Production of Lactosucrose by Sterigmatomyces elviae mutant Using Whole Cell Immobilization

국문 초록 (Abstract)

Lactosucroses ((4)^G-β-Galactosylsucrose)는 저칼로리성이고, 충치예방의 효과가 있고, 혈청 내 콜레스테롤과 지방을 감소시키고, 동물성장을 촉진하며 Bifidus균의 증식에 도움을 주는 등 여러 가지 생리활성 기능을 나타내는 기능성 건강식품으로 그 수요가 증가하고 있는 감미료이다. lactosucroses는 다른 올리고당에 비해 정장작용이 3배정도 효과가 있고 sucrose와 감미도가 가장 비슷하며 낮은 pH에서 분해되지 않는 내산성을 나타내기 때문에 청량음료나 주류에 첨가되는 식품첨가제로 주목 받고 있다. lactosucroses는 Arthrobacter sp, sprobomyces sp, Rahnella sp과 같은 미생물로부터 생산되어진 β-fructofuranosidase (EC 3.2.1.26)의 작용에 의해 sucrose와 lactose로부터 생산되어진다.
이 연구의 목적은 Sterigmatomyces elviae Mutant로부터 생산되는 lactosucrose의 생산을 위한 배지 및 배양조건 최적화를 하는데 있으며, whole cell enzyme 고정화 방법을 이용한 연속 생산은 높은 생산성의 lactosucrose를 얻기 위해 수행되어졌다.
유과올리고당 생산을 위한 배지 최적화는 통계학적인 실험 및 분석을 실시하였다. Response surface methodology는 애매한 상호작용을 보이는 인자들의 최적화를 위해 사용되어질 수 있다. 배지조성 및 배양조건은 RSM를 이용, Sterigmatomyces elviae Mutant에 의해 lactosucroses 생산을 개선하기위해 최적화 되어졌다. RSM을 이용한 central composite design은 연구에 의해 사용되어졌다. Lactosucroses 생산을 위한 5단계, 5가지 변수의 설계가 얻어졌다. 배지조성의 최적 농도는, sucrose 1.21%, yeast extract 0.98%, bactopeptone 2.92%, and ammonium sulfate 0.536% 로 나타났으며, 그때, 탄소원과 ammonium sulfate가 lactosucroses 생산을 위해 가장 중요한 인자임을 나타내었다. 최적화된 배양조건은 10%접종량과 pH6.7, 배양온도 37℃로 결정되었다. 통계학적 방법에 의해 최적화되었을 때 Lactosucrose의 생산량과 균체량은 39.13%과 16.66%로 증가되었다. 알지네이트를 이용한 whole cell enzyme 고정화에 의한 연속생산을 최적화한 결과 반응온도는 50℃, 반응pH는 6.0, 기질농도는 40%이고 반응 유속은 1.26ml/min 이였으며 그때의 lactosucrose의 최대 생산량은 208g/L였다. 이러한 조건에서 lactosucrose의 생산은 38일 이상 34.6%의 높은 생산이 유지되었다. 다른 균주와 생산성을 비교할 때 안정성 및 생산량에서 Sterigmatomyces elviae Mutant가 가장 좋은 생산성 및 안정성을 나타내었다.
올리고당은 그 수요가 급증하고 있으며, 특히 lactosucrose의 경우 아직 그 연구가 미비하여 실제 산업적으로도 유용하게 사용되어질 수 있을 것으로 생각된다.

다국어 초록 (Multilingual Abstract)

Lactosucroses ((4)^G-β-Galactosylsucrose) have attracted attention in response to an increasing demand for so-called health foods with functional properties such as calorie free, non-cariogenic. Lactosucrose is used as food additives in refresh drinks and alcoholic liquors.
This study is to optimize the culture medium and conditions for the production of lactosucrose by Sterigmatomyces elviae mutant. Continuous production using whole cell enzyme immobilization was also carried out with aim of obtaining a high productivity of lactosucrose.
A statistical experimental design and analysis were used for the medium optimization for the lactosucroses production. Among culture media, optimal concentration of sucrose, yeast extract, bacto peptone and ammonium sulfate were found to be 1.21%, 0.98%, 2.92% and 0.536% respectively. The sucrose and ammonium sulfate have an important effect on the production of lactosucrose. The optimized culture conditions were determined to be 10% inoculum size, pH6.7 and 37℃. The production of lactosucrose and cell mass was increased about 39.13% and 16.66% by using statistical method.
Optimized condition for the continuous production by the immobilization of whole cell enzyme using calcium alginate were determined to be at 50℃, pH 6.0, Brix 40 and the flow rate of 1.26ml/min. The maximum concentration of lactosucrose was 208g/L. In this condition, production of lactosucrose was maintained at high concentration of 34% over 52 days. Sterigmatomyces elviae mutant showed the best productivity and stability when comparing with different microorganisms.

목차 (Table of Contents)

• Abstract = ⅰ
• 국문초록 = ⅱ
• Contents = ⅳ
• List of Figures = ⅸ
• List of Tables = ⅹⅱ
• Part 1. OverView = 1
• Ⅰ. Introduction = 2
• 1. Lactosucrose = 2
• 1.1. General description of lactosucroses = 2
• 1.2. Functions and applications of lactosucroses = 3
• 1.3. Reaction mechanism of lactosucrose = 4
• 2. Experimental design for improvement of lactosucrose Production = 5
• 2.1. Statistical methods for optimization = 5
• 2.1.1. Analysis of variance = 12
• 2.1.2. Response surface methodology(RSM) = 13
• 2.1.2.1. Advantages of RSM = 13
• 3. Immobilization = 13
• 3.1 Definitions = 13
• 3.2. Immobilization technique = 15
• 3.2.1. Entrapment = 15
• 3.3. Applications of immobilization = 15
• 3.4. Advantage and Disadvantages of whole cell immobilization = 16
• 3.5. Various immobilization methods = 18
• 3.6. Immobilization by entrapment method using sodium alginate = 19
• 4. Production of lactosucrose = 20
• 4.1. Production of lactosucroses by immobilization method = 20
• 4.2. Production of high-content lactosucrose = 20
• 5. Market trend = 21
• 6. Objective = 21
• Part 2. Optimization Medium and Culture Condition Using Statistical Methods = 23
• Ⅰ. Materials and Methods = 24
• 1.1. Microorganisms = 24
• 1.2. Medium and culture conditions = 24
• 1.3. Selection of microorganism = 25
• 1.4. Mutagenesis = 25
• 1.4.1. N-methyl-N’-nitro-N-nitrosoguanidine mutation = 25
• 1.4.2. Isolation of high-level lactosucrose producing mutants = 25
• 1.5. Experimental designs = 26
• Ⅱ. Results and discussion = 28
• 2.1.Selection of microorganism and mutation = 28
• 2.1.1. Selection of microorganism = 28
• 2.1.2. Mutation = 28
• 2.2 Optimization of medium and culture condition for lactosucrose production = 33
• 2.2.1. Optimization of medium component using statistical analysis = 33
• 2.2.1.1. 2^(5-1) Fractional Factorial design experiment Ⅰ = 33
• 2.2.1.2. Determination of the influencing factors in total components of culture medium by analysis of variance (ANOVA) = 33
• 2.2.1.3. Determination of optimal concentration of medium concentration by using the response surface methodology (RSM) = 38
• 2.2.2. Optimization of culture conditions for lactosucrose production = 57
• 2.2.2.1. Effect of culture temperature = 68
• 2.2.2.2. Effect of agitation speed = 68
• 2.3. Comparison of basal medium and optimization medium = 68
• 2.3.1. Cultivation using optimized medium = 68
• 2.3.2. Comparison of cell weight and lactosucrose production in different optimization step = 72
• 2.4. Cultivation using 2.5L bioreactor = 72
• 2.4.1. Effect of aeration on the cell weight and lactosucrose production = 72
• Ⅲ. Conclusion = 76
• Part 3. Immobilization of Sterigmatomyces elviae Mutant by Entrapping Cells in Calcium Alginate for Production of Lactosucrose = 77
• Ⅰ. Materials and Methods = 78
• 1.1. Microorganisms = 78
• 1.2. Medium and culture conditions = 78
• 1.3. Immobilization = 78
• 1.4. Analytical methods = 80
• 1.5. Experimental Design = 80
• Ⅱ. Results and discussion = 83
• 2.1. Determination of optimal cell and alginate concentration for immobilization of S. elviae = 83
• 2.1.1. Effect of pH on lactosucrose producton = 92
• 2.1.2. Effect of temperature on lactosucrose production = 92
• 2.1.3. Effect of substrate concentration on the lactosucrose production = 92
• 2.2. Continuous production of lactosucrose using whole cell immobilization = 96
• 2.2.1. Effect of flow rate on the lactosucrose production = 96
• 2.2.2. Comparison of continuous production in the various microorganisms using whole cell immobilization = 96
• Ⅲ. Conclusion = 99
• Part 4. Physical Property of Lactosucrose = 100
• Ⅰ. Materials and Methods = 101
• 1.1. Materials = 101
• 1.2. Methods = 101
• 1.2.1. Viscosity = 101
• 1.2.2. Water activity = 101
• 1.2.3. pH stability = 101
• 1.2.4. Thermal stability = 102
• Ⅱ.Results and discussion = 102
• 2.1. Measurement of viscosity = 102
• 2.2. Measurement of water activity = 102
• 2.3. Measurement of pH stability = 106
• 2.4. Measurement of Thermal stability = 106
• Ⅲ. Conclusion = 109
• References = 110