Weissella cibaria 의 만니톨탈수소효소에 대한 생화학적 특성과 스타터 김치의 적용 = Biochemical Characterization of Mannitol Dehydrogenase from Weissella cibaria and Starter Kimchi Application

다국어 초록 (Multilingual Abstract)

Mannitol is a naturally occurring six-carbon sugar alcohol. It is used as a bodying agent, humectant, anticaking agent and antioxidant and has a sweet cool taste. Because of these characteristics, mannitol is widely applied in the food industry.
This study was performed to characterize the mannitol dehydrogenase (MDH) from Weissella cibaria to prepare for the preparation of starter kimchi containing mannitol. Also, physicochemical, microbiological and sensory characteristics of starter kimchi were investigated. Starter kimchi and regular kimchi were prepared in the presence of added 1% sucrose. Then, the kimchi were fermented at 15℃ for 3 d and stored at 4℃. The amount of mannitol and characteristics of kimchi were analyzed for 18 d with sensory evaluation. Mannitol producing ability in MRS broth and kimchi were measured using HPLC. Wei. cibaria was cultured in optimal conditions and then MDH was produced from Wei. cibaria. MDH was partially purified and biochemical properties were characterized.
The amount of produced mannitol in MRS broth with 5% fructose was 2.27%. The mannitol concentration of starter kimchi and regular kimchi were 0.62% and 0.44%, respectively. These results suggest that Wei. cibaria could be used in mannitol-containing kimchi as a starter microorganism.

목차 (Table of Contents)

• Ⅰ. Introduction 1
• Ⅱ. Materials and Methods 4
• 1. Identification of mannitol-producing strain 4
• A. Bacterial strain 4
• B. Gram staining 4
• C. Scanning electron microscopy 4
• D. 16S rDNA sequencing 4
• 2. Characterization of strain 5
• A. Cell viability at low temperature 5
• B. Mannitol production in MRS broth 5
• 1) Determination of mannitol by HPLC 5
• 2) Mannitol standard curve 5
• 3) Mannitol productivity according to fructose concentration 6
• 4) Mannitol producing ability at low temperature 6
• 3. Purification and characterization of mannitol dehydrogenase (MDH) 6
• A. Microorganism and culture conditions 6
• B. Assay of MDH activity 6
• C. Determination of protein concentration 10
• D. Purification of mannitol dehydrogenase 13
• 1) Preparation of crude enzyme extract 13
• 2) Molecular filtration using Centriprep 13
• 3) DEAE column chromatography 16
• 4) Hydroxyapatite column chromatography 16
• 5) Gel filtration column chromatography 16
• E. Identification of mannitol dehydrogenase 17
• 1) SDS-PAGE 17
• 2) Molecular mass determination 17
• F. Characterization of mannitol dehydrogenase 17
• 1) Enzyme kinetics 17
• 2) Effect of temperature on stability and activity 18
• 3) Effect of pH on stability and activity 18
• 4) Specificity of substrate and coenzyme 18
• 5) Effect of metal ions and some chemicals on the MDH activity 19
• 4. Preparation of starter kimchi 19
• A. Preparation of kimchi 19
• B. Analytical methods 22
• 1) pH 22
• 2) Titratable acidity 22
• 3) Lactic acid bacteria counts 22
• 4) Quantification of Wei. cibaria using real-time PCR 22
• A) Isolation of DNA from kimchi 22
• B) Primer and probe 23
• C) Real-time PCR 23
• D) Standard curve 25
• D. Mannitol production in kimchi 25
• E. Sensory evaluation 25
• F. Statistical analysis 27
• Ⅲ. Results and Discussion 29
• 1. Identification of mannitol-producing strain 29
• A. Gram reaction 29
• B. Scanning electron microscopy 29
• C. 16S rDNA phylogenetic tree 29
• 2. Properties of Wei. cibaria 34
• A. Cell viability at low temperature 34
• B. Mannitol production in MRS broth 34
• 1) Mannitol productivity according to fructose concentration 34
• 2) Mannitol producing ability at low temperature 34
• 3. Purification and characterization of mannitol dehydrogenase 38
• A. Purification of mannitol dehydrogenase 38
• 1) Summary of the purification procedures 38
• 2) SDS-PAGE 38
• 3) Molecular mass determination 42
• B. Characterization of mannitol dehydrogenase 42
• 1) Enzyme kinetics 42
• 2) Effect of temperature on stability and activity 42
• 3) Effect of pH on stability and activity 46
• 4) Specificity of substrate and coenzyme 46
• 5) Effect of metal ions including some chemicals on the MDH activity 46
• 4. Preparation of starter kimchi 46
• A. Analytical methods 46
• 1) pH 46
• 2) Titratable acidity 50
• 3) Lactic acid bacteria 50
• 4) Quantification of Wei. cibaria using real-time PCR 54
• B. Mannitol production in kimchi 54
• C. Sensory evaluation 54
• Ⅳ. Conclusion 58
• References 59
• Abstract in Korean 63