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 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 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 Thesis Title cleaning 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 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 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 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 Thankyou very much and best regards Thai Nguyen, September 25 Student Trinh Thi My Duyen TABLE OF CONTENT 3.2.1 3.2.7 Cloning of Screened Gene into yT&A-Vector following Transformed into DH5a 25 3.2.7.1 3.2.7.2 3.2.7.3LIST OF FIGURES 3.2.7.4 3.2.7.5 3.2.7.6Figure 3.8 Phylogenetic tree gene and homologous rate of J2-16S-F3R3 strain 38 3.2.7.7Figure 3.9 Phylogenetic tree gene and homologous rate of J6-16S-F3R3 strain 38 3.2.7.8Figure 3.10 Phylogenetic tree gene and homologous rate of Ht4-16S-F3R3 strain 38 3.2.7.9Figure 3.11 Phylogenetic tree gene and homologous rate of Ht6-16S-F3R3 strain 39 3.2.7.10 Figure3.12 The level of anti-Staphylococcus aureus 40 10 3.2.7.465 6: J6+HT4 3.2.7.466 7: J6+HT6 3.2.7.467 8: MRS broth 3.2.7.468 Figure3.13 The level of anti- Pseudomonas aeruginosa 3.2.7.469 the Experiments performed with Pseudomonas aeruginosa had 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 3.2.7.470 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 3.2.7.471 3.2.7.472 3.2.7.473 3.2.7.474 3.2.7.476 Table 3.3 The anti-Staphylococcus aureus level 3.2.7.475 Figure3.3 The anti- Staphylococcus aureus level of J2, J6, HT4, and HT6 strain 3.2.7.477 3.2.7.478 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 indicatebacterial invasion on the entire agar plate surface Therefore, J2 and J6 have better antibacterial properties than Ht4 and Ht6 3.2.7.479 3.2.7.480 3.2.7.481 3.2.7.482 3.2.7.484 Table 3.4 The anti-Pseudomonas aeruginosa 3.2.7.483 Figure 3.15 The anti-Pseudomonas aeruginosa level of J2, J6, HT4, and HT6 strain 3.2.7.485 3.2.7.486 bacterial Form table 3.4, this is the clearest demonstration of the dependence on concentration for inhibiting pathogenic bacteria More speciíically, 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 3.2.7.487 5.1 PART V CONCLUSIONS Conclusion 3.2.7.488 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: 3.2.7.489 HT4: Lactobacillus rhamnosus 3.2.7.490 HT6: Lactobacillus paracasei 3.2.7.491 J2: Lactobacillus pentosus or Lactobacillus Plantarum subsp plantarum 3.2.7.492 J6: Lactobacillus Plantarum 3.2.7.493 HT1 and J3: not specified 3.2.7.494 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 3.2.7.495 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 3.2.7.496 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 3.2.7.497 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 3.2.7.498 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 REFERENCES 3.2.7.499 3.2.7.500 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 surveillanceprogram (SCOPE) Diagn Microbiol Infect Dis 1997;28:211-219 3.2.7.501 Jarvis WR, Martone WJ Predominant pathogens in hospital infections J Antimicrob Chemother 1992;29:19-24 3.2.7.502 Vosahlikova S, Drevinek P, Cinek O, Pohunek P, Maixnerova M, Urbaskova P, et al High genotypic diversity of Pseudomonas aeruginosa strains isolatedfrom patients with cystic fibrosis in the Czech Republic Res in Microbiol 2007;158:324-329 3.2.7.503 Brook I Otitis media: microbiology and management J Otolaryngol 1994;23:269-275 3.2.7.504 Lyczak JB, Cannon CL, Pier GB Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist Microbes Infect 2000;2:1051-1060 3.2.7.505 Burgess andpharmacodynamics DS Use to of pharmacokinetics optimize antimicrobial treatment of Pseudomonas aeruginosa infections Clin Infect Dis 2005;2:S99-104 3.2.7.506 Paul R Rhomberg, Ronald N Jones, Helio S Sade Results from the meropenem yearly susceptibility test information collection (MYSTIC) program:report of the 2001 data from 15 United States medical centers Int J Antimicrob Agents 2004;23:52-59 3.2.7.507 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 3.2.7.508 FD Lowy N Engl J Med., 1998, 339, 520-532 3.2.7.509 I Fedtke; F Gotz; A Peschel Int J Med Microbiol., 2004, 294, 189-194 3.2.7.510 Immun., DP Greenberg; AS Bayer, AL Cheung, JI Ward Infect 1989, 57, 1113-1118 3.2.7.511 Herrmann Infect Immun., 2002, 70, 2933-2940 3.2.7.512 D Joh; ER Wann; B Kreikemeyer; P Spezial Matrix Biol., 1999, 18, 211-223 3.2.7.513 Chem., PW Park; J Rosenbloom, WR Abrams, RP Mecha J Biol 1996, 271, 15803-15809 3.2.7.514 JM Patti; BL Allen; MJ McGavin; M Hook Annu Rev Microbiol., 1994, 48, 585-617 3.2.7.515 GM Costa; C Pizzi; C Leone; A Borghi; E Cordioli; R Bugiardini Cardiologia., 1999, 44, 675-678 3.2.7.516 I Raad Lancet, 1998, 351, 893-898 3.2.7.517 S Rajan; L Saiman Semin Respir Infect., 2002, 17, 47-56 3.2.7.518 MB Melchior; H Vaarkamp; J Fink-Gremmels, Vet J., 2006, 171, 398- 407 3.2.7.519 2012, 25, 10-16 J Parra-Ruiz; C Vidaillac; MJ Rybak Rev Esp Quimioter., 3.2.7.520 Microbiol 3.2.7.521 H Rohde; JK Knobloch; MA Horstkotte; D Mack Med Immunol (Berl), 2001, 190, 105-112 3.2.7.522 P Landini Res Microbiol, 2009, 160, 259-266 3.2.7.523 TTV Doan; HKT Nguyen J App Pharm Sci., 2013, 3, 33- 3.2.7.524 Gordon 36 D, lactobacilluspreparation Macrae J, for Wheater DM use A with antibiotics Lancet 1957;272:899-901 3.2.7.525 Vandenbergh PA Lactic acid bacteria, their metabolic products, and interference with microbial growth FEMS Microbiol Rev 1993;12:221-238 3.2.7.526 Carson CF, Riley TV Non-antibiotic therapies for infectious diseases 3.2.7.527 Commun Dis Intell 2003;27:143-146 3.2.7.528 Gibbs PA Novel uses for lactic acid fermentation in foodpreservation J Appl Bacteriol Symp Suppl 1987;63:51S-58S 3.2.7.529 R Fuller, “Probiotics for farm animals,” in Probiotics a Critical Review, pp 15-22, Horizon Scientific, Wymondham, UK, 1999 3.2.7.530 R Fuller, “Probiotics in man and animals,” Journal of Applied Bacteriology, vol 66, no 5, pp 365-378, 1989 3.2.7.531 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 3.2.7.532 Krốckel L (2006) Use of Probiotic Bacteria in Meat Products 3.2.7.533 Fleischwirtschaft 86:109-113 3.2.7.534 Reviews 220-229 Rao, N S S (1982) Biofertilizers Interdisciplinary Science 7(3), 3.2.7.535 Khachi (2014) 3.2.7.536 Vikas Ghumare, Manisha Rana, Omkar Gavkara and Babita Jr of Industrial Pollution Control (2014) 30(2) 199-200 ... 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. .. Major Environmental Science and Management Student name Trinh Thi My Duyen Student ID DTN155429007 Study on the evaluation of probiotics as environmental Thesis Title cleaning agents Assoc Prof... 2.3) and warp the upper part of the flask Some holes on the warp were needed to decrease the pressure when heating Then the flask was placed in the microwave for minutes to dissolve the particles