Acid proteases 생산을 위한 Aspergillus oryzae 고정화 기법의 최적화

최명락,김진만 여수대학교 1998 論文集 Vol.13 No.2

Aspergillus oryzae spores were entrapped in cellulose sponge cubes for production of acid proteases. It grown as a cubic mycelium form in the cavity of carrier. The results obtained were summarized as follows: Immobilization of mycelium in 0.3cm(0.3×0.3×0.3cm) cellulose sponge was more desirable than that of 0.5cm(0.5×0.5×0.5cm) cellulose sponge. Because 0.3cm length of sponge gives the higher surface area per unit volume. In the case of immobilized mycelium weight and free mycelium in medium, the optimal volume of cellulose sponge was 5ml. 0.025% magnesium sulfate in medium appeared maximal acid proteinase and acid carboxypeptidase production by 45.2nkat and 73.5nkat, respectively.

Aspergillus oryzae 酸性 proteinase의 再固定化

崔明洛 麗水水産大學校 産業技術硏究所 1993 産業基術硏究所 論文集 Vol.2 No.-

The present study is an attempt to investigate the immobilized carrier of Aspergilius oryzae cell wall. Optimum conditions for immobilization of Triton X-100 soluble acid proteinase on fractionated cell wall were pH 3.5 and 35℃. The activity of the immobilized enzyme(IME) had similar to or more than that of NATIVE cell wall fraction-bound acid proteinase(CWE). Crosslinking with glutaraldehyde on a adsorbed layer increased the immobilization of acid proteinase. Optimum pH of IME was 2.3. Optimum temperature, the stability of pH and temperature in IME were similar to those of native CWE. Considering bioreactor, the characterization of IME may prevent microbial contamination under acid pH condition during processing.

Lipoxygenase-3 缺乏 大豆系統의 選拔과 特性

정규화,최명락 여수대학교 1998 論文集 Vol.13 No.2

Carotene bleaching test와 SDS-polyacrylamide 전기영동을 통하여 수집한 대두 유전자원 723계통으로 부터 lipoxygenase-3 동위효소가 결핍된 두 계통(N0. 67. No. 217)을 선발하였다. 비린냄새가 아주 적은 돌연변 선발계통은 생리적 이상이 발견되지 않았으며 지방산조성 및 아미노산조성도 정상계통과 큰 차이가 없었다. 연구결과는 경제적 이용성을 갖는 대두품종의 육성에 보탬이 될 뿐만 아니라 주요작물의 유전자원수집과 보존의 중요성을 말해준다. 723 soybean germplasm collections were screened for genotypes missing lipoxy-genase-3 isozyme(linoleate : oxygen oxidoreductase, E.C. 1.13.11.12). Two lines 'No. 67' and 'No. 217' were found to lack lipoxygenase-3 by both carotene bleaching test and improved SDS-polyacrylamide gel electrophoresis. The mutant seeds showing very low beany flavor germinated, grew, and matured normally. Their food composition was similar to that of the normal type of soybean. These results encourage us to breed soybean cultivars having a great economical value for the utilization of soybean and demonstrate the importance of collecting and maintaining extensive germplasm collections of our major crops.

미세조류의 Methane 발효특성

강창민,최명락 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.5

미세조류 Chlorella vulgaris를 기질로 하여 메탄발효에 의한 회분실험 및 연속실험을 실시한 결과, 다음과 같은 결론이 얻어졌다. 회분실험에서 조류의 분해는 경과 일수 10일을 기준하여 2단계로 구분 가능하고, 조류의 성분 중에서 분해되기 쉬운 부분은 최초의 10일간에 거의 분해되었다. 30일간의 회분실험에 있어, 기질 농도 1400~36000 mg/ℓ까지는 경시변화와 함께 가스 생성 속도가 직선적으로 증가하여, 적정 기질농도는 이 범위였다. 연속실험에 있어서는 중간대사물질인 용해성 유기물이 빠르게 소비되어, 반응조내에 축적되지 않았다. 즉 고형 유기물의 가수분해속도가 공정 전체를 제어했다. 생성되는 휘발성 유기산의 농도의 크기는 acetic acid>propionic acid>n-butylic acid>i-valeric acid>n-valeric acid>i-butylic acid의 순서였다. 조류의 분해가능성 유기물의 가수분해속도는 외견상 1차반응식으로 표현 가능하다. 또 가수분해속도정수는 VSS를 기준하여 K_h :0.23~0.28 day^-1, 난분해성 물질 비율(S_e/S_i)·100% : 72.2~84.2%가 구해졌다. 한편, 조류의 가용화를 위해 열처리, 알칼리처리 및 초음파처리에 의한 검토가 요구된다. This study was carried out to examine degradation characteristics of microalgae Chlorella vulgaris in methane fermentation. We measured COD and VS reduction, gas and methane productivity, VFA (volatile fatty acid), respectively. Then we calculated material balance and hydrolysis rates in soluble and solid material. The substrate concentration was controlled from 14 gCOD_cr/ℓ to 64 gCOD_cr/ℓ in batch cultures, and HRT (hydraulic retention time) controlled from 2 days to 30 days in continuous experiments. The results were as follows. In batch culture, accumulated gas productivity increased with the increase of the substrate concentration. The SS and VSS was removed all about 30% increase of substrate concentration and the most of the degradable material removed during the first 10 days. The curve of gas and methane production rate straightly increased until substrate concentration is 26 gCOD_cr/ℓ. In continuous culture experiments, the removal rates at HRT 10days were 20% for total COD and TOC, respectively. At longer HRT, there was no increase in the removal efficiency. At HRT 15 days, the removal rates were 30% for SS and VSS, respectively. Soluble organic materials were rapidly degraded, and so there was no accumulated. Soluble COD concentration was not increase regardless of HRT-increasing. That meaned the hydrolysis was one of the rate-limiting stage of methane fermentation. The first-order rate constants of hydrolysis were 0.23~0.28 day^-1 for VSS, and 0.07~0.08 day^-1 for COD.

키토산 올리고당의 항균효과

김진만,최명락 여수대학교 1998 論文集 Vol.13 No.2

N-glucosamine monomer unit가 7∼14개의 oligochitosan을 제조하고 이의 항균력을 이용한 식품 및 농수산용으로의 응용 가능성을 조사하였다. 식품변패의 지표 미생물인 Escherichia coli를 비롯하여 Bacillus spp. Serratia spp. 및 Pseudomonas spp.등과 같은 여러 가지 식품의 변질에 관여하는 미생물 균주, Rhizopus spp. 등의 식물 연부병 원인균 등 주요 지표 미생물에 대한 항균능력을 검정하였다. 항균 스펙트럼을 조사한 후 각 세균에 대한 MIC(g/100 ml of medium)을 산정한 결과 0.001-0.02의 농도를 나타내었으나 효모 및 곰팡이 균주에 대해서는 다소 높은 경향을 나타내었다. 또한 양식환경에서 자주 출현하여 폐해를 일으키며 특별한 방제 대책이 없는 스쿠치카 섬모충과 oligochitosan을 접촉시켰을 경우 세포의 파열이 신속히 일어남이 관찰되어 이를 응용한 기생충 및 어병균의 방제방법이 가능할 것이다. Antimicrobial activities of chitosan oligomers of seven or more residues were studied to look into their potential use for agricultural and aquacultural purpose. Minimal inhibitory concentrations(MIC) for microbial culture were various depending on microorganism and those of yeast and fungi, to the extent, higher those of bacteria. Also the oligochitosan has been shown cell lysis activity against Scuctiocilate spp.

다핵균 및 단핵균으로부터 고protease 생산균주의 Screening

송상호,임현수,최명락,유은정,김성영 여수대학교 1998 論文集 Vol.13 No.2

4 multinucleate and 7 mononuclear fungi were investigated for screening of high protease productive strains. The protease activity of mononuclear strains was generally greater than that of multinucleate strains. For acid proteinase, Aspergillus awamori AOK 1007 and Asp. oryzae shoyu 2 as mononuclear strains were selected in glucose-polypeptnoe medium. For acid carboxypeptidase, two mononuclear strains were screened as Asp. awamori AOK 1007 and Asp. usamii mut. shirousami AOK 1011 in glusose-sodium glutanate medium. In this result, mononuclear fungi appeared higher protease production than that of multinuclear fungi. So, mononuclear fungi may gave many merits to improve protease activity by using of mutation or cell fusion technology.

갓김치 ether 추출물의 발효 과정상의 생리 기능성 변화

유은정,임현수,최명락,송상호 여수대학교 1999 論文集 Vol.14 No.2

일반 김치의 경우 17±1℃에서 2일 사이에 pH가 4.0에 이르는 것에 비해 갓김치의 경우 18℃에서 저장한 경우에 14일 경에 pH가 4.0(Fig.1)에 이르러 일반 김치 보다 저장성이 3~5배 높은 것으로 나타났으며 총균수에서도 비슷한 양상을 나타내었다. 또한 간암세포에 대한 항암성에서 IC30의 농도가 발효기간별 시료 모두 1000㎍이상으로 나타났으며 발효 0일째 시료가 1000㎍에서 50%의 간암세포 사멸성을 나타내서 가장 항암효과가 우수한 것으로 나타났고, 간정상세포에 대해서는 1000㎍ 이상에서도 28% 이하의 사멸성을 나타내서 정상세포에 대해 큰 영향을 미치지 않는 것으로 나타났다. 또한 항산화 효과에 있어서는 초기(0~2일) 발효시 수소공여능이 53%로 나타나서 다른 식품 추출물의 항산화 효과에 비해서는 비교적 높은 효과를 나타내었다. 그러나 발효 14일에는 15%로 감소되어서, ethyl ether에 의해 추출되어 항산화 효과를 나타내었던 물질들이 발효가 진행됨에 따라 감소된 것으로 사료되었다. The anticancer and antioxidative effects of Mustard Leaf Kimch ether extracts according to various fermentation period were investigated. pH of the Mustard Leaf Kimchi was reached at 4.0 on 14th day, so the period reaching well-fermentating state was up to 3~5 times longer than that of ordinary Kimchi. The anticancer effect on HepG2 by the addition of fermented Mustard Leaf Kimchi ether extracts was higher than that on CCL-13. IC30 of Mustard Leaf Kimchi in HepG2 was showed more than 1000㎍.Especially, 50% of death ratio was showed in HepG2 cells by the addition of ether extracts(0 day). the other side. 28% of death ratio was shown in CCL-13 up to 1000㎍ of ether extracts. As well as the Mustard Leaf Kimchi ether extracts(0-2 days) showed higher antioxidative effect than of others. The hydrogen donor activity was 53% at initial fermentation period, but it was decreased at 14% on 14th day. So, ether soluble antioxidative materials in Mustard Leaf Kimchi was decreased during fermentation.

부착성 동물세포의 Scale-up을 위한 방법의 개발

김승모,송상호,최명락,유은정,임현수 여수대학교 1997 論文集 Vol.11 No.2

This study was a protocol of serial propagation for anchorage-dependent animal cell. Vero. by the optimization of trypsin and EDTA concentration in T-25 flask. Commen enzymetic treatment in PBS solution has been modified for a high degree of cell datachment from T-25 flask and for transfer of a maximum number of viable cells from one culture to the next during the scale up process. Culture of Vero cells were washed with Dulbecco's modified Eagle's medium(DMEM) and then exposed to DMEM containing trypsin(0.025%) and EDTA(0.16%). Up to 90% of cells detached from bottom of flask and the viability of cells was greater than 93%. The protocol using the developed method was successfully applied to the next 10 times scale up process. Consequently, the developed method changed multiple steps of processes to one step, so it promised th easy scale-up and low cost demand of animal cell culture.

효소적 방법으로 제조된 키토산 올리고당의 첨가에 따른 김치의 숙성 및 저장 기간 연장에 관한 연구

임현수,김진만,유은정,최명락 여수대학교 1998 論文集 Vol.13 No.2

효소로 처리된 키토산 올리고당의 첨가에 의한 김치의 숙성 기간 연장 효과를 알아보기 위하여 배추김치에는 0.2-0.4%, 2일째에 0.4%를 첨가하여 20℃에서 10일간 관찰한 결과 0.2% 첨가시 대조군에 비해 2일, 0.4% 첨가시는 4일, 2일째에 0.4%를 첨가한 경우는 8일 이후로 연장 효과가 2-4배 있었으며, 0.4% 이상에서는 미생물 생육자체가 억제되므로 풍미 및 유기산의 생성을 고려할 때 2일째 첨가된 0.4%가 젖산균의 수는 어느정도 유지하면서 stationary phage를 연장시킴으로서 더 좋은 결과를 나타냈다. 갓 김치의 경우 0.1%-0.5% 첨가시 대조군에 비해 1.2-1.4배의 연장 효과가 있어서 배추 김치에 비해서는 낮은 효과를 나타냈다. 따라서, 효소로 처리된 키토산 올리고당의 첨가에 의해 배추 김치는 2-4배의 연장 효과, 갓김치는 1.2-1.4배의 연장 효과를 나타내어 미생물 증식 억제에 의한 김치의 숙성 및 저장 기간 연장 효과를 기대할 수 있었다. To prolong the fermenting and preservation period of Kimchi, chitosanoligosaccharide was prepared by chitosanase. Various concentrations of chitosanoligosaccharide were added into Chinese cabbage Kimchi and Gat Kimchi. Chinese cabbage Kimchi was formulated with various chitosanoligosaccharide concentrations(0.2-0.4%) and fermented at 20℃. And Gat Kimchi was prepared with various chitosanoligosaccharide(0.1-0.5%) concentrations during 26 days at 4℃. In this results, the fermentation period was increased 2-2.5 and 1.2-1.4 folds more than control(0% of chitosanoligosaccharide) for Chinese cabbage Kimchi and Gat Kimchi, respectively. Conclusively, adding of chitosanoligosaccharide into Kimchi significantly prolonged the fermentation and preservation period.

국균세포벽을 모델로 한 새로운 고정화담체의 개발

유은정,김종덕,송상호,임현수,최명락,정규화 여수대학교 1997 論文集 Vol.11 No.2

The purpose of this investigation was to develop a new carrier using chitin and glucan as the model for Aspergillus oryzae cell wall. In case of the ratio of chitin and glucan(3/1), the activity of the immobilized protease had higher than that of model(chitin/glucan=1/3) and carriers were formed gel. In case of soluble chitin and glucan treated with calcium carbonate, there were a 1.8-fold increase in activity of immobilized protease compared with untreated chitin. The scanning electron microscope image showed that a pore size of this carrier had a homogenized pore size and a smooth surface.