1-Deoxynojirimycin을 생산하는 Bacillus subtilis S10 배양액의 혈당강하 효과

국문 초록 (Abstract)

본 연구는 α-glucosidase의 강력한 효소저해제인 1-deoxynojirimycin(DNJ)을 고효율로 생산하는 균주의 선발 및 동정, 배양최적화 및 균 배양액의 혈당강하효과 등에 관하여 조사하였다. 먼저 토양으로부터 α-glucosidase에 대해 강력한 저해능을 나타내는 9균주를 대상으로 이 중에서 DNJ 생산성이 가장 우수한 한 개 균주를 최종 선발하였고, 이 균주를 16S rDNA 염기서열분석으로 균주동정을 하여 Bacillus subtilis S10이라 명명하였다. 본 균주의 배양액을 이온교환수지법(Amberlyst 15 H? form, Dowex 1×2-100 OH? form, Amberlite CG-50 NH₃? form)에 의해 DNJ를 정제하고, 정제된 DNJ를 LC-MS로 분석한 결과 표준 DNJ와 동일한 물질임을 확인하였다. DNJ 대량생산을 위한 S10균주의 배양최적탄소원 및 최적질소원과 이들의 최적농도를 조사한 결과 각각 1% galactose, 1.6% polypeptone임이 밝혀졌으며, 확립된 최적화 조건에서의 DNJ 생산량은 0.75 g/L이었다. 고혈당 유도 마우스를 대상으로 각종 혈당강하제의 효과를 알아보기 위해 혈당치를 비교한 결과 무처리구의 혈당치 510±10.9 ㎎/dL에 비해 acarbose 처리구는 139.4±33.1 ㎎/dL, 누에분말 처리구는 209.1±19.6 ㎎/dL, 그리고 S10 균주 배양액 처리구는 208.6±39.8 ㎎/dL로써 무처리구에 비해 각각 72.7%, 59.0%, 그리고 59.1%의 혈당 억제율을 나타냈다. 이상의 연구결과 S10균주 배양액은 누에분말과 같은 수준의 고혈당 억제 및 완화효과가 있음이 확인되었으며 향후 S10균주의 대량생산을 통해 식후혈당조절을 위한 건강기능식품 개발이 기대된다 하겠다.

다국어 초록 (Multilingual Abstract)

1-Deoxynojirimycin (DNJ) is a strong α-glucosidase inhibitor which inhibits hyperglycemia in animals. To select the Bacillus strains highly producing DNJ, 4,000 strains were isolated from soil and grain samples. By the inhibitory activity against α-glucosidase, nine Bacillus strains were selected and then identified by 16S rDNA sequencing. B. subtilis S10 was finally selected as the best strain for the production of DNJ. Various carbon sources and nitrogen sources in culture medium were evaluated for the highest production of DNJ. As the results, the optimized concentration of carbon source and nitrogen source was 1.0% galactose and 1.6% polypeptone and the concentration of DNJ produced was 0.75 g/L. The effect of culture supernatant of B. subtilis S10 on lowering blood glucose level was investigated in streptozotocin (STZ)-induced diabetic mice model. Mice were randomly assigned to control group (saline) and three test groups such as acarbose group, silkworm powder group and B. subtilis S10 group. After eight-week oral feeding, blood glucose levels of the B. subtilis S10 and silkworm powder groups were respectively 209.1±19.6 ㎎/dL (59.1%) and 208.6±39.8 ㎎/dL (59.0%) lower than 510±10 ㎎/dL of the control group. These results indicated that the culture supernatant of B. subtilis S10 was able to reduce the blood glucose level in STZ-induced diabetic mice.

목차 (Table of Contents)

• Abstract
• 서론
• 재료 및 방법
• 결과 및 고찰
• 요약
• 문헌

참고문헌 (Reference)

1 Yagi M, "The structure of moranoline a piperidine alkaloid from Morus species" 50 : 571-572, 1976

2 Bischoff H, "The mechanism of α-glucosidase inhibition in the management of diabetes" 18 : 303-311, 1995

3 Hardick DJ, "The biosynthesis of 1-deoxynojirimycin in Bacillus subtilis var niger" 49 : 6707-6716, 1993

4 Asano N, "Sugar-mimic glycosidase inhibitors: natural occurrence, biological activity and prospects for therapeutic application" 11 : 1645-1680, 2000

5 Yamada H, "Screening of α-glucosidase Ⅱ inhibitor from Chinese herbs and its application on the quality control of mulberry bark" 47 : 47-55, 1993

6 Paek NS, "Production of 1-deoxynojirimycin by Streptomyces sp. SID9135" 7 : 262-266, 9135

7 Aasno N, "Polyhydroxylated pyrrolidine and piperidine alkaloids from Adenophora triphylla var. japonica (Campanulaceae)" 53 : 397-382, 2000

8 Aasno N, "Polyhydroxylated alkaloids isolated from mulberry trees (Morus alba L.) and silkworms (Bombyx mori L.)" 49 : 4208-4213, 2001

9 Ezure Y, "Moranoline (1-deoxynojirimycin) fermentation and its improvement" 49 : 1119-1125, 1985

10 Murao S, "Isolation and characterization of a new trehalase inhibitor, S-GI" 44 : 219-221, 1980

11 Werner F, "Inhibitors, obtained from bacilli, for glycoside hydrolases"

12 Scofield AM, "Inhibition of mammalian digestive disaccharidases by polyhydroxy alkaloids" 39 : 29-32, 1986

13 Evans SV, "Glycosidase inhibition by plant alkaloids which are struc-tural analogues of monosaccharides" 24 : 1953-1955, 1985

14 Schmidt DD, "Glucosidase-inhibitoren aus Bazillen" 66 : 584-585, 1979

15 Hardick DJ, "Glucose is a precursor of 1-deoxynojirimycin and 1-deoxymannonojirimycin in Streptomyces subrutilus" 48 : 6285-6296, 1992

16 Lee HS, "Effect of several sericultural products on blood glucose lowering for alloxan-induced hyperglycemic mice" 40 : 38-42, 1998

17 Park SW, "Effect of acarbose in NIDDM insufficiently treated with diet alone" 18 : 263-269, 1994

18 Lembcke B, "Effect of 1-deoxynojirimycin derivatives on small intestinal disaccharidase activities and on active transport in vitro" 31 : 120-127, 1985

19 Watanabe K, "Different effects of two α-glucosidase inhibitors, acarbose and voglibose, on serum 1,5-anhydroglucitol (1,5AG) level" 18 : 183-186, 2004

20 Kim JW, "Determination of 1-deoxynojirimycin in Morus alba L. leaves by derivatization with 9-fluorenylmethyl chloroformate followed by reversed- phase high-performance liquid chromatography" 1002 : 93-99, 2003

21 Kim SH, "Comparison of glucose-lowering activity of the extracts from Kangwon-do mountain mulberry leaves (Mori Folium) and silk worm" 5 : 391-395, 1997

22 Asano N, "Calystegine B4, a novel trehalase inhibitor from Scopolia japonica" 293 : 195-382, 1996

23 Kim MS, "Blood glucose lowering effects of mulberry leaves and silkworm extracts on mice fed with high-carbohydrate diet" 31 : 117-125, 1998

24 Breitmeier D, "Acarbose and 1-deoxynojirimycin inhibit maltose and maltooligosaccharide hydrolysis of human small intestinal glucoamylase-maltase in two different substrate-induced modes" 346 : 7-14, 1997