 and Ht6-10 strains were sequenced with 16S-F3R3 gene sequences with the following results: The analytical gene sequence is about 1100 bp compared to the estimated size of 1100 bp, but in the sequence, there are many valuable useful differences that can be used to classify The J2-5 strain had a 99.3% homologous rate with the DNA sequence of Lactobacillus pentosus or Lactobacillus Plantarum subsp 98.6% 37 download by : skknchat@gmail.com is the homologous rate of J6-7 with Lactobacillus Plantarum strain The Ht4-4 strain had 99.7% homologous rates similar to Lactobacillus rhamnosus strain The data show that Ht6-10 had 97.9% like Lactobacillus paracasei strain J3-1 and Ht1-6 are two strains that have not been determined and the result when sequencing is "vector" Therefore, these four strains identified are beneficial bacteria that have been published in various countries in the NCBI data bank Figure 3.8 Phylogenetic tree gene and homologous rate of J2-16S-F3R3 strain Figure 3.9 Phylogenetic tree gene and homologous rate of J6-16S-F3R3 strain Figure 3.10 Phylogenetic tree gene and homologous rate of Ht4-16S-F3R3 strain 38 download by : skknchat@gmail.com Figure 3.11 Phylogenetic tree gene and homologous rate of Ht6-16S-F3R3 strain Table 3.1 Identify strains in the study on gene bank No Strains Primer Size (bp) Similar J2: Lactobacillus pentosus or J2 16S F3R3 1100 Lactobacillus plantarum subsp plantarum J6 16S F3R3 1100 Lactobacillus plantarum Ht4 16S F3R3 1100 Lactobacillus rhamnosus Ht6 16S F3R3 1100 Lactobacillus paracasei 4.7 The results of the antibacterial activity assay 4.7.1 Antibacterial of mix strains of bacterial Table 3.2 The antibacterial level of mix strains is measured in units of millimeters 39 download by : skknchat@gmail.com 1: All strains 2: J2+J6 3: HT4+HT6 4: J2+HT4 5: J2+HT6 6: J6+HT4 7: J6+HT6 8: MRS broth Figure3.12 The level of anti-Staphylococcus aureus For bacterial resistance Staphylococcus aureus, J2-J6 and J6-Ht4 are working best with mm of circle measured Follow by J6-Ht6 with 2.5mm of the antibacterial circle The remaining groups have antibacterial circles no wider than 2mm J2-Ht4 shows the least antibacterial by a mere 0.5mm Figure3.13 The level of anti- Pseudomonas aeruginosa 40 download by : skknchat@gmail.com Experiments performed with Pseudomonas aeruginosa had the best results belong to both J6-Ht4 and j6-Ht6 group (3mm) J2-Ht6 is the worst-performing group and it seems that the bacterial concentration is too few to be able to fight off pathogenic bacteria compared to the rest In short, all bacterial groups have the ability to fight off pathogenic bacteria Among them, the three groups J2-J6, J6-Ht4 and J6-Ht6 are the best about the antibacterial ability 4.7.2 Antibacterial of each strain Table 3.3 The anti-Staphylococcus aureus level Figure3.3 The anti- Staphylococcus aureus level of J2, J6, HT4, and HT6 strain From table 3.5, the two bacteria J2 and J6 show that almost the same about the big of an anti-Staphylococcus aureus circle Whilst Ht4 and Ht6 indicate 41 download by : skknchat@gmail.com bacterial invasion on the entire agar plate surface Therefore, J2 and J6 have better antibacterial properties than Ht4 and Ht6 Table 3.4 The anti-Pseudomonas aeruginosa Figure 3.15 The anti-Pseudomonas aeruginosa level of J2, J6, HT4, and HT6 strain Form table 3.4, this is the clearest demonstration of the bacterial dependence on concentration for inhibiting pathogenic bacteria More specifically, higher concentrations lead to stronger antibacterial and vice versa All four strains of bacteria exhibit anti- Pseudomonas aeruginosa properties The size of the antibacterial circle is almost the same at the same concentration This suggests that all four strains of bacteria will have nearly the same antibacterial efficacy 42 download by : skknchat@gmail.com PART V CONCLUSIONS 5.1 Conclusion There are a total of strains of bacteria in both of the Joint and Health bacteria groups After performing experiments and sending samples for sequencing, the results received four strains were identified as: HT4: Lactobacillus rhamnosus HT6: Lactobacillus paracasei J2: Lactobacillus pentosus or Lactobacillus Plantarum subsp plantarum J6: Lactobacillus Plantarum HT1 and J3: not specified Following the sequencing are antibacterial activities, which is the main purpose of this experiment Based on the results, the strains of J2 and J6 are better antibacterial than Ht4 and Ht6 (with both pathogenic bacteria) Especially when the concentration of bacteria is higher, the inhibition of bacteria increases For the experiment of mixing strains of bacteria, the results showed good cooperation between strains of bacteria to inhibit pathogenic bacteria Based on the results on the table illustrate how the bacterial inhibition levels of random mix strains and four strains have been identified Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus pentosus or Lactobacillus Plantarum subsp plantarum and Lactobacillus Plantarum Overall, in this experiment, all of the bacterias have the ability to control pathogenic bacteria The width of the antibacterial cycle around the hole depends on the concentration of bacteria 43 download by : skknchat@gmail.com In table 3.4, the result shows the size of the antibacterial-cycle Compare with the similar about an experiment, time, protocol, method and equipment, the mixture of J6 and Ht4 indicated the best result of eliminating with both Staphylococcus aureus bacteria and Pseudomonas aeruginosa bacteria with about 3mm of eliminate-cycle width J2 and Ht4 group show the shortest antiStaphylococcus aureus cycle around the hole on the agar plate by a mere 0.5mm Only the J2-Ht6 groups did not show its anti-Pseudomonas aeruginosa ability The size of the antibacterial cycle of the remaining groups no bigger than 3mm In conclusion, four lactobacillus strains have ability resistant to the two types of pathogenic bacteria, which is actually potential bacteria for producing friendly cleaning agents 5.2 Recommendations - Based on the antibacterial properties of probiotic bacteria, which are beneficial bacteria, it is necessary to continue research and development - Testing of this product to purify the environment from this probiotic bacterium in the future - Experimental survey and compare the superiority of probiotics compared with conventional detergents - It is therefore suggested the idea of the bacterial strain or a group of bacteria strains that have the best cleaning as an environmental cleaner 44 download by : skknchat@gmail.com REFERENCES Pfaller MA, Jones RN, Marshall SA, Coffman SL, Hollis RJ, Edmond MB, et al Inducible amp C beta-lactamase-producing gram-negative bacilli from bloodstream infections: frequency, antimicrobial susceptibility, and molecular epidemiology in a national surveillance program (SCOPE) Diagn Microbiol Infect Dis 1997;28:211–219 Jarvis WR, Martone WJ Predominant pathogens in hospital infections J Antimicrob Chemother 1992;29:19–24 Vosahlikova S, Drevinek P, Cinek O, Pohunek P, Maixnerova M, Urbaskova P, et al High genotypic diversity of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis in the Czech Republic Res in Microbiol 2007;158:324–329 Brook I Otitis media: microbiology and management J Otolaryngol 1994;23:269–275 Lyczak JB, Cannon CL, Pier GB Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist Microbes Infect 2000;2:1051–1060 Burgess DS Use of pharmacokinetics and pharmacodynamics to optimize antimicrobial treatment of Pseudomonas aeruginosa infections Clin Infect Dis 2005;2:S99–104 Paul R Rhomberg, Ronald N Jones, Helio S Sade Results from the meropenem yearly susceptibility test information collection (MYSTIC) program: 45 download by : skknchat@gmail.com report of the 2001 data from 15 United States medical centers Int J Antimicrob Agents 2004;23:52–59 Patzer JA Dzierzanowska Increase of imipenem resistance among Pseudomonas aeruginosa isolates from a Polish pediatric hospital Int J Antimicrob Agents; 29 1993;2007:153–158 FD Lowy N Engl J Med., 1998, 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Macrae J, Wheater DM A lactobacillus preparation for use with antibiotics Lancet 1957;272:899–901 Vandenbergh PA Lactic acid bacteria, their metabolic products, and interference with microbial growth FEMS Microbiol Rev 1993;12:221–238 Carson CF, Riley TV Non-antibiotic therapies for infectious diseases Commun Dis Intell 2003;27:143–146 Gibbs PA Novel uses for lactic acid fermentation in food preservation J Appl Bacteriol Symp Suppl 1987;63:51S–58S R Fuller, “Probiotics for farm animals,” in Probiotics a Critical Review, pp 15–22, Horizon Scientific, Wymondham, UK, 1999 R Fuller, “Probiotics in man and animals,” Journal of Applied Bacteriology, vol 66, no 5, pp 365–378, 1989 FAO/WHO, Report on Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria, 20 Kröckel L (2006) Use of Probiotic Bacteria in Meat Products Fleischwirtschaft 86:109-113 Rao, N S S (1982) Biofertilizers Interdisciplinary Science Reviews 7(3), 220–229 47 download by : skknchat@gmail.com Vikas Ghumare, Manisha Rana, Omkar Gavkara and Babita Khachi (2014) Jr of Industrial Pollution Control (2014) 30(2) 199-200 48 download by : skknchat@gmail.com ... 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. .. The component of the ligation reaction 25 Table 2.8 The component of restriction enzyme digestion reaction 30 Table 3.1 Identify strains in the study on gene bank 39 Table 3.2 The. .. 32 4.1 The result of the culture of bacteria 32 4.2 The result of gram staining 32 4.3 The results of the DNA extraction of six strains in the study 33 4.4 The results of PCR