Earticle

Lentinula edodes has traditionally been cultivated on hardwood logs, mainly oak (Quercus spp.) in order to obtain fruiting bodies for human consumption. These mushrooms have many nutritional and medicinal benefits. This study was conducted to investigate the characteristics of mycelial growth against to carbon sources, nitrogen sources, and phosphate in 15 breeding lines of L. edodes. Mycelial growth of L. edodes had an advantage over fructose and glucose in monosaccharides, sucrose and maltose in disaccharides, and starch in polysaccharride as carbon sources. Yeast extract was the most effective nitrogen source for mycelial growth. K2HPO4 was good for the mycelial growth while (NH4)2HPO4 repressed it. The optimum culture media for mycelial growth of L. edodes was GYA (glucose 2.0%, yeast extract 0.2%, agar 1.8%)and optimum temperature was 25℃.

The choice of the culture medium is an important factor for the mass production of mycelia in submerged cultures. The influence of liquid medium on the mycelial dry weight of Paecilomyces tenuipes was investigated in this study. The regression equation is expressed as Y=-1292.94187+17.78612X1+18.92425X2+2.11464X3-0.019375X1X2-0.006276X1X3+0.008177X2X3- 0.070169X1 2-0.292175X2 2-0.008818X3 2, where Y represents the value of the mycelial dry weight (g/L), X1 is the particle size of wood sawdust in liquid medium (mesh), X2 is the concentration of the wood sawdust in liquid medium, and X3 is incubation time (h). The medium was optimized using a response surface methodology, and the optimal medium contained 30 g of wood sawdust (140 mesh), 20 g of glucose, and 10 g/L of peptone. Under these conditions, the mycelial dry weight reached 38.1 g/L (actual value). The culture medium containing wood sawdust is simple and easy to use, highly efficient, and eco-friendly, and its effectiveness in large preparations of P. tenuipes mycelia with low material costs has been demonstrated.

Autocrine or paracrine mediators released by the early embryo are implicated in the support of embryonic development. Their mechanisms and optimal embryo density in the medium, however, are uncertain. This study was conducted to establish the optimal embryo density and culture medium volume in mouse parthenogenetic embryo culture. In experiment 1, culture of parthenogenetirally activated oocytes at a concentration of 2~4 embryos/ uL significantly improved development to the blastoryst stage (72%≤) compared with culture at the lower (0.2~1 embryos㎕, 0~37.5%) and the higher (5~6 embryos㎕, 30~53%) concentration for 120 h when the oocytes were cultured in a 5㎕ drop under mineral oil In experiment 2, the embryos cultured at a concentration of 2~4 embryos㎕ in a 10㎕ drop (81.1%) showed significantly higher blastocyst rates than those in a 5㎕ drop (68.5%). This study optimizes in vitro culture condition by modifying embryo density and the volume of culture medium It may give appropriate level of autocrine and/or paracrine factors to enhance viability and subsequent normal development of mouse parthenogenetic embryos in vitro.

본 연구는 4차 산업 대응 옴니채널 유통 환경 속에서, 중소기업 대상의 범용 솔루션 라스트마일 딜리버리 시스템 을 개발 구축함으로서 중소기업에 배송효율 극대화 및 운송 서비스 향상으로 물류경쟁력 강화를 달성할 수 있는 클라우드 기반 사용료 방식의 초기투자비용 및 운영비를 절감할 수 있도록 하여 중소기업이 쉽게 접근 가능한 범용 솔루션 개발 및 구축하여 활용하고 배포되어 범용화를 실현하는 것이 그 목적이다. 라스트마일 딜리버리 시스템을 구축한 중소기업은 시뮬레이션을 통한 운영전략 수립, 물량변동에 대응하여 효율적인 노선 운영이 가능하고 배차계 획 수립 소요시간이 단축되며 최적화된 배송 경로를 통한 고객 만족 및 브랜드 이미지 제고를 통해 고객 서비스에서 도 우위를 선점할 수 있다. 본 연구의 라스트마일 딜리버리 시스템이 구축되고 범용화를 실현한다면 적은 비용으로 중소기업들의 배송 경쟁력을 강화시키고 고객들의 니즈에 맞는 서비스를 제공할 수 있어 물류와 유통 산업의 전반적 인 성장의 기여가 기대되어진다.

In this research, we developed and built a universal solution last mile delivery system for small and medium sized enterprises in the quaternary omni-channel distribution environment. The objective is to develop, utilize, and even distribute and generalize general-purpose solutions that small and medium sized enterprises can easily access by reducing initial investment and operating costs. Small and medium sized enterprises that have built a last delivery system can efficiently operate routes in response to simulation and volume changes, reduce the time required for dispatching plans, and improve customer satisfaction and brand image through optimized delivery routes. You can dominate the service. By setting up regional areas, centralizing capacity management, and by realizing integrated dispatch of orders from other customers, the customer order acceptance rate can be improved. By using optimal transportation plans, you can increase the satisfaction of the courier, efficiently operate the vehicle, and reduce fuel costs by reducing the mileage. Even if stores and centers and added, it is easy to expand and apply, improving the logistics operation efficiency.

Production of the antioxidant substances by Bacillus polyfermenticus SCD was investigated using shake-flaskfermentation. The one-factor-at-a-time method was first employed to determine the key ingredients for optimal mediumcomposition, then further investigation of the medium composition was performed using response surface methodology(RSM). The antioxidant activity was measured using 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assays. After screeningvarious elements, fructose, tryptone, and MgSO4·7H2O were chosen as the main factors for study in the statisticalexperimental design. Central composite design (CCD) was then used to determine the optimal concentrations of these 3components. Under the proposed optimized medium containing 2.8% fructose, 1.34% tryptone, 0.015% MgSO4·7H2O, 0.5%NaCl, and 0.25% K2HPO4, the model predicted an antioxidant activity of 80.5% (R2=0.9421). The actual experimental resultswere in agreement with the prediction.

본 연구에서는 홍국균 (Monascus pilosus)을 이용하여 이차대사산물인 monacolin-K를 대량생산하기 위하여 탄소원 또는 질소원으로 콩 분말을 기질로 하여 배지를 최적화하고자 하였다. 생산배지에서 탄소원으로 콩 분말을 적용한 실험에서는 soluble starch대신에 콩 분말을 전량 또는 50% 대체하였을 경우 전 실험구간에서 monacolin-K 생산량이 대조구의 50% 미만으로 생산되거나 전혀 생산되지 않았다. Soluble starch 대신에 sucrose를 사용한 실험의 경우 monacolin-K와 건조균체량은 각각 262 mg/L, 31.6 g/L로 대조구의 218 mg/L, 30.2 g/L보다 생산량이 증가하였다. 따라서 생산배지의 탄소원으로 sucrose를 변경하였다. 생산배지의 질소원으로 사용하는 yeast extract (15 g/L), malt extract (40 g/L), beef extract (30 g/L) 대신에 3종류의 콩 분말로 대체 실험을 한 결과, 대조구에서 monacolin-K는 262 mg/L를 생산되었고, yeast extract 50% 대신에 DSM 콩 분말을 사용한 실험구에서는 monacolin-K를 215 mg/L을 생산되어 가장 좋은 결과를 냈다. 따라서 생산배지에 yeast extract 50% 대신에 DSM 콩 분말을 적용하기로 하였다. Plackett-Burman Design 실험계획법에 의한 실험결과 monacolin-K의 생산에 영향을 미치는 인자는 yeast extract, beef extract, (NH4)2SO4 순으로 영향을 주는 것으로 나타났다. Monacolin-K 생산에 영향을 미치는 주요인자 3가지로 중심합성계획에 의한 반응표면분석을 수행한 결과 최적배지조성은 sucrose 96 g/L, DSM 7.5 g/L, yeast extract 8.4 g/L, (NH4)2SO4 12.0 g/L, malt extract 40 g/L, beef extract 26.7 g/L, Na2HPO4 0.5 g/L, L-Histidine 3.0 g/L, KHSO4 1.25 g/L 이고, monacolin-K 생산 예상량은 369.9 mg/L 이었다. 최적배지를 이용하여 홍국균을 27℃, 200 rpm 조건에서 플라스크 배양 한 결과 monacolin-K 생산량과 건조균체량은 각각 418 mg/L, 27.1 g/L이었다. 결과적으로 monacolin-K 생산과 균체량의 감소 없이 콩 분말을 이용한 배지성분의 변화는 어느 정도 가능함을 알 수 있었다. 현재 알려진 고가의 질소원을 대체하기 위해서는 초기부터 균의 형태구조에 영향을 미치는 배지와 발효환경을 고려하여야 할 것으로 판단되었다.

During the last decade, monacolin-K biosynthesized by fermentation of red yeast rice (Monascus strains) was proved to have an efficient cholesterol lowering capability, leading to rapid increase in the market demand for the functional red yeast rice. In this study, the production medium composition and components were optimized on a shake flask scale for monacolin-K production by Monascus pilosus (KCCM 60160). The effect of three different soybean flours on the monacolin-K production were studied in order to replace the nitrogen sources of basic production medium (yeast extract, malt extract and beef extract). Among the several experiments, the production medium with dietary soybean flour to replace a half of yeast extract was very good for monacolin-K production. Plackett-Burman experimental design was used to determine the key factors which are critical to produce the biological products in the fermentation. According to the result of Plackett-Burman experimental design, a second order response surface design was applied using yeast extract, beef extract and (NH4)2SO4 as factors. Applying this model, the optimum concentration of the three variables was obtained. The maximum monacolin-K production (369.6 mg/L) predicted by model agrees well with the experimental value (418 mg/L) obtained from the experimental verification at the optimal medium. The yield of monacolin-K was increased by 67% as compared to that obtained with basic production medium in shake flasks.

에리스리톨을 산업적으로 대량생산하기 위해서는 에리스리톨의 생산성을 높이고 부산물인 글리세롤의 생성을 억제하는 배지 및 발효 공정의 최적화가 필요하다. 에리스리톨 생성을 위한 최적 탄소원은 포도당이고 최적 농도는 400 g/L이며 최적 온도는 3였으며 이 때 산소는 과잉으로 공급해 주어야 함을 알았다. 에리스리톨의 수율과 생산성을 향상시키기고 배지가격을 줄이기 위해 효모 추출물의 사용을 5 g/L에서 3 g/L로 줄이고 urea 2.72g/L K2HPO4 1.79 g/L, MgSO4. 7H2O 0.18 g/L를 첨가해 줌으로써 에리스리톨 수율을 31.4%에서 45.2%로 에리스리톨 생산성을 0.747 g/L/h에서 1.071 g/L/h로 향상시켰다. 또한 글리세롤의 생산량도 96.6 g/L에서 45.7 g/L로 줄었다. 이 최적 배지를 바탕으로 5L 발효기에서의 재현성과 pH에 대한 영향을 알아보기 위해 실험을 한 결과 pH를 조절한 경우에 에리스리톨 생산성이 낮아졌다. 5L 발효에서는 다량의 거품이 발생되는 것을 관찰하였다.

Optimization of the medium and fermentation conditions for erythritol production has been studied. We have found that the optimal carbon source was glucose at the concentration of 400 g/L. The optimal temperature was 30℃ with excessive aeration. Improved erythritol productivity was achieved by reducing the yeast extract from 5 g/L to 3g/L while adding 2.7 g/L urea, 1.79g/L $K2HPO4, and 0.18g/L MgSO. 7$H2O. The erythritol productivity increased from 0.747 g/L/h to 1.071 g/L/h and the yield increased from 31.4% to 45.2%. The byproduct glycerol was reduced from 96.6g/L to 45.7g/L as well. We have performed 5L fermentation with and without the pH control. The erythritol productivity with the pH control was about 30% lower than that without pH control. Excessive foaming of 5L fermentation has been observed during fermentation.

Aspergillus terreus에 의한 이타콘산 생산 발효공정에서 생산균주의 성장을 어느 정도 제한시킴으로써 배양생리적 특성이 이타콘산 생합성 쪽으로 치우치도록 통계적 방법을 적용하여 itaconic acid의 생산배지 조성을 최적화하는 연구를 수행하였다. 이타콘산은 TCA회로를 거쳐 합성된 cis-aconitic acid의 디카르복실화 반응에 의해 생합성되는 고부가 화학원료물질이다. 우선 One factor at a time (OFAT) 방법을 이용하여 이타콘산의 생산성 증가에 크게 영향을 미치는 중요한 탄소원들로 sucrose, glucose, fructose와 soluble starch를 확인할 수 있었고, 질소원들로는 cottonseed flour와 soybean meal을 찾을 수 있었다. Fractional factorial design을 통하여 이들 6가지 요인들 간의 상호작용의 정도를 확인한 결과 sucrose와 cottonseed flour간의 상호작용의 정도가 가장 컸고, 나머지 요인들 간의 상호작용의 정도는 작거나 혹은 이타콘산 생산에 오히려 부정적인 결과를 나타냈다. 또한 full factorial design (FFD) 실험을 통해 생산배지에 KH2PO4와 MgSO4가 과량 첨가되면 이타콘산의 생산성이 심각하게 저해됨을 알 수 있었다. FFD의 1차모델식을 근간으로 하여 최급상승법 (steepest ascent method, SAM)을 적용하여 sucrose, cottonseed flour, KH2PO4 및 MgSO4의 최적 농도로 향하는 가장 가파른 기울기를 구함으로써, 신속하고 효율적으로 최적 농도지점에 대한 정보를 얻을 수 있었다. SAM이 제시해주는 농도 부근에서 반응표면분석 (response surface method, RSM)을 적용하여 각 배지성분의 농도를 최적화 시키기 위해, 2개의 중요한 요인인 sucrose와 cottonseed flour를 이용하여 중심합성계획 (central composite design, CCD) 실험을 수행하였다. 그 결과 이타콘산의 최적 배지조건은 sucrose 90.4 g/L, cottonseed flour 53.8 g/L인 것으로 관찰되었고, 이 농도에서 이타콘산의 생산성은 초기 사용된 배지에서의 생산성에 비해 약 7배 증가한 4360 mg/l로 나타났다. 이로부터 탄소원 (C)으로 사용한 sucrose와 질소원 (N)으로 사용한 cottonseed flour 간의 C/N 비율이 이타콘산의 생산성에 큰 영향을 미친다는 것을 확인할 수 있었다.

Statistical optimization of the production medium was carried out in order to find an optimal medium composition in itaconic acid fermentation process. Itaconic acid utilized in the manufacture of various synthetic resins is a dicarboxylic acid biosynthesized by fungal cells of Aspergillus terreus in a branch of the TCA cycle via decarboxylation of cis-aconitate. Through OFAT (one factor at a time) experiments, six components (glucose, fructose, sucrose, soluble starch, soybean meal and cottonseed flour) were found to have significant effects on itaconic production among various carbon- and nitrogen-sources. Hence, using these six factors, interactive effects were investigated via fractional factorial design, showing that the initial concentrations of sucrose and cottonseed flour should be high for enhanced production of itaconic acid. Furthermore, through full factorial design (FFD) experiments, negative effects of KH2PO4 and MgSO4 on itaconic acid biosynthesis were demonstrated, when excess amounts of the each component were initially added. Based on the FFD analysis, further statistical experiments were conducted along the steepest ascent path, followed by response surface method (RSM) in order to obtain optimal concentrations of the constituent nutrients. As a result, optimized concentrations of sucrose and cottonseed flour were found to be 90.4g/L and 53.8g/L respectively, with the corresponding production level of itaconic acid to be 4.36 g/L (about 7 fold higher productivity as compared to the previous production medium). From these experimental results, it was assumed that optimum ratio of the constituent carbon (sucrose) and nitrogen (cottonseed flour) sources was one of the most important factors for the enhanced production of itaconic acid.

광범위한 약리작용을 가지고 있어 질병의 치료 및 예방 에 효과가 있는 천연물 중 가장 주목받고 있는 약용버섯은 분류학상 균류 (Fungi) 중 진균류 (Eumycetes)에 속하며 대부분은 담자균류 (Basidiomycetes)의 일종이다 [1,2]. 최근 담자균류 유래 약용버섯들의 항암효과가 과학적으로 입증 되고 있으며 [3,4], 약용버섯 추출물 중에서 특별히 면역증 강작용에 의해 탁월한 항암효능과 당뇨병 개선효과를 보이 는 생리활성물질은 β-D-glucan 구조를 갖는 수용성 단백다 당체 (향후 “단백다당체”로 명명함)로 구성되어 있음이 밝 혀졌다 [5,6,7,8]. 표고버 섯균사체 추출물의 경우 면역증 강효과로 인해 기능성 식품과 암치료 (보조)제 의약품으로 사용되고 있다. 이 버섯의 자실체에서 추출한 단백다당체 역시 항암성 면역증강제인 Lentinan 주사제로 판매되고 있 다. 이 외에도 액상배양된 운지버섯 균사체에서 추출한 단백 다당체 (PSK 및 Krestin)는 항암성 면역증강제로, 치마버섯 균사체 배양시 생합성되는 세포외다당체 (exopolysaccharides) 역시 동일한 효능의 주사제 (Shizophyllan)로 판매되고 있다. 학명이 Inonotus obliquus로 불리는 차가 (Chaga, Tchaga) 버섯은, 나무줄기나 그루터기에 자생하는 백색부후균의 일종 으로, 1년에 0.9 cm 이하로 자라는 매우 귀한 약용버섯이다. Inonotus obliquus 유래의 대표적인 생리활성물질인 단백다 당체의 경우 항당뇨 효과 [9] 및 면역증강 효과로 인한 항암 효과가 뛰어난 것으로 이미 오래 전에 입증된 바 있는데 [10,11,12], 이러한 단백다당체는 특별히 고안된 정제과정을 거쳐 정제될 경우 건조중량의 5% 이하의 함량을 나타내는 미량성분이다 [13,14]. Inonotus obliquus는 최소 5년 이상 성장해야 포자가 형성되고, 통상 10~20년 이상 자라야 채집 이 가능한 것으로 알려져 있다. 이처럼 차가버섯 자실체의 까다로운 성장조건으로 인해 생산성이 매우 낮으므로, 정품 의 야생 차가버섯을 구입하기가 쉽지 않고, 그 가격 또한 매 우 비싼 실정이다. 한편 차가버섯 자실체의 고체배양에 의해 단백다당체를 생산하는 방법은 대량생산 시 품질관리가 힘 들며 자실체 재배를 위한 많은 노동력과 시간이 요구됨에 따라 생산성도 떨어지는 단점이 있다 [15,16]. 반면에 차가버섯 균사체 액상배양을 통한 단백다당체 생산공정은 기존의 자실체 배양을 통해 생산하는 방법에 비 해 보다 잘 규명된 조건하에서 대량 생산이 가능하며 배양 기간 또한 단축시켜 생산성을 크게 높일 수 있는 장점이 있다 [17,18,19]. Inonotus obliquus 균사체의 액상배양에 의한 단백다당체의 산업적인 대량생산을 위해서는 고생산성 균주의 확보가 핵심적인 관건이다. 고생산성 균주를 개발 하기 위해서는 최종 생산배양에서 선별된 균주들의 단백다 당체 생산성을 확인해야 하는데, 이를 위해서는 다음의 일련 의 배양공정을 거쳐야 한다: 단일 콜로니를 얻기 위한 원형 질체 형성 및 재생, 고체성장배지에서의 계대배양 (고체성 장배장), 정치배양, 플라스크에서의 액상 성장배양을 거쳐 최종적으로 플라스크 또는 발효조애서의 액상 생산배양. 이 일련의 배양공정에서 균주개발 시 가장 큰 장애요인은 고체 성장배지에서의 계대배양 (이후 “고체성장배양”)인데. 그 이유는 이 배양 단계에서 특별히 장기간의 배양 시간이 요구 되어 신속한 균주개발이 매우 어렵기 때문이다. 본 연구에 서는 고체성장배양 시의 이러한 문제점을 극복하고자, 배양 환경의 최적화에 대한 연구를 중점적으로 수행하였다. 본 연구팀은 실제로 원형질체 형성을 통한 균주 개량에 관한 연구를 통해, 고체성장배양에서 훌륭한 성장 특성을 보이 는 균주들이 최종 액상 생산배양에서도 단백다당체의 생산 성이 높음을 확인한 바 있다 [20]. Inonotus obliquus 균사체와 같은 균사형성 고등균류의 경우 배지성분에 따라 배양 생리적 및 배양 형태적 특성이 확연히 달라진다. 따라서 고생산성 균주의 신속 선별을 위 해서는 고체성장배지에 포함되는 영양원의 성분 및 조성의 최적화가 필수적이다. 배지 최적화를 수행할 때 단일 요소 에 대해서만 변화를 줄 경우, 영양원 간의 상호작용을 제 대로 파악할 수 없으므로 최적 배지조성을 찾기 힘들며, 여러 요소를 전체적으로 조사할 경우 과다한 실험양으로 인한 한계에 직면하게 된다. 이러한 어려움을 해결하기 위 해 본 연구에서는 유용대사산물의 생물공학적 대량생산을 위해 주로 사용되는 통계적 배지최적화 방법을 도입하였다 [21,22,23]. 즉 고체성장배지의 조성을 최적화하기 위해 균 체의 성장속도에 큰 영향을 미치는 주요 배지성분들을 우 선 찾아내고, 그 성분들의 교호작용을 확인하기 위한 다양 한 통계적 실험방법들을 순차적으로 적용한 후, 최종적으 로 반응표면분석법을 적용하여 균사체의 성장속도가 최대 가 되는 배지조성을 결정하였다.

The protein-bound innerpolysaccharides (IPS) produced by suspended mycelial cultures of Inonotus obliquus have promising potentials as an effective antidiabetic as well as an immunostimulating agents. To enhance IPS production, intensive strain improvement process should be carried out using large amount of UV-mutated protoplasts. During the whole strain-screening process, the stage of solid growth-culture was found to be the most time-requiring step, thus preventing rapid screening of high-yielding producers. In order to reduce the cell growth period in the solid growth-stage, therefore, solid growth-medium was optimized using the statistical methods such as (i) Plackett-Burman and fractional factorial designs (FFD) for selecting positive medium components, and (ii) steepest ascent (SAM) and response surface (RSM) methods for determining optimum concentrations of the selected components. By adopting the medium composition recommended by the SAM experiment, significantly higher growth rate was obtained in the solid growth-cultures, as represented by about 41% larger diameter of the cell growth circle and higher mycelial density. Sequential optimization process performed using the RSM experiments finally recommended the medium composition as follows: glucose 25.61g/L, brown rice 12.53 g/L, soytone peptone 12.53 g/L, MgSO4 5.53 g/L, and agar 20 g/L. It should be noted that this composition was almost similar to the medium combinations determined by the SAM experiment, demonstrating that the SAM was very helpful in finding out the final optimum concentrations. Through the use of this optimized medium, the period for the solid growth-culture could be successfully reduced to about 8 days from the previous 15~20 days, thus enabling large and mass screening of high producers in a relatively short period.

가능한 한 대량의 균주를 테스트하여 고생산성을 지닌 균주를 신속히 선별하기 위해서는 소규모 액상배양 방법의 확립이 필수적인데, 강력한 지질저하제인 monacolin-K의 생산균주인 균사형성 Monascus의 경우 포자나 균사체의 형성이 활발하지 않은 배양 생리적 특성으로 인해 소규모 (miniature) 액상배양이 매우 어려운 문제점이 있다. 본 연구에서는 monacolin-K 고생산성 균주개량의 효율성을 큰 폭으로 증가시키고자, 기존의 플라스크 액상배양 방법을 소규모화시킨 tube 배양 방법을 개발함으로써 단기간에 보다 많은 양의 균주를 테스트하고자 하였다. 이차대사산물인 monacolin-K의 생합성은 각각의 배양단계에서의 생산균주의 배양형태가 중요한데, 특별히 최종 생산배양에서 생산성 증가에 심각한 영향을 미치는 배양형태인 직경 1mm 이하의 pellet 모양을 유도하기 위해서는 성장배양 시에 반드시 고농도의 균사모양이 유도되어야 하는 것으로 관찰되었다. 50 ㎖ tube (7 ml의 조업부피)를 이용하는 소규모 액상 성장배양의 경우 solid seed 배양 단계에서의 포자형성배지의 조성을 통계적 방법을 통해 최적화하고, 또한 성장배지 성분에 brown rice 가루 20 g/L를 첨가할 때,원하는 균사모양의 배양형태가 유도됨을 확인할 수 있었다. 한편 7 ml의 조업부피의 tube를 이용한 소규모 성장배양 방법의 개발로 인해, 기존의 flask 배양을 이용할 때보다 단기간에 훨씬 많은 변이주의 생산성을 조사하게 되어 균주개량 속도를 큰 폭으로 향상시킬 수는 있었으나, 선별된 개별 균주의 monacolin-K의 생산성은 오히려 전체적으로 감소하는 경향을 보여 주었다. 이러한 결과는 소규모 배양방법을 확립하기 위해 새로이 개발한 포자형성배지와 성장배지의 조성변화로 인해 기존에 확립된 생산배지에서는 생산균주의 monacolin-K 생합성 능력이 최대로 발휘되지 않았기 때문에 발생한 것으로 판단되었다. 따라서 본 연구를 통해 확립된 소규모 성장배양에 적합한 최적의 생 산배지 조성을 확립하고자 통계적 방법인 Plackett-Burman design을 적용하여 monacolin-K 생산성 향상에 영향을 주는 glycerol, malt extract, yeast extract, brown rice의 4가지 성분들을 최종 선별하였고, 또한 이들의 최적농도 결정을 위해 반응표면분석 실험을 수행하였다. 이와 같이 결정된 최적생산배지를 사용하여 생산배양을 수행한 결과, monacolin-K의 생산성이 2배 이상 증가하고, 배양 형태 또한 용존산소와 영양분의 전달이 매우 용이한 직경 1mm 이하의 pellet 모양을 유지함을 확인할 수 있었다.

It is crucial to develop a miniaturized cultivation method for large and rapid screening of high-yielding mutants of monacolin-K, a powerful anti-hypercholesterolemic secondary metabolite biosynthesized by the fungal cells of Monascus ruber. In order to investigate as many strains as possible in a short time, a miniaturized fermentation method especially suitable for the cultivation of the filamentous Monascus mutants was developed using 50 ㎖ culture-tube (7 ㎖ of working volume) instead of the traditional 250 ㎖ flask (50 ㎖ of working volume). Generally, in filamentous fungal cell fermentations, morphologies in growth and production cultures should be maintained as thick filamentous and compact-pelleted (usually less than 1 mm in diameter) forms, respectively, for enhanced production of secondary metabolites in final production cultures. In this study, we intended to induce the respective optimal morphologies in the miniaturized culture system for the purpose of rapid screening of overproducers. Miniaturized growth culture system was successfully developed due to the mass production of spores in the statistically optimized solid medium. When large amounts of spores were inoculated into the growth cultures, and brown rice flour (20 g/L) was also supplemented to the growth medium, dense filamentous morphologies were successfully induced in the growth cultures performed with the 50 ml culture tubes. It was implied that the amounts of spores inoculated into the growth tube-cultures and the growth medium components should be the key factors for the induction of the filamentous forms in the growth fermentations. Furthermore, in order to statistically optimize production medium, multiple experiments based on Plackett-Burman design and response surface method (RSM) were carried out, resulting in more than 2 fold enhanced production of monacolin-K in the final production cultures with the optimized production medium. Notably, under the production culture conditions with the statistically optimized medium, optimal pellet sizes below 1 mm in diameter were reproducibly induced, in contrast to the thick and viscous filamentous morphologies observed in the previous production cultures.

dagA as β-agarase degrades agarose into neoagarotetraose and neoagarohexaose as major products. Neoagarooligosaccharides including above two have a good potential as active ingredient in the pharmaceutical, food, and cosmetic industries. The major objective of this study was to develop an optimized medium for the production of dagA by employing Streptomyces lividans. Two very popular statistical methods of Plackett-Burman design (PBD) and the response surface method (RSM) with a central composite design (CCD) were employed to select key components and to optimize their concentrations, respectively. Among 7 nutrients tested, one carbon source of glucose, one nitrogen source of yeast extract, and MgCl2 · 6H2O were selected through PBD as medium components whose concentrations were to be optimized. Through the RSM experiment, the optimum concentrations of glucose 25 g/L, yeast extract 11 g/L and MgCl2 · 6H2O 5 g/L were identified. With the optimized medium composition, 7.42 unit/ml of dagA was produced in 3 days of cultivation.

Invertase (EC 3.2.1.26)는 sucrose의 β-D-fructofuranoside 결합을 가수분해하여 glucose와 fructose의 혼합당류인 invert sugar를 생산하는 효소이다. Invert sugar는 sucrose에 비해 감미도가 높고 비결정성이기 때문에 식품공업이나 제과공업 에서 널리 사용되고 있다 [1-3]. Invertase는 미생물과 고등 식물 등의 다양한 기원으로부터 생산되는데, 미생물에서는 주로 효모인 Saccharomyces 속을 중심으로 Rhizopus oryzae, Amylomyces rouxii, Zymomonas mobilis, Aspergillus niger, Rhodotorula glutinis, 등으로부터 생산되는 것이 알려져 있으며 [4-8], 효소생산 최적화 [9,10], 효소정제 및 특성규 명 [11,12], 고정화 [13-16], 및 효소의 분자 생물학적 연구 등 [17]의 분야에서 비교적 연구가 활발히 진행되어 왔다. Bacilus flexus는 연쇄상간균으로 의료분야와 포장 분야에서 이용되는 생분해 가능한 내열성 플라스틱인 polyhydroxyalkanoate (PHA)의 생산 [18], 또는 알칼리성 amylase 생산 [19] 등에 이용되는 미생물이다. 본 연구에서는 국내 여러 지역의 토양 시료로부터 invertase 생산 미생물을 스크리닝한 결과, invertase를 비교적 고 농도로 생산하는 Bacilus flexus 균주를 분리하여 이를 동 정하였고, invertase 생산조건을 플라스크 배양을 통해 최 적화하였다.

In the present study, we isolated a new bacterial strain producing invertase (EC 3.2.1.26) and determined optimized culture condition in flask culture. The strain was identified as Bacilus flexus determined by the 16S rDNA sequencing method. The invertase was produced only in the sucrose medium as the sole carbon source. Potassium nitrate was an adequate nitrogen source for enzyme production, whereas meat peptone showed the highest bacterial growth. Enzyme production was increased about 2-fold when MgSO4·7H2O was supplemented to the growth media. The optimum temperature was found to be 30℃ for both enzyme production and bacterial growth. Invertase exhibited pH optima in the range 5.0-6.0 and have a temperature optimum at 40℃, similarly to other invertases found from different microbial sources. Several mineral ions (K and Fe) stimulated the invertase activity, whereas some bioelements (Ag, Mg, and Mn) inhibited enzyme activity. Under the optimized culture condition, the maximum enzyme production (over 250 units/mL) was achieved at 20 h. To the best of our knowledge, this is the first time to report on invertase production by Bacilus flexus.

Covalent cross-links between a number of proteins and peptides explain why transglutaminase may be widely used by food processing industries. The objective of this work was optimization of the fermentation process to produce transglutaminase from a new microbial source, the Streptomyces sp. P20. The strategy adopted to modify the usual literature media was: (1) fractional factorial design (FFD) to elucidate the key medium ingredients, (2) central composite design (CCD) to optimise the concentration of the key components. Optimization of the medium resulted in not only an 86% increase in microbial transglutaminase activity as compared to the media cited in the literature, but also a reduction in the production cost. Optimal fermentation conditions - namely temperature and agitation rate - were also studied, using CCD methodology. Usual conditions of 30°C and 100 rpm were within the optimal area. All other parameters for enzyme production were experimentally proven to be optimum fermentation conditions.

활성오니의 주요 미생물인 Zoogloea ramigera의 증식과 zooglan 생산에 영향을 미치는 배양조건에 대하여 연구하였다. Z. ramigera의 증식과 zooglan 생산에 영향을 미치는 성분의 영향을 살피기 위해 특정성분을 제한한 결과 phosphate 제한시 zooglan의 생산이 현격하게 감소하였다. 탄소원으로 포도당 이외에 유당을 이용한 zooglan 생산이 우수하였다. 질소원으로 사용된 NH4CI 와 (NH4)2SO4 가 세포 증식을 향상시켰다. 포도당과 NaNO3의 비가 60일 경우 포도당의 초기농도가 456g/L일 때 zooglan 생산이 18.5g/L로 가장 높았다.

Effects of growth conditions on the growth of Zoogloea ramigera and the production of zooglan were investigated. The production of zooglan was greatly reduced in the phosphate-limiting medium. NH4CI and (NH4)2SO4 improved cell growth when they were used as a nitrogen source. The medium containing 45 g/L of glucose and 27 g/L of NaNO3 resulted in the highest production of zooglan at 18.5 g/L.

Optimal media and cultural conditions for the production of prodigiosin-like pigment were established using Serratia sp. KH-95. Glucose and phosphate(K2PO4) stimulated the cell growth, but inhibited the production of pigment at concentration levels of above 10 g/L and 2.0 g/L, respectively. Addition of soy been oil or rice oil to the production medium accelerated cell growth up to more than 2-3 times, but the production of prodigiosin increased about 15-20% in spite of the good cell growth. The effect of pH on the production of pigment was investigated in a 5 liter-bioreactor. When the pH of culture broth was maintained below 8.0, most of pigment was attached to the surface of cells. When the pH of culture broth was above 8.5, however, about 70% of total pigment was suspended in the supernatant of the broth. The cell growth and production of pigment were inhibited at dissolved oxygen concentration of below 10% of air-saturation.

For production of biomass and protease, statistical experimental methods were applied to optimize production–medium composition using a Bacillus licheniformis SCK 121057. Firstly, medium components thought to have positive effects on biosynthesis of biomass and protease were selected by preliminary experiments; one factor at a time method. Factors showing significant effects were chosen by the Plackett-Burman design. Fractional factorial design (FFD) was then used to investigate the interactive effects of the selected medium components on productivity of biomass and protease. The steepest ascent method (SAM) and response surface methodology (RSM) were employed to determine the optimal concentrations of each component for maximal production of biomass and protease. Using the optimized medium, 4.02 g/L of biomass and 161.27 U/ml of protease were produced, almost three times higher than that from the initial medium conditions. Finally, fermentation conditions were examined for production of biomass and protease in a laboratory fermentor using the optimized production medium. The maximum biomass (6.54 g/L) and protease (240.06 U/ml) were obtained at 42℃, 200 rpm, 2 vvm and pH 8.

Exopolysaccharides(EPS) from Aureobasidium pullulans are used for health foods and cosmetics. In order to better exploit EPS production from A. pullulans, medium composition was optimized using stastical methods. And, effect of each nutrient of basal medium was investigated by preliminary studies (one-factor-at-a-time). Maximum growth and EPS production were observed at various initial pH 3-5, while growth and EPS production were also very low at pH 2 and pH 7. Among various nitrogen sources (Ashbya gossypii extract, beef extract, corn steep liquor, corn steep, powder, malt extract, soytone peptone and yeast extract) tested, the highest mycelial growth and EPS production were achieved in the medium containing Ashbya gossypii extract. Also, among various carbon sources, EPS production and the growth of A. pullulans was the best on sucrose. The Plackett–-Burman design was used to evaluate the effects of 7 variables selected from preliminary studies, and three statistically significant variables namely, sucrose, NaNO3, and Ashyba gossiipii extract, were selected. Then, a three-level Box-Behnken design was employed to optimize the medium composition for EPS production in shake-flask. Using this methodology, the quadratic regression model of producing EPS was built and the optimal combinations of media constituents and culture conditions for maximum EPS production (about 30g/L) were determined.

The medium components were optimized by using a statistical experimental design approach for increasing chitinase production from the Antarctic bacterium Sanguibacter antarcticus KOPRI 21702. To increase the production of psychrophilic chitinase, significant carbon and nitrogen sources were determined and other elements were chosen by using a two-level Plackett-Burman statistical design technique. In the first optimization step, glycerol and peptone were selected as main carbon and nitrogen sources, respectively. In the second step, the influence of fifteen components included in Marine Broth medium on chitinase production was evaluated. As a result, NaCl, Fe(C6H5O7), MgCl2, Na2SO4, H3BO3, and CaCl2 were found to have a positive influence on the chitinase production, while KCl, KBr, and NH4NO3 had a negative influence. In the third step, the concentrations of NaCl, Fe(C6H5O7) and MgCl2 were further optimized. The maximum chitinase activity obtained was 89 U/L using 10 g/L glycerol, 5 g/L peptone, 23 g/L NaCl, 0.01 g/L Fe(C6H5O7), and 2.5 g/L MgCl2. [This work was supported by a grant from the Korea Polar Research Institute (PE10050).]

Glucan formed by linking of many glucose molecules has several interesting in properties, and it is structurally divided into two types of α-glucan and ß-glucan depending on the mode of bonding. Among them, ß-glucan is a powerful immune stimulator known to activate macrophages in the immune system. Especially, to enhance production of ß-glucan (exopolysaccharides, EPS), we have optimized media for EPS by Aureobasidium pullulans. The optimization of culture medium was investigated for the production of ß-glucan by statistical experimental design. In the previous study, among organic nitrogen sources, it was found that Ashbya gossypii extract was pivotal factor for the production of ß-glucan. After adaptive nefactor- at-a-time (OFAT) method experimentation was carried out, the relative importance of medium components for maximum glucan production was evaluated by using Plackett-Burman design (PBD). As the result, it was showed that sucrose, ammonium sulfate and A. gossypii extract were significant components for EPS production. Also, on the basis of PBD analysis, Box-Behnken design and Response Surface Method (RSM) were carried out to search for optimum medium of EPSs productionby using statistical analysis system (SAS) program. Using the optimized parameters from RSM, theoretical maximum production of ß-glucan resulted in about 60 g/L. Under optimized medium, EPS production of the optimum medium was ten times higher than one (5.34 g/L) of the basal medium.