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학술연구/단체지원/교육 등 연구자 활동을 지속하도록 DBpia가 지원하고 있어요.
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논문 기본 정보
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- 2021
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In this study, the structure of the oligosaccharide produced from Weissella cibaria was analyzed, and the effectiveness of this oligosaccharide as prebiotics was evaluated. In order to select lactic acid bacteria producing glycosyltransferase, strains producing viscous colonies on phenylethyl alcohol with 20%(w/v) sucrose (PES) agar plate were selected after isolating lactic acid bacteria from various sources. Among 974 lactic acid bacteria strains, 187 strains showed viscous colony on PES agar plate. Glycosyltransferase activity was measured via the incubation of the enzyme for different lactic acid bacteria at 37 ℃ with sucrose and maltose as donor and acceptor molecules respectively. Each of the enzyme reaction samples was spotted onto a TLC plate. Also, glycosyltransferase activity of the reaction sample was measured using the modified Somogyi-Nelson method, which measures the amount of reducing sugar. Among 187 lactic acid bacteria strains, 55 strains showed high glycosyltransferase activities. Weissella confusa CCK931, W. cibaria YRK005 and Leuconostoc lactis SBC005 showed the highest enzyme activities of 24.53, 22.37, and 20.30 U/mL, respectively.
Oligosaccharides produced from W. cibaria YRK005 were purified using size exclusion chromatography and the result was confirmed through Thin layer chromatography (TLC). YRK-oligosaccharides are gluco-oligosaccharides consisting of only glucose. YRK-oligosaccharides had 6.2% α-1,4, 93.8% α-1,6 linkages, and the branching ratio of α-1,4 and α-1,6 linkages was 0.1. Also, the molecular weight of YRK-oligosaccharides was found to be 11.22 ⅹ 102 Da.
To determine whether YRK-oligosaccharides were resistant to mammalian digestive enzymes, they were passed through simulation saliva, gastrointestinal tract and confirmed by TLC and HPAEC-PAD. As a result, oligosaccharides were not degraded by salivary amylase, but slightly degraded in gastrointestinal tract. In order to confirm the prebiotics effect of YRK-oligosaccharides, various carbons sources were added to the medium from which the dextrose was removed. The viable cell count of four probiotics strains, Lactobacillus acidophilus, Lactobacillus pentosus, Lactococcus lactis, Bifidobacterium adolescentis were significantly higher than modified medium with fructo-oligosaccharides known as prebiotics. In particular, at 24 h incubation, all four strains showed better growth in m-MRS media containing YRK-oligosaccharides than in m-MRS supplemented with positive controls glucose and FOS.
Oligosaccharides produced from W. cibaria YRK005 were purified using size exclusion chromatography and the result was confirmed through Thin layer chromatography (TLC). YRK-oligosaccharides are gluco-oligosaccharides consisting of only glucose. YRK-oligosaccharides had 6.2% α-1,4, 93.8% α-1,6 linkages, and the branching ratio of α-1,4 and α-1,6 linkages was 0.1. Also, the molecular weight of YRK-oligosaccharides was found to be 11.22 ⅹ 102 Da.
To determine whether YRK-oligosaccharides were resistant to mammalian digestive enzymes, they were passed through simulation saliva, gastrointestinal tract and confirmed by TLC and HPAEC-PAD. As a result, oligosaccharides were not degraded by salivary amylase, but slightly degraded in gastrointestinal tract. In order to confirm the prebiotics effect of YRK-oligosaccharides, various carbons sources were added to the medium from which the dextrose was removed. The viable cell count of four probiotics strains, Lactobacillus acidophilus, Lactobacillus pentosus, Lactococcus lactis, Bifidobacterium adolescentis were significantly higher than modified medium with fructo-oligosaccharides known as prebiotics. In particular, at 24 h incubation, all four strains showed better growth in m-MRS media containing YRK-oligosaccharides than in m-MRS supplemented with positive controls glucose and FOS.
목차
ABSTRACT………………………………………………………………..ⅠList of Tables……………………………………………………………….ⅰList of Figures………………………………………………………….…..ⅱ1. Introduction……………………………………………………………...11.1. Probiotics……………………………………………………………..11.2. Prebiotics………………………………………………………..……31.3 Market status of probiotics and prebiotics…………………………….61.4. Oligosaccharides………………………………………………..…….81.5. Glycosyltransferase…………………………………………….……112. Materials and Methods…………………………………………………132.1. Screening of glycosyltranferase-producing lactic acid bacteria….…132.1.1. Screening of lactic acid bacteria using PES media…………..…132.1.2. Screening of lactic acid bacteria using TLC……………………152.1.3. Glycosyltransferase activity………………………………….…162.2. Identification of Weissella cibaria YRK005………………….…….182.2.1. Isolation of W. cibaria YRK005…………………….……….…182.2.2. Genomic DAN isolation of W. cibaria YRK005………….……192.2.3. Identification of W. cibaria YRK005…………………………..202.2.4. Scanning electron microscopy……………………………….…212.2.5. Biochemical characteristics……………………………….……222.3. Determination of YRK-oligosaccharides structure …………..……..232.3.1. Purification of oligosaccharides……………………….…….….232.3.2. HPAEC-PAD analysis………………………………….……….242.3.3. Size exclusion HPLC analysis………………………….………252.3.4. Analysis of sugar composition…………………………….……272.3.5. Composition of glycosidic bonds……………………………....272.3.6. Proton nuclear magnetic resonance spectroscopy……………...282.4. Prebiotic effects……………………………………………………...292.4.1. Acid tolerance test of YRK-oligosaccharides…………………..292.4.2. Bile tolerance test of YRK-oligosaccharides…………………...292.4.3. In vitro digestive model………………………………………...302.4.4. Probiotic strains………………………………………………...322.4.5. Media and growth conditions…………………………………..332.4.6. Growth of probiotics on YRK-oligosaccharides………….……352.5. Statistical analysis3. Results and Discussion……………………………………………….…363.1 Screening of lactic acid bacteria using PES media……………….….363.2. Screening of lactic acid bacteria using TLC…………………..….…363.3. Glycosyltransferase activity…………………………………………373.4. Identification of Weissella cibaria YRK005……………………......393.4.1. Phenotypic characteristics………………………………………393.4.2. Phylogenetic analysis………………………………………..….403.5. Determination of YRK-oligosaccharides structure………………….443.5.1. Purification of oligosaccharides………………………………...443.5.2. Analysis of molecular mass…………………………………….463.5.3. Monosaccharides composition………………………………….473.5.4. 1H-NMR spectroscopy………………………………………….513.5.5. Glycosidic bond composition by hydrolase………..….….…….543.6. Prebiotic effects………………………………………………...……563.6.1. Acid and bile tolerance of YRK-oligosaccharides……………..563.6.2. Resistance to simulated saliva and gastrointestinal digestion.…603.6.3. Prebiotic effects of YRK-oligosaccharides………………….…63Conclusions………………………………………………………………...68References………………………………………………………………….69국문 초록...................................................................-.................................77
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