S-Space College of Agriculture and Life Sciences (농업생명과학대학) Dept. of Agricultural Biotechnology (농생명공학부) Theses (Master's Degree_농생명공학부)
Selection and Characterization of Lactic Acid Bacteria with Improved Antimicrobial activity to Calf Diarrhea-Inducing pathogen by Genome Shuffling
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- Choi, Yunjaie
- Issue Date
- 서울대학교 대학원
- 학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2012. 2. Choi, Yunjaie.
- Consuming probiotics such as lactic acid bacteria on a regular basis can promote the physiological function of animal and help prevent many diseases. While various Lactobacillus species are being applied as probiotics, of these, L. plantarum has a long history of safe use for preparation of fermented foods and it is also an important member of the GI tract microflora (Jai K. Kaushik, 2009). Due to good properties of lactic acid bacteria, there have been plenty of studies of lactic acid bacteria breeding for ages long.
The object of this study is to breed lactic acid bacteria with the object of improving its antimicrobial activity to pathogen E.coli K99 that can cause calf diarrhea. The first stage of this study is to examine the basic characters of wild type lactic acid bacteria [Lactobacillus salivarious 29 (LS29), Pediococcus acidilactia 175 (PA175), Lactobacillus plantarum 177 (LP177)] such as bacterial identification, growth curve, pH curve, and antimicrobial ability. In this first stage, comparison of antimicrobial ability between lactic acid bacteria to pathogens that can cause calf diarrhea were focused. For the attainment of this aim and purpose, 'antipahogenic ability standard index' which is a table representing original antimicrobial activities of wild-type lactic acid bacteria was made. To make the antipathogenic ability standard index, pathogen-topagar-assay that analyzes the antimicrobial ability of lactic acid bacteria to pathogen based on clear zone diameter were repeatedly performed. A candidate of lactic acid bacteria, Lactobacillus plantarum 177 that will be breeded by mutation breeding methods were selected upon the basis of antipathogenic ability standard index data. The second stage of this study is to breed by UV mutagenesis using the cantidate lactic acid bacteria that have selected based on antimicrobial ability by antipathogenic ability standard index in the first stage. In these UV mutagenesis step, lactic acid bacteria were irradiated by UV ray and then the cells survived after irradiation were swabbed. Then sequentially the swabbed cells were cocultured with pathogen and selected at late death phase in view of lactic acid bacteria. Those rounds of panning were repeated three times. And the selected UV mutants were screened by pathogen-topagar-assay. The third stage of this study is to breed by genome shuffling mutagenesis using the candidate lactic acid bacteria that have selected at the previous stage. In this genome shuffling mutagenesis step, lactic acid bacteria were mutated by genome shuffling method and then the cells survived after regeneration were swabbed. Then sequentially the swabbed cells were cocultured with pathogen and selected at late death phase in view of lactic acid bacteria. and the selected genome shuffling mutants were screened by pathogen-topagar-assay. Finally, the three Lactobacillus plantarum genome shuffling mutants (LP GS1, LP GS2, and LP GS3) were selected on the basis of their better antimicrobial activity against E.coli K99 (major cause of calf diarrhea) compared with wild-type L. plantarum.
The finally selected 3 genome shuffling mutants were verified whether antimicrobial activity of these mutants increased. Additional research such as coculture with E.coli K99 and Salmonella typhimurium, Growth curve, pH curve was conducted to characterize the three GS mutants selected by GS mutagenesis. Growth curve pH curve were also measured to check whether basic characters of cell were changed or not. The results show that genome shuffling mutants inhibit the pathogen better than wild type and UV mutants without any change on physiological character of the bacteria.
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