Lactobacillus casei KH-1의 분리동정 및 젖산생산의 최적화 = Isolation and characterization of Lactobacillus casei KH-1 and optimization of lactic acid production

국문 초록 (Abstract)

생분새성 고분자 합성물질의 주원료가 되는 젖산을 경제적으로 생산하기 위해 김치로부터 호모발효젖산균 KH-1을 분리하였다. 전산균 KH-1은 연쇄간균의 형태였고, 16S rRNA sequencing에 의해 계통발생학적 분석의 결과 Lactobacillus casei와 99%의 동질성을 보였다. L. casei KH-1은 glucose를 젖산으로 98% 전환시키는 호모발효균으로서 D(-)와 L(+)-이성질체를 각각 7%와 93%를 포함하는 racemic mixtrure로 젖산을 생산하였다. 젖산발효를 위한 최적의 배지조건을 검토하기 위해 glucose, yeast extract와 corn steep liquor으로 구성된 임의의 합성배지에서 균의 성장과 젖산생산에 대한 동력학적 해석과 구성성분의 최적조건을 결정하였다. MRS배지의 질소원을 저렴한 원료 즉, yeast extract와 corn steep liquor를 1:1로 첨가한 배지로 대신하여 최대의 젖산수율 (1.09 g·g^(-1))을 얻어 젖산발효에 효과적인 질소원의 대체가 이루어졌다. Box-Behnken반응표면모델로 설계되 17개 회분식실험을 수행한 실험데이터로부터 각각 Gombertz and Leudeking and Piret(LP) model과 관련되 모수, U_(max),(X_(max), a와 b를 추정하였고 yeast extract, corn steep liquor와 glucose가 L. casei KH-1의 성장과 젖산생산에 미치는 영향이나, 영향인자들 사이의 교호관계를 반응표면모델을 통하여 분석하였다. 또한 Design-Expert을 사용하여 결정된 L. casei KH-1이 성장과 젖산생산에 yeast extract, corn steep liquor와 glucose의 최적조건은 각각 1.276, 3.505, 2.390%와 0.697, 1.708, 2.215%였다. 배지의 원료비용을 더욱더 절감하기 위하여 Tricoderma harzianum FJ1에 의해 생산된 셀룰라아제를 사용하여 폐지(신문지와 종이박스)를 당화시켜 얻은 당류를 L. casei KH-1의 젖산발효에 이용하였다. 이 때 신문지와 종이박스의 당화율은 각각 61와 36%정도였고, 이를 이용하여 각각 5.587, 9.600 g/L의 젖산을 얻었다. MRS배지를 이용하여 얻는 젖산수율 0.962 g·g^(-1) 보다 높은 1.559 g·g^(-1)의 젖산생산수율을 얻는 종이박스당화액은 젖산생산에 효과적인 기질이었다.

다국어 초록 (Multilingual Abstract)

A homofermentative lactic acid bacterium KH-1 isolated from kimchi showed 99% homology with Lactobacillus casei from analysis of 16S rRNA sequencing. The conversion ratio of lactic acid from glucose by L. casei KH-1 was 98% in anaerobic condition, and the lactic acid was composed as racemic mixture of D(-)-and L(+)-lactic acid, 7% and 93%, respectively. The optimal conditions of yeast extrct, corn steep liquor and glucose concentration for the growth and lactic acid production of strain were investigated and the effect of these conditions were assessed using a response surface methodology. A Box-Behnken was used as an experimental design for the allocation of treatment combination as seventeen pH-controlled batch cultures. The effects of yeast extract, corn steep liquor and glucose were very signifcant for the maximum specific growth rate, max, and the maximum biomass concentration, Xmax. Although the experiments were performed in pH-controlled batch culture for L. casei KH-1, the growth- and nongrowth-associated production rate parameters, a and b were significant on response surface model. It was turned out that the growth and lactic acid production of L. casei KH-1 were strongly affected by the glucose and that the importance of the tested media composition was demonstrated. The estimated optimal conditions of the growth and lactic acid production of L. casei KH-1 were 1.276, 3.505, 2.390% and 0.697, 1.708, 2.215% of yeast extract, corn steep liquor and glucose, respectively. To reduce material cost of medium, waste paper(newspaper and box paper) saccharified by cellulases of Tricoderma harzianum FJ1 was used in the lactic acid fermentation of L. casei KH-1. Saccharification rates of newspaper and box paper were 61 and 36%, respectively, and produced lactic acid concentration was 5.587 and 9.600 g/L, respectively. The gram yield of lactic acid produced per gram of glucose(YP/S) was higher in the saccharification broth derived newspaper than MRS medium. That is to say saccharification broth derived from newspaper was more effective substrate for lactic acid production.

목차 (Table of Contents)

• Korean Summary = I
• List of Figures = III
• List of Table = V
• 1. Introduction = 1
• 2. Theory = 3
• A. Lactic acid = 3
• B. Cellulose = 5
• C. Physiological characteristics of Lactobacillus casei in lactic acid fermentation = 6
• D. Degradation of cellulose = 9
• E. Response surface methodology (RSM) = 12
• 3. Materials and Methods = 13
• A. Isolation and characteristics of a homofermentative lactic acid bacterium = 13
• 1) Composition of medium = 13
• 2) Cultivation conditions = 17
• a) Preculture conditions = 17
• b) Batch culture conditions = 17
• 3) Screening of a lactic acid producer = 17
• 4) Isolation of strain KH-1 = 19
• a) Scanning electron microscopy (SEM) = 19
• b) 16S rRHA sequenceing of strain KH-1 = 22
• B. Kinetic analysis of growth and lactic acid production in pH-controlled batch cultrue of Lactobacillus casei KH-1 = 24
• 1) Microorganism, growth media, and culture conditions = 24
• 2) Descriptive modeling = 24
• 3) Experimental design and statistical analysis = 25
• C. Lactic acid production from waste paper = 26
• 1) A cellulase producer used = 26
• 2) Composition of medium = 27
• 3) Characterization of Trichoderma harzianum = 27
• 4) Production of cellulase by Trichoderma harzianum FJ1 = 30
• 5) Conditions of saccharification = 30
• 6) Lactic acid fermentation using reducing sugar broth from waste paper = 30
• D. Analytical methods = 31
• 1) Cell dry weight(CDW, g/L) = 31
• 2) Residual glucose concentration (RGC, g/L) = 31
• 3) Lactic acid concentration (LAC, g/L) = 32
• 4) L(+), D(-)-lactic acid concentration = 32
• 5) Determination of enzyme activity = 32
• a) Cellulase activity = 32
• b) CMCase activity = 33
• c) β-glucosidase activity = 33
• d) Avicelase activity = 33
• 4. Results and Discussion = 34
• A. Isolation and characteristics of a homofermentative lactic acid bacterium = 34
• 1) Isolation of strain KH-1 = 34
• 2) Characteristics of growth and lactic acid production by L. casei KH-1 = 37
• 3) Effects of initial pH on the lactic acid production = 40
• 4) Sugar utilization of L. casei KH-1 = 42
• 5) Effects of nitrogen sources on the lactic acid production = 44
• B. Kinetics analysis of growth and lactic acid production in production in pH-controlled batch cultures of Lactobacillus casei KH-1 = 47
• 1) Maximum specific growth rates(μ_(max)) = 47
• 2) Maximum biomass concentratin(X_(max)) = 52
• 3) Production rate parameters (a, b) = 55
• C. Validating the optimum point of the factors = 62
• D. Lactic acid production from waste paper = 66
• 1) Saccharification of waste paper as cellulose biomass = 66
• 2) Lactic acid fermentation using saccharification broth derived from waste paper = 69
• 3) Effects of nitrogen sources on the lactic acid production using saccharification broth derived from newspaper = 72
• 5. Conclusion = 75
• 6. Reference = 77
• Nomenclature = 84
• Abstract = 85