• Title/Summary/Keyword: Nattokinase

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Evaluation of Nattokinase for Antithrombotic Effect and Pharmacological Efficacy by a Biological Test and Clinical Trial (동물 및 인체시험을 통한 Nattokinase의 항응고 작용 및 섬유소 용해능 평가)

  • Kim, Jae-Bum;Yoo, Chul-Bae;Shin, Hyun-Man;Jung, Joon-Ki;Jang, Hyung-Wook
    • KSBB Journal
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    • v.26 no.5
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    • pp.393-399
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    • 2011
  • Bacillus subtilis natto producing high level of a fibrinolytic enzyme was selected and Ultra Nattokinase$^{(R)}$ was manufactured by fermentation and purification. It was performed the evaluation of the antithrombotic effect of Ultra Nattokinase$^{(R)}$ (20,000 FU/g) with rat blood plasma. The maximum aggregation (inhibition ratio) was 71% (0%), 69% (2.8%), 62% (12.7%), 16% (77.5%) and 9% (87.3%), respectively, in the order of 0, 5, 10, 50 and 100 mg/mL of Ultra Nattokinase$^{(R)}$ solutions. Ultra Nattokinase$^{(R)}$ had antithrombotic effect, which was associated with the suppression of collagen-induced platelet aggregation. Ultra Nattokinase$^{(R)}$ in the topic of the FDP (fibrinogen degradation products) in blood coagulation tests showed a significant increasing trend. And based on the daily record of meal 39 people of ITT (what ?) group consisted with 19 people of NP (what ?) group and 20 people of PN (what ?) group except four people, two people who took vitamin K affecting the experiment and two people who took alcohol, finding to be taken Ultra Nattokinase$^{(R)}$ showed an increase in the FDP value after four weeks. In addition, FDP value of 41 people of ITT group except two people having metabolic syndrome was increased by Ultra Nattokinase$^{(R)}$.

The Evaluation of Antithrombotic and Fibrinolytic Activities of Nattokinase from Bacillus subtilis Natto (Bacillus subtilis Natto가 생산하는 Nattokinase의 항혈전 및 피브린 용해능 효능평가)

  • Lee, Da-Lyung;Hong, Sung-Yu;Jang, Yang-Su;Jang, Hyung-Wook;Maeng, Chang-Jae;Yoo, Chul-Bae;Baek, Dae-Heoun
    • KSBB Journal
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    • v.27 no.6
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    • pp.375-380
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    • 2012
  • We previously reported that Ultra nattokinase$^{(R)}$ showed high fibrinolytic activity and revealed antithrombotic effect in rat blood plasma based on its ability to suppress collagen-induced platelet aggregation. This research was carried out to verify the clot lysing activity and blood flow enhancing effects of Ultra nattokinase$^{(R)}$ via monitoring and comparing the antithrombotic effects in rat artery between oral administration of Ultra nattokinase$^{(R)}$ and maltodextrin. SD rats were fed with 1.11 mg/kg of Ultra nattokinase$^{(R)}$ for 4 weeks. The effect on arterial thrombosis was then evaluated using an antithrombotic model after induction by $FeCl_3$. Detected fibrinolytic activity was proportional to the content of Ultra nattokinase$^{(R)}$ and statistical extents of the antithrombotic activity was enhanced strongly twice rather than control group. The PT and the aPTT, however, showed only a small difference between two groups. The results suggest that Ultra nattokinase$^{(R)}$ can effectively treat thromboembolism and enhance blood flow, and that Ultra nattokinase$^{(R)}$ can also prevent venous occlusion by aiding clot lysis.

Nattokinase, ${\gamma}-GTP$, Protease Activity and Sensory Evaluation of Natto Added with Spice (향미성 Natto의 Nattokinase, ${\gamma}-GTP$, Protease 활성도와 관능적 평가)

  • 김복란;이상영
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.24 no.2
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    • pp.228-233
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    • 1995
  • To make Natto, tradiational Japanese food fermented by Bacillus natto, more acceptable to Koreans, garlic(2%) and/or red pepper oleoresin(0.2%) were mixed with Natto. Through out the fermentation period, the changes in enzyme activities and sensory evaluation were compared with those of conventional Natto. Nattokinase activities were detected from 12 hour fermentation in all samples. After that period, steady increased in Nattokinase activity was observed. The activity of nattokinase decreased slightly when garlic and/or red pepper oleoresin was added. Changes in ${\gamma}-glutamyl$ transpeptidase(${\gamma}-GTP$) was not significant among samples and the similar tendency was observed in nattokinase activity. With addition of garlic, production of protease reached maximum after 8 hour of fermentation whereas it took 16 hour when red pepper oleoresin was added. However, after 24 hour of fermentation, any significant differences in protease activity were not observed. Sensory evaluation indicated that the tastes of Natto with either garlic and red pepper oleoresin or red pepper oleoresin only were much more acceptable than conventional Natto or one with garlic only.

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Separation of Nattokinase from Bacillus subtilis Fermentation Broth by Expanded Bed Adsorption with Mixed-mode Adsorbent

  • Lu Miao-Hua;Lin Dong-Qiang;Wu Yuan-Chun;Yun Jun-Xian;Mei Le-He;Yao Shan-Jing
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.10 no.2
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    • pp.128-135
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    • 2005
  • Mixed-mode hydrophobic/ionic matrices exhibit a salt-tolerant property for adsorbing target protein from high-ionic strength feedstock, which allows the application of undiluted feedstock via an expanded bed process. In the present work, a new type of mixed-mode adsorbent designed for expanded bed adsorption, Fastline $PRO^{\circledR}$, was challenged for the capture of nattokinase from the high ionic fermentation broth of Bacillus subtilis. Two important factors, pH and ion concentration, were investigated with regard to the performance of nattokinase ad-sorption. Under initial fermentation broth conditions (pH 6.6 and conductivity of 10 mS/cm) the adsorption capacity of nattokinase with Fastline PRO was high, with a maximum capacity of 5,350 U/mL adsorbent. The elution behaviors were investigated using packed bed adsorption experiments, which demonstrated that the effective desorption of nattokinase could be achieved by effecting a pH of 9.5. The biomass pulse response experiments were carried out in order to evaluate the biomass/adsorbent interactions between Bacillus subtilis cells and Fastline PRO, and to demonstrate a stable expanded bed in the feedstock containing Bacillus subtilis cells. Finally, an EBA process, utilizing mixed-mode Fastline PRO adsorbent, was optimized to capture nattokinase directly from the fermentation broth. The purification factor reached 12.3, thereby demonstrating the advantages of the mixed-mode EBA in enzyme separation.

Effects of Feeding Nattokinase as Natural Feed Additives on Milk Production and Blood Metabolites in Lactating Dairy Cows (천연 사료첨가제 Nattokinase 공급에 따른 젖소의 산유능력 및 혈액성상에 미치는 영향)

  • Lim, Dong-Hyun;Park, Joong-Kook;Kim, Hyeon-Shup;Ki, Kwang-Seok;Lee, Hyun-June;Kwon, Eung-Gi;Kim, Mi-Kyoung;Kim, Chang-Hyun;Kim, Sang-Bum
    • Korean Journal of Organic Agriculture
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    • v.19 no.4
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    • pp.553-563
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    • 2011
  • This experiment was conducted to determine the effect of nattokinase (NK) additives on milk production and composition, and blood metabolites in dairy cows. The two kinds of nattokinase with high fibrinolytic activity were produced by two strains of bacteria, Bacillus amyloliquefacines (NK1) and Bacillus subtilis (NK2). Total fifteen Holstein cows (average $1.83{\pm}0.37$ parity; average milk yield $23.2{\pm}3.2$ kg/d) were randomly assigned to three treatments (5 animals per treatment). Cows were fed TMR supplemented with 0g, 100g and 100g for control, NK1 and NK2 treatment, respectively for 4 weeks. Milk yield was significantly higher (p<0.05) for NK1 (22.89 kg/d) than for control (21.07 kg/d) and NK2 (21.36 kg/d). Somatic cell counts in NK treatments were significantly lower than that in control group (58,000 vs. 21,000 and 35,000 cells/ml, control vs. NK1 and NK2). Serum ALT levels in all treatment were similar to the range of 32.00~35.83 IU/L, but AST levels in NK1 (85.67 IU/L) was significantly decreased compared with those in control and NK2 (121.67 and 117.67 IU/L respectively). Serum T-CHO levels in NK1 (145.33 mg/dl) was significantly decreased (p<0.05) compared with that in control (179.00 mg/dl) and NK2 (176.17 mg/dl). This finding showed that NK1 additives could possibly have a positive effect in lactation performance of mid-lactation dairy cows by increasing milk yield, reducing somatic cell count, improving liver function and decreasing cholesterol in blood.

Identification of Novel Bacillus subtilis IDCC 9204 Producing a High-Level Fibrinolytic Enzyme and Properties of NK-IL9204 (고농도 혈전용해효소를 생산하는 신규 Bacillus subtilis IDCC 9204의 분리 및 NK-IL9204의 효소학적 특성)

  • Lee, Seung-Hun;An, Gwangmin;Kim, Heu-Hang;Kang, Jae-Hoon;Kang, Dae-Jung
    • Korean Journal of Food Science and Technology
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    • v.44 no.5
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    • pp.600-606
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    • 2012
  • A Bacillus sp. that produces fibrinolytic enzyme was isolated from Cheonggukjang, a traditional Korean soybean-fermented food. According to 16S rRNA gene base sequencing, the bacillus was identified as a variety of Bacillus subtilis, and named Bacillus subtilis IDCC 9204. Fibrinolytic enzyme NK-IL9204 was stable up to $60^{\circ}C$ and within pH range of 5-10. Purified NK-IL9204 was detected through fibrin zymography. The molecular weight and isoelectric point of the enzyme were estimated to be 27.7 kDa and 6.7 by SDS-PAGE and 2D electrophoresis, respectively. Its amino acid sequence was similar to that of nattokinase (identities 99.5%) and different from that of nattokinase BPN (identities 86.4%). The plasma fibrinolytic activity of NK-IL9204 was measured by euglobulin clot lysis times (ECLT). The NK-IL9204 was orally administered to SD rats for 3 weeks (1,000 FU/rat/day). The ECLT was significantly shortened by supplementation of NK-IL9204.

Nattokinase Crude Extract Inhibits Hepatocellular Carcinoma Growth in Mice

  • Yan, Yongmin;Wang, Yanjing;Qian, Jiali;Wu, Sihui;Ji, Yi;Liu, Yanxiao;Zeng, Jian;Gong, Aihua
    • Journal of Microbiology and Biotechnology
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    • v.29 no.8
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    • pp.1281-1287
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    • 2019
  • Nattokinase (NK, E.C. 3.4.21.62) is a serine protease produced by Bacillus subtilis natto that shows promise for the treatment of thrombotic disease. In this study, we assessed the effects of NK on the development of hepatocellular carcinoma (HCC), a principal malignancy of the liver that causes morbidity and mortality worldwide. Crude extracts of NK (NCE) were isolated from fermentation medium by centrifugation and separated into three fractions (<10 K, 100~30 K and >30K). Orthotopic HCC mouse models were established and NCE was administered by oral gavage. H&E staining was performed to examine the pathology of HCC livers. Immunohistochemistry and immunofluorescence were used to evaluate FOXM1, CD31, CD44 and vimentin expression in the liver. Compared to PBS groups, NCE increased the survival rates of HCC-bearing mice to 31% and decreased ascites. Low-intensity ultrasound imaging showed that the hypoechoic mass area was lower in NCE-treated mice and that tumor growth significantly decreased. IHC staining showed that the expression of FOXM1 was inhibited by NCE treatment. Immunofluorescence results revealed lower levels of CD31, CD44 and vimentin in the NCE groups. Taken together, these data demonstrate that NCE from Bacillus subtilis natto improves survival and inhibits tumor growth in HCC mice.

Cloning and High Expression of Nattokinase Gene from Bacillus subtilis BB-1 (Bacillus subtilis BB-1으로부터 나토키나아제 유전자 크로닝 및 대량발현)

  • Lee Young-Hoon;Lee Sung-Ho;Park Ki-Hoon;Choi Young-Ju;Jeong Yong-Kee;Gal Sang-Wan
    • Journal of Life Science
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    • v.16 no.2 s.75
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    • pp.274-281
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    • 2006
  • A fibrinolytic enzyme gene was isolated from Bacillus subtilis BB-1 by PCR method. Primers for PCR cloning were designed according to pre-identified gene for fibrinolytic enzymes from B. subtilis. The primer sequences were 5'-CGG ATC CGT GAG AGG CAA AAA GGT G-3' and 5'-TGA ATT CTT AAT GTG CTG CTG CTT GTC C-3' as concensus sequences of the fibrinolytic genes of Bacillus species. The PCR product was 1,145 bp and the sequence homology was 99% with nattokinase gene isolated from Japanese natto. The cloned fibrinolytic gene was reconstructed in Bacillus-E. coli shuttle vector, pEB for bulk-production. The fibrinolytic enzyme was purified by FPLC from the cloned B. subtilis 168. The optimum pH and temperature of the enzyme were 7.0 and $35^{\circ}C$, respectively. The fibrinolytic enzyme did not show any activity toward to skim milk, gelatin, casein and blood agar plate. The enzyme specific polyclonal antibody was prepared in rabbit for further assays such as detection of the gene expression in plant cells. This means that the enzyme may be used for health-care such as thrombosis without any hamful effects in the blood vessel.