식물(植物) 병원성(病源性) 미생물(微生物)에 미치는 Pseudomonas fluorescens, KR-164의 영향(影響)

이영환,김영일,이재평,김용웅,김용재,이재와,Rhee, Young-Hwan,Kim, Yeong-Yil,Lee, Jae-Pyeong,Kim, Yong-Wong,Kim, Yong-Jae,Lee, Jae-Wha 한국토양비료학회 1990 한국토양비료학회지 Vol.23 No.1

The antagonistic fluorescent pseudomonas, which was isolated from continuous cropping rhizosphere of pepper and cucumber, was identified as Pseudomonas fluorescens (P.f.). For further study, transformant was derived from the isolated P.f. after spontaneous mutation to give antibiotic resistance to nalidixic acid and rifampicin as marked strain. Both P.f. and transformant strains were used for this study and the results obtained were summarized as follows. 1. One of the most effective antagonistic strain, KR164, was selected against F. solani, F. oxysporum, R. solani and this strain was identified and classified as Pseudomonas fluorescens biotype IV. 2. Transformant, KR1641, was derived from strain KR164 and both strains had the same biological and biochemical characteristics. 3, Mycelial lysis and abnormal mycelia of plant pathogenic fungi were microscopically observed after simultaneous culture of fungus and given bacterial strain. 4. The length of chinese cabbage to the autolyzed became longer with given bacterial strain in dark culture. 5. Percentage of germination, number of leaves, length of height, and length of root in chinese cabbage in pot experiment were improved by inoculation of given bacterial strain. 6. The number of given bacterial strain kept generally stable until 34 days after inoculation of itself in pot experiment. Inoculation of given bacterial strain did affect the number of plant disease fungi to be decreased but did not affect the number of other bacteria, Bacillus, in pot experiment. 고추, 오이 등의 시설원예(施設園藝) 연작토양(連作土壤)에서 토양전염성(土壤傳染性) 식물병원균(植物病院菌)에 길항력(拮抗力)을 갖는 Pseudomonas fluorescens를 분리(分離), 동정(同定)하고 이 균주(菌株)를 nalidixic acid와 rifampicin에 내성을 갖는 spontaneus multant를 유도(誘導)하여 표식균주(標識菌株)를 만들었다. 이들 표식균주(標識菌株)와 모균주(母菌株)의 생화학적 특성(特性), 병원성 사장균(絲狀菌)에 대한 길항력(拮抗力), 암소배양(暗所培養), 작물(作物) pot 실험(實驗) 결과(結果)를 요약(要約)하면 다음과 같다. 1. 고추, 오이 등의 시설원예(施設園藝) 연작지(連作地) 토양(土壤)에서 형광성(螢光性) Pseudomonas를 분리(分離)하여 Rhizoctonia solani, Fusrium oxysporum, Fusarium solani에 생육(生育) 저해력(沮害力)이 우수한 KR-164를 선발(選拔)하고 이들의 분류학적(分類學的) 성질(性質)을 검토(檢討)한 결과(結果) Pseudomonas fluorescens biotype IV로 분류(分類), 동정(同定)하였다. 2. KR-164에서 표식균주(標識菌株)인 KR-1641 변이주(變異株)를 유도하였고, 이들 표식균주(標識菌株)와 모균주(母菌株)의 생화학적(生化學的) 성질(性質)은 동일(同一)하였다. 3. 분리균주(分離菌株)와 식물(植物) 병원성 사장균(絲狀菌)을 대치배양(對置培養)하여 검경한 결과(結果) 여러 형태(形態)의 균사이장(異狀)이 관찰(觀察)되었다. 4. 분리균주(分離菌株)를 배추 암소배양(暗所培養)에 처리(處理)한 결과(結果) 무처리구(無處理區)에 비해 생육기간(生育其間)이 현저하게 연장(延長) 되었다. 5. 분리균주(分離菌株)의 접종(接種)이 대조구(對照區)와 사장균(絲狀菌) 접종구(接種區)에 비해 배추의 발아율(發芽率) 및 엽수, 초장, 근장 등의 생육(生育)을 향상(向上)시켰다. 6. 토양(土壤)에 접종(接種)된 표식균주(標識菌株)는 접종(接種) 35일까지 안정(安定)하였으나 병원성 사장균(絲狀菌)은 현저하게 감소(減少)하였고, 표식균주(標識菌株)는 근권(根圈) 유용미생물(有用微生物)의 생육(生育)에 뚜렷한 영향(影響)을 미치지 않았다.

젓갈에서 분리한 Lactobacillus spp.로 제조한 요구르트의 이화학적 특성

이영환,나한주,이용규,신승이,김종현,Rhee, Young-Hwan,Na, Han-Ju,Lee, Yong-Kyu,Shin, Seung-Yee,Kim, Jong-Hyun 한국응용생명화학회 1997 Applied Biological Chemistry (Appl Biol Chem) Vol.43 No.1

젓갈류로 부터 Lactobacillus cases 2주와 L. pentosus 1주를 분리, 동정한 후 이들 유산균의 요구르트 제조 Starter로서의 이용 가능성을 고찰하고자, 이들 균주를 Starter로 이용하여 요구르트를 제조한 결과 요구르트 고유의 pH값은 $4.03{\sim}4.26$, 산도는 $1.049{\sim}1.217%$, 점도는 $1,772{\sim}2,232\;cps$, 그리고 생균수는 $1.4{\times}10^9{\sim}1.6{\times}10^9\;cfu/ml$였다. 요구르트의 완충능은 (요구르트 100 ml 기준) 1.0N HCl의 경우 12.50 ml 부터 14.06 ml 소모 되었고 1.0N NaOH는 9.46 ml 부터 13.06 ml 소모 되었다. 한편, 요구르트 제조시 균주 첨가 후 24시간 부터 72시간 동안 경시적으로 ${\beta}-galactosidase$ 활성도를 조사한 결과 48시간에 최고의 효소활성을 보였으며 이후에는 감소하였다. 또한 산성조건에서 2시간 동안 경시적으로 ${\beta}-galactosidase$ 활성도와 유산균 생존율을 측정한 결과 pH 3.5에서 2시간 후에는 $20{\sim}60%$ 정도 효소활성이 감소하였으며 pH 2.5와 pH 1.5에서 효소활성은 거의 없었다. 한편, 유산균의 생존율은 pH 3.5에서 2시간 후 거의 변화가 없었으나 pH 2.5와 pH 1.5에서는 각각 $1.9{\times}10^6{\sim}1.8{\times}10^8,\;1.0{\times}10^3{\sim}2.4{\times}10^5\;cfu/ml$ 수준이었다. Three strains of lactic acid bacteria were isolated from fish and shrimp pickles. Two strains were identified as Lactobacillus casei, and one strain as L. Pentosus, respectively. All three strains were used as starters in producing yogurts. The physico-chemical characteristics of yogurts were examined. The original pH, titratable acidity, visicosity and viable cell counts of the yogurts were $4.03{\sim}4.26,\;1.049{\sim}l.217%,\;1,772{\sim}2,232\;cps\;and\;1.4{\times}10^9{\sim}1.6{\times}10^9\;cfu/ml$, respectively. In evaluating buffer capacity, $12.50{\sim}14.06\;ml$ 1.0N HCl was consumed to titrate 100 ml of yogurt to pH value 2 units below the original pH value and $9.46{\sim}13.06\;ml$ of 1.0N NaOH was consumed to pH value 4 units above the original pH value. The ${\beta}-galactosidase$ activity reached maximum at 48 hrs, and reduced gradually during fermantation. After 2 hr incubation of yogurts at $37^{\circ}C$ under different pH conditions, ${\beta}-galactosidase$ activities of three strains were reduced to 50% at pH 3.5, but there were no remaining activities neither at pH 2.5 nor at pH 1.5. Under the same pH conditions the number of viable cells decreased to $1.9{\times}10^6{\sim}1.8{\times}10^8\;cfu/ml$ at pH 2.5 and $1.0{\times}10^3{\sim}2.4{\times}10^5$ at pH 1.5, respectively. However, no significant difference was found at pH 3.5.

Electroporation 에 의한 근권 미생물 Pseudomonas fluorescens 의 형질전환

이영환(Young Hwan Rhee),김종현(Jong Hyun Kim) 한국응용생명화학회 1995 Applied Biological Chemistry (Appl Biol Chem) Vol.38 No.5

The antagonistic rhizobacteria, Pseudomonas(P.) fluorescens against F. oxysporum and R. solani were isolated and selected, and then, their biological and physiological characteristics were investigated. The possibility and optimum condition of the electroporation of antagonistic rhizobacteria with Ps70, one of the selected one, and plasmid pSV2-neo was studied. Its optimum condition was found with HGEB which contains 1 mM (pH 7.0) hepes and 10% glycerol at setting of 200 resistance, 25 ㎌ capacitance, and 2.5 ㎸ applied voltage. In addition, the transformation efficiency obtained with pSV2-neo was compared to other plasmids with different sizes. The applied voltage, the buffer composition and the parallel resistor (time constant) were shown to have the greatest effect on transformation efficiency in electroporation. And the rest of the selected rhizobacteria were also successfully transformed with pSV2-neo by electroporation.

김치에서 분리한 유산균 Lactobacillus plantarum 의 이화학적 특성 및 β - galactosidase 활성

이영환(Young Hwan Rhee),강미선(Mi Seon Kang) 한국응용생명화학회 1996 Applied Biological Chemistry (Appl Biol Chem) Vol.39 No.1

Three strains of inhibitory lactic acid bacteria (No. 49, No. 61, No. 75) against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escheirchia coli(ATCC33694) and Bacillus subtilis(ATCC6633) were isolated from kimchi, and then, identified to be Lactobacillus plantarum after examinations of their biological and physiological characteristics. To investigate a possible application of these three lactobacilli in milk fermentation industry, we made yogurts and then evaluated their β-galactosidase activities at various; incubation time(from 24 hrs to 72 hrs). The result of experiment was that β-galactosidase activities were reached maximum at 48 hrs and that reduced gradually with the lapse of time. And the β-galactosidase activity of lactobacilli, and their viable cell counts at 37℃ for 2 hrs under various pH conditions were investigated. β-galactosidase activities of 3 strains were reduced 50% at pH 3.5, but there were no remaining activities at pH 2.5, and pH 1.5, respectively. The frequency of the survival cell of lactobacilli in yogurt were 0.12∼0.75% at pH 2.5. 6.3×10^(-5)∼2.7×10^(-3)% at pH 1.5, respectively, but there was no significant difference at pH 3.5. The values of original pH, titratable acidity as lactic acid, viscosity, and viable cells of yogurts were 4.08∼4.30, 1.05∼1.25%, 1,818∼2,124 cps and 7.3×10^8∼3.0×10^9 cfu/㎖, respectively. To estimate buffer capacity of yogurt, the volume of 1.0 N HCl to 2 unit below original pH of yogurt(100 ㎖) was 11.98∼13.02 ㎖ and the volume of 1.0N NaOH to 4 unit above original pH of yogurt(100 ㎖) was 10.82∼12.86 ㎖.

김치에서 분리한 유산균 Lactobacillus plantarum 의 β - galactosidase 특성에 관한 연구

이영환(Young Hwan Rhee),강미선(Mi Seon Kang) 한국응용생명화학회 1996 Applied Biological Chemistry (Appl Biol Chem) Vol.39 No.1

To investigate a possible application of three strains of lactic acid bacteria(strain No. 49. No. 61. No. 75) from kimchi in milk fermentation industry, the optimal condition for production of intracellular β-galactosidase from Lactobacillus(L.) plantarum and its enzymatic properties were examined. The preferable carbon source of the medium for strain No. 49 in production of β-galactosidase was MRS broth with 1.0% lactose instead of dextrose of pH 65. for strain No. 75 with 1.0% galactose and for strain No. 61 with 3.0% lactose at pH 7.5, respectively. The maximum enzyme production from strain No. 49, No. 75 was observed after 48 hours culture at 30℃ in a medium containing the appropriate carbon source, from strain No. 61 after 48 hours culture at room temperature. The optimum temperature for β-galactosidase activity from L. plantarum was 60℃ for strain No. 49, 37℃ for strain No. 61 and 50℃ for strain No. 75, respectively. The heat stability of enzyme activities for all three strains remained 90% at 45℃. The optimal pH was pH 6.5 and enzyme activities were most stable at pH for all three bacteria.

개 전이성 생식후 종양 세포의 성숙기 및 퇴축기에 따른 미세구조

박남용,이영환,Park, Nam-Yong,Rhee, Young-Hwan 한국현미경학회 1987 Applied microscopy Vol.17 No.1

Naturally occurring canine transmissible venereal tumors of genital organs in mature and regressive stages from 6 dogs were examined by transmission electron microscope. The tumor cells at the stage of maturation were comprised of large round and ovoid cells with prominent nuclei and nucleoli, a few spindle-shaped cells, and irregularly shaped cells. The mature round cells were characterized by the presence of a central ovoid to irregularly round nucleus with a large eccentric nucleolus, vesicular endoplasmic reticulum, round to oval swollen mitochondria with few cristae, Golgi's apparatus, and plasma membranes with numerous microvilli. As the tumor degenerated, the tumor cells were increased in the number of spindle-shaped, fibroblast-like and irregularly shaped cells, collagen bundles, and mainly lymphocytes, in contrast to those of the stage of maturation. Regressing tumor cells were characterized by the swelling and vacuolation of mitochondria and endoplasmic reticulum, membrane-bound granules, lamellar complex, tubular structures, and dense bundles of collagen. It was suggested that transformation might occur in the course of tumor growth causing morphological change from the round to the fibroblast-like cells, and that there was the evidence of cell-mediated tumor cell lysis by lymphocyte infiltration.

김치에서 분리한 유산균 Lactobacillus plantarum의 이화학적 특성 및 ${\beta}-galactosidase$ 활성

강미선,이영환,Kang, Mi-Seon,Rhee, Young-Hwan 한국응용생명화학회 1996 Applied Biological Chemistry (Appl Biol Chem) Vol.39 No.1

김치에서 분리한 유산균 중에서 Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli(ATCC33694) 그리고 Bacillus substilis(ATCC 6633) 등 5종의 미생물에 대한 항균성이 우수한 3주(No. 49, No. 61, No. 75)를 최종 선발하여 동정한 결과 Lactobacillus plantarum으로 동정 되었다. 이들 3 균주를 starter로 사용하여 요구르트 등 여러 발효식품에 응용할 수 있는지 검토하기 위하여, 이들 균주로 요구르트를 제조한 후 요구르트의 ${\beta}-galactosidase$ 활성도를 균주 첨가 후 24시간 부터 72시간 동안 경시적으로 조사하였다. 그 결과 48시간 후에 효소활성은 최고에 도달하였으며 그 후 시간이 경과함에 따라 감소하였다. 또한 2시간 동안 경시적으로 요구르트 고유의 pH값보다 낮은 산성조건에서 ${\beta}-galactosidase$ 활성도와 생존율을 측정한 결과 pH 3.5에서 2시간 후에는 3균주 모두 50% 정도 활성이 감소하였으나 pH 2.5와 pH 1.5에서 ${\beta}-galactosidase$ 활성은 거의 없었다. 유산균의 생존율은 pH 3.5에서 2시간 후 거의 변화가 없었고, pH 2.5에서는 각각 $0.12%{\sim}0.75%$ 그리고 pH 1.5에서는 생존율이 거의 없었다. 한편, 제조 요구르트의 이화학적 성질을 조사한 결과 요구르트 고유의 pH 값은 4.08 부터 4.30사이이었으며, 적정산도는 $1.05{\sim}l.25%$ 점도는 $1,818{\sim}2,124\;cps$, 생균수는 $7.3{\times}10^8{\sim}3.0{\times}10^9\;cfu/m{\ell}$였다. 유산균이 살아있는 상태로 장내에 도달할 수 있는지의 여부를 간접적으로 나타내는 완충능을 측정하기 위하여 1.0N HCl로 요구르트 $100\;m{\ell}$를 고유의 pH값에서 2단위 낮은 pH값까지 적정한 결과 소모된 HCl량은 $11.98{\sim}13.02\;m{\ell}$이었고 1.0 N NaOH로 4단위 높은 pH값까지 적정하는데 소모된 NaOH량은 $10.82{\sim}12.86\;m{\ell}$로 시판 요구르트 보다 높은 완충능을 보여 주었다. Three strains of inhibitory lactic acid bacteria (No. 49, No. 61, No. 75) against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escheirchia coli(ATCC33694) and Bacillus subtilis(ATCC6633) were isolated from kimchi, and then, identified to be Lactobacillus plantarum after examinations of their biological and physiological characteristics. To investigate a possible application of these three lactobacilli in milk fermentation industry, we made yogurts and then evaluated their ${\beta}-galactosidase$ activities at various; incubation time(from 24 hrs to 72 hrs). The result of experiment was that ${\beta}-galactosidase$ activities were reached maximum at 48 hrs and that reduced gradually with the lapse of time. And the ${\beta}-galactosidase$ activity of lactobacilli, and their viable cell counts at $37^{\circ}C$ for 2 hrs under various pH conditions were investigated. ${\beta}-galactosidase$ activities of 3 strains were reduced 50% at pH 3.5, but there were no remaining activities at pH 2.5, and pH 1.5, respectively. The frequency of the survival cell of lactobacilli in yogurt were $0.12{\sim}0.75%$ at pH 2.5, $$6.3{\times}10^{-5}{\sim}2.7{\times}10^{-3}% at pH 1.5, respectively, but there was no significant difference at pH 3.5. The values of original pH, titratable acidity as lactic acid, viscosity, and viable cells of yogurts were $4.08{\sim}4.30,\;1.05{\sim}1.25%,\;1,818{\sim}2,124\;cps\;and\;7.3{\times}10^8{\sim}3.0{\times}10^9\;cfu/m{\ell}$, respectively. To estimate buffer capacity of yogurt, the volume of 1.0 N HCl to 2 unit below original pH of yogurt($100\;m{\ell}$) was $11.98{\sim}13.02\;m{\ell}$ and the volume of 1.0N NaOH to 4 unit above original pH of yogurt($100\;m{\ell}$) was $10.82{\sim}12.86\;m{\ell}.

김치에서 분리한 유산균 Lactobacillus plantarum의 ${\beta}-galactosidase$ 특성에 관한 연구

강미선,이영환,Kang, Me-Seon,Rhee, Young-Hwan 한국응용생명화학회 1996 Applied Biological Chemistry (Appl Biol Chem) Vol.39 No.1

김치에서 분리한 유산균 Lactobacillus(L.) plantarum 3 균주(strain No.49, No.61, No.75)의 ${\beta}-galactosidase$의 생성을 위한 최적 조건을 검토하고, 효소학적 특성을 조사하였다. 효소 ${\beta}-galactosidase$의 최적 생성 조건은 strain No. 49의 경우 탄소원을 dextrose대신에 lactose를, strain No. 75 균주는 galactose를 1.0% 함유한 MRS broth에서, 초기 pH를 6.5로 하여 $30^{\circ}C$에서 48시간 배양하였을 때로 판명되었다. Strain No. 61 균주의 경우는 lactose를 3.0% 함유한 MRS 배지의 초기 pH를 7.5로 하여 실온에서 48시간 배양하였을때가 최적생성조건이었다. 효소의 최적 활성 온도는 $60^{\circ}C,\;37^{\circ}C,\;50^{\circ}C$로 3균주 모두 L. plantarum임에도 불구하고 각각 다른 온도에서 최고 활성을 보였다. 특히, strain No. 61, No. 75는 반응 온도가 상승함에 따라 활성의 감소가 관찰된 반면 strain No. 49는 $60^{\circ}C$의 높은 온도에서 가장 높은 활성을 보여 높은 온도에서도 효소활성이 유지 되는 점은 주목할 만하였으며, 3균주 모두 $45^{\circ}C$까지 90% 이상의 안정성을 나타내었다. 한편 효소의 최적 pH는 3 균주 모두 pH 6.5이었으며, pH 6.0에서 효소활성이 가장 안정하였다. To investigate a possible application of three strains of lactic acid bacteria(strain No. 49. No. 61. No. 75) from kimchi in milk fermentation industry, the optimal condition for production of intracellular ${\beta}-galactosidase$ from Lactobacillus(L.) plantarum and its enzymatic properties were examined. The preferable carbon source of the medium for strain No. 49 in production of ${\beta}-galactosidase$ was MRS broth with 1.0% lactose instead of dextrose of pH 65. for strain No. 75 with 1.0% galactose and for strain No. 61 with 3.0% lactose at pH 7.5, respectively. The maximum enzyme production from strain No. 49, No. 75 was observed after 48 hours culture at $30^{\circ}C$ in a medium containing the appropriate carbon source, from strain No. 61 after 48 hours culture at room temperature. The optimum temperature for ${\beta}-galactosidase$ activity from L. plantarum was $60^{\circ}C$ for strain No. 49, $37^{\circ}C$ for strain No. 61 and $50^{\circ}C$ for strain No. 75, respectively. The heat stability of enzyme activities for all three strains remained 90% at $45^{\circ}C$. The optimal pH was pH 6.5 and enzyme activities were most stable at pH for all three bacteria.

Transfer of foreign Genes into the Bradyrhizobium japonicum and their Inoculation Effects on Soybean Plants

김용웅,김길용,이영환,김광식,Kim, Yong-Woong,Kim, Kil-Yong,Rhee, Young-Hwan,Kim, Kwang-Sik 한국토양비료학회 1992 한국토양비료학회지 Vol.25 No.4

접종근류균(接種根瘤菌)의 소장(消長)을 유전공학적(遺傳工學的)으로 조제(調劑)한 Bradyrhizobium japonicum RJB6 $str^rnal^rneo^r$을 이용(利用)하여 연구(硏究)하였다. 전남(全南)일대로 부터 분리(分離) 동정한 균(菌)에 먼저 streptomycin과 nalidixic acid에 저항성(抵抗性)을 갖는 spontaneous mutant를 유도하고 이어 pSUP2021를 함유(含有)하고 있는 E. coli와 conjugation하여 neomycin 저항성(抵抗性) gene(Tn5)을 도입(導入)하였다. Southern hybridization한 결과(結果) 4.9kb상에서 Tn5를 확인(確認)했다. 서로 다른 균밀도(菌密度)로 파종(播種)한 40일후, 크로로필을 제외한 근류수(根瘤數), 근류신선중(根瘤新鮮重), 간장 및 질소함량이 low cell suspension 처리구보다 heavy cell suspension 처리구에서 약간 높았다. 표식균주(標識菌株)의 회수율(回收率)은 heavy cell suspension 처리구에서 12%인 반면, low cell suspension 처리구에서는 5%에 지나지 않았다. The fate of inoculum strain of Bradyrhizobium japonicum was studied by using genetically marked strain. RJB6 $str^rnal^rneo^r$. A spontaneous mutant of B. japonicum isolated from nodules was made to have antibiotic resistance against streptomycin and nalidixic acid. In order to make genetically marked strain, neomycine resistant gene(Tn5) was introduced into this spontaneous mutant by conjugation with E. coli containing pSUP2021. The southern hybridization was carried out to confirm the plasmid insertion. Hybridization of chromosome DNA using pSUP2021(Tn5) as a probe showed that Tn5 was located on the 4.9kb fragment of chromosome. Soybean seeds were planted into a soil previously cultivated with soybean and inoculated with different cell densities of marked strain. Fourty days after planting, the inoculation effects on nodule number, nodule fresh weight, plant height and nitrogen content in the plot inoculated with heavy cell suspension was a little better than those in the plot with low inoculation. The recovery percentage of the marked strains was about 12% in the plot inoculated with heavy density cell suspension, while 5% in the plot inoculated with low cell suspension.

04 포스터 발표 : 환경 화학 분야(PC) ; PC-11 : 복숭아혹진딧물에 대한 살충활성을 지닌 Lipopeptide 구명

양시영 ( Si Young Yang ),이영환 ( Young Hwan Rhee ),김인선 ( In Seon Kim ) 한국환경농학회 2015 한국환경농학회 학술대회집 Vol.2015 No.-

Green peach aphid (myzus persicae) has been known as a sucking insect taking nutrients from host plant and caused a significant reduction in crop production and quality. The aim of the present study is to isolate a microorganism exhibiting insecticidal activity against M.persicae and identify the insecticidal metabolites. A total of 13 bacterial isolates were obtained from red chili-pepper paste with vinegar. Among them, an isolate showed significant insecticidal activity against M.persicae. The supernatant of isolate showed 100.0% of aphid mortality and exhibited low surface tension of 33.8 mN/m after 96h incubation in LB medium. The insecticidal metabolites exhibited LC 50 value of 85.6 μg/mL. The metabolites was subjected to prep- HPLC and major four peaks (A, B, C, D) were detected at 14.8, 16.0, 17.4, 19.2 min. LC-MS analysis of the four peaks detected (M+H)+ peaks at m/z 1022(A), 1022(B), 1008(C), 1036(D), respectively. LC-MS/MS analysis of the fraction A, B showed presence of C-14-Glu-Leu-Leu- Val-Asp-Leu-Leu and fraction C showed the presence of C-14-Glu-Leu-Leu-Val-Asp-Leu-Val and fraction D showed presence of C-15-Glu-Leu-Leu-Val-Asp-Leu-Leu. LC 50 value of the each metabolite was revealed at 19.6, 29.9, 51.3 and 16.3 μg/mL, respectively.