9 04 Vol. No. Serial No.6 China Brewing Research Report sanning electron microscope SEM g ml = 4 4h UV-800PC 0000r/min 5min SLY-B 0μL - LHS-50SC 7 6h.

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1 DNAout [].. 5g/L g/l nattokinase NK g/l K HPO 4 0.5g/L KH PO 4 0.g/L [-4] MgSO 4 ph7.0~7. LB 0g/L 5g/L [5-7] 0g/L NaCl 5g/L ph7.0 urokinase UK streptokinase SK tissue-type plasminogenactivator t-pa 5 w/v [8-] BN mg / 560U/ 40IU/ Biowest. 材料与试剂.. BN-05 王常苏, 孙晓彤, 余洁, 高冰 BN-05 BN %.5% BN ±0. IU/g 7% TS0. doi:0.969/j.issn A Identification of nattokinase high activity strain BN-05 and optimization of the fermentation technology WANG Changsu, SUN Xiaotong, YU Jie, GAO Bing (.School of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 400, China;.Xinhong Institute of Food and Brewing, Wuhan 4005, China) Abstract: Nattokinase high activity strain BN-05 was preliminarily identified and the nattokinase prodution technology with solid-substrate fermentation was optimized in order to improve the content of nattokinase. BN-05 was determined as Bacillus subtilis by morphological and molecular identification. The optimum condition of technology was as follows: degree of soybean fragmentation, fermentation temperature 6, inoculum 5.5% and glucose addition.5%. At the optimizing conditions, the activity of nattokinase fermented by BN-05 could reach ±0. IU/g (wet weight), which was 7% times of the former culture conditions. Key words: Bacillus subtilis; nattokinase; solid-substrate fermentation; enzyme activity 0g/L 5g/L 0g/L 5g/L ph7.4~7.6. 仪器与设备 AvcmJ-E FTGENE Bacillus natto BN-05 5D PCR Techne Hitachi S-000N

5% BN-05 4 75.6±0. IU/g 7% TS0. doi:0.969/j.issn.054-507.04.0.0 A 054-507 04 0-009-05 Identification of nattokinase high activity strain BN-05 and optimization of the fermentation technology WANG Changsu, SUN Xiaotong, YU Jie, GAO Bing (.

2 9 04 Vol. No. Serial No.6 China Brewing Research Report sanning electron microscope SEM g ml = 4 4h UV-800PC 0000r/min 5min SLY-B 0μL - LHS-50SC 7 6h. 实验方法.. LB BN-05.. LB 7 4h SEM 6S rrna DNA PCR 6sf95 TGACGAGTGGCGGACGGGTG 6sr94 CCATGGTGTGACGGGCGGTGTG SEM NCBI Blast [-4] MEGA5.0.. LB 4h 00mL/50mL 7 80r/min h 660nm OD.. 5 Fig. SEM analysis of strain BN-05 0~5 ~5h 5 0min 5 8%.. 6S rrna 7 48h h PCR BN-05 6S rrna 5bp - GeneBank KC7005 BLAST 6S rrna 99% MEGA5.05 L 4 9 Paenibacillus borealis..4 - Atrup Bacillus subtillis [5] % 0.05mol/L BN-05 Tris-HCl ph7.8. 种子生长曲线 0mL 50.5mg/L BN r/min 9cm 00μL 0IU/mL h 660nm OD h mm 0~8h 8~6h 6h. BN-05 菌种鉴定 BN-05 SEM Bacillus subtillis AJ0 BN-05 6~8h 8h

SEM analysis of strain BN-05 0~5 ~5h 5 0min 5 8%.. 6S rrna 7 48h h PCR BN-05 6S rrna 5bp - GeneBank KC7005 BLAST 6S rrna 99% MEGA5.05 L 4 9 Paenibacillus borealis.

3 y= x R = 固态发酵产纳豆激酶的条件优化.4. Bacillus subtilis Bootstrap MEGA S rrna Fig. Phylogenetic tree inferred by neighbor-joining method based on 6S rrna gene sequence using MEGA version 5.05 % 7 48h h 5 5 Fig. 5 Effect of different carbon source on NK activity BN-05 Fig. Growth curve of the BN-05 seed 5 [6]. 尿激酶的标准曲线 0±0. IU/g 00IU/mL 400IU/mL 800IU/mL 000IU/mL 00IU/mL 400IU/mL 600IU/mL IU/mL 000IU/mL 400IU/mL 0μL 0min 7 6h y x 4 4 Fig. 4 Standard curve of urokinase activity 6 Fig. 6 Effect of fragmentation of fermented soybean on NK activity

5 Effect of different carbon source on NK activity BN-05 Fig. Growth curve of the BN-05 seed 5 [6]. 尿激酶的标准曲线 0±0.

4 94 04 Vol. No. Serial No.6 China Brewing Research Report ±.05 IU/g.4. L 4 9 L 9( 4 ) % 4% 6% 8% 0% orthogonal experiment 7 Table Factors and level of L 9( 4 ) solid fermentation producing NK A /% B /% C D / Fig. 7 Effect of different inoculum size on NK activity 7 6% 6 560±05.4 IU/g %~6% NK 8%~0% % Table Results of solid fermentation producing NK orthogonal experiment A B C D / IU g Table Variance analysis of solid fermentation producing NK orthogonal experiment F F A B C D Fig. 8 Effect of different culture temperature on NK activity 4 C>D>B>A C D C D B A %.5% 5 NK 864±95. IU/g ±0. IU/g

8 Table Results of solid fermentation producing NK orthogonal experiment A B C D 4 5 7 98.000 994.667 4 05

5 BN-05 99±9.87 IU/g 7% BN-05 Haematol, 990, 84():9-4. [8] FUJITA M, ITO Y, HONG K, et al. Characterization of nattokinase degraded products from human fibrinogen or cross-linked fibrin[j]. Fibri- nolysis, 995, 9(): %.5% natto: A popular soybean fermented food in Japan[J]. Biochem Biophys Res Commun, 99, 97(): [7] SUMI H, HAMADA H, NAKANISHI K, et al. Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinase[j]. Acta [9] IWAI K, NAKAYA N, KAWASAKI Y, et al. Antioxidative functions of natto, a kind of fermented soybeans: effect on LDL oxidation and lipid metabolism in cholesterol-fed rats[j]. J Agr Food Chem, 00, 50(): BN ±0. IU/g % [0] URANO T, IHARA H, UMEMURA K, et al. The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis cleaves and inactivates plasminogen activator inhibitor type[j]. J Biol Chem, 00, 76 (7): [] FURIE B, FURIE B C. Mechanisms of thrombus formation[j]. N Engl J Med, 008, 59(9): [] KUMADA K. Isolation of Bacillus subtilis natto which shows high fibrinolytic activity[j]. Igakutu Seibutsugaku, 99, 6(6): []. [J] [] DEEPAK V, KALISHWARALAL K, RAMKUMARPANDIAN S, et al. Optimization of media composition for nattokinase production by Bacillus []. [P] subtilis using response surface methodology[j]. Bioresour Technol, 008, 99(7): [4] SUMI H, HAMADA H, TSUSHIMA H, et al. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese natto: A typical and popular []. B-0 6S rdna [J] soybean food in the Japanese diet[j]. Experientia, 987, 4(0): 0- [4]. GY-4 [D].. [5] TAI M W, SWEET B V. Nattokinase for prevention of thrombosis [J]. Am J Health Syst Pharm, 006, 6():-. 0. [5] ASTRUP T, M 譈 LLERTZ S. The fibrin plate method for estimating fibrinolytic activity[j]. Arch Biochem Biophys, 995, 40(): [6] FUJITA M, NOMURA K, HONG K, et al. Purification and characterization [6]. of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese [J] !!!!!!!!!!!!!!!!!!!!!!!"!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!" 98 CN -88/TS ISSN BM47 RCCSE CSCD [email protected] /8508!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!"!!!!!!!!!!!!!!!!!!!!!!!"

Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinase[j]. Acta [9] IWAI K, NAKAYA N, KAWASAKI Y, et al.

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g 16 6 2018 11 Chinese Journal of Bioprocess Engineering Vol. 16 No. 6 Nov. 2018 doi 10. 3969 /j. issn. 1672-3678. 2018. 06. 004 1 2 1 1 1. 330200 2. 330006 4 Box-Behnken 1 25 g /ml 121 92 min 2 6. 284%