• KSBB Journal
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• 제27권6호
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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.

• KSBB Journal
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• 제26권5호
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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$^{(R)}$.

• 한국식품영양과학회지
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• 제24권2호
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• pp.228-233
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• 1995

일본의 전통 발효 식품인 Natto의 맛 개선을 위한 연구 목적으로 증자대두에 Bacillus natto(NN-1)를 접종하고 마늘 2%와 고추 oleoresin 0.2%를 각각 첨가한 후, $40^{\circ}C$에서 24시간 발효시켜 제조한 Natto에서의 nattokinase 활성은 전 시험구에서 발효 12시간 부터 나타나기 시작하여 점차적으로 증가하였으며 24시간 발효하였을 때의 활성은 마늘과 고추 oleoresin 첨가에 의해 활성도가 약간 낮았다. ${\gamma}-GTP$ 활성은 24시간 발효하였을 때 전 시험구간에 활성도가 비슷하였으며 마늘 2%와 고추 oleoresin 0.2% 첨가에 의해 ${\gamma}-GTP$ 활성에는 영향을 주지 않았다. Protease 활성은 마늘 첨가구에서 발효 8시간에 가장 높은 활성을 나타내었으며 발효 24시간에는 전 시험구간에서 비슷한 활성을 나타내었다. 관능검사 결과, 맛에서는 oleoresin 첨가구와 마늘, 고추 oleoresin 첨가구가 유의성이 인정되었으므로 Natto를 제조할 때 고추 oleoresin 0.2%와 마늘 2%, 고추 oleoresin 0.2%를 혼합하여 Natto를 발효시키는 것이 한국인의 입맛에 적합한 Natto의 맛 개선에 바람직한 것으로 생각된다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권2호
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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.

• 한국유기농업학회지
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• 제19권4호
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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.

• 한국식품과학회지
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• 제44권5호
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• pp.600-606
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• 2012

콩을 소재로한 전통 발효식품으로부터 혈전용해능이 뛰어난 균주를 분리하였으며, B. subtilis로 동정되었다. 따라서 이를 B. subtilis IDCC 9204(특허균주기탁: KCTC-11471 BP), 그 혈전용해 효소는 NK-IL9204로 명명하였다. B. subtilis IDCC 9204가 생산하는 고역가의 NK-IL9204를 단백질 분석법에 기초하여 분석한 결과, 분자량은 27.7 kDa의 homogenous enzyme으로 확인되었다. 또한 기존에 알려진 일본의 발효식품인 낫도 유래의 B. subtilis var. natto가 생산하는 nattokinase 와의 sequence 분석을 진행한 결과, 99.5% homology가 일치하는 serine protease계열의 nattokinase로 확인되었다. 그러나 NK-IL9204는 물리 화학적인 조건에서 B. subtilis var. natto가 생산하는 nattokinase와 다소 차이를 나타내었으며 본 실험에서는 B. subtilis var. natto가 생산하는 nattokinase보다 상대적으로 높은 열 안정성과 pH 안정성을 나타내었다. In vitro 실험에서 NK-IL9204는 최적 반응온도 $40^{\circ}C$, 열 안정성은 $90^{\circ}C$까지 효소활성을 유지하였으며, 최적 반응 pH는 pH 8로 알칼리-혈전용해효소의 특성을 나타내었으며, 약산성에서 강알칼리 영역까지 넓은 pH 구간 안정성을 갖는 것이 특징이다. NKIL9204의 in vivo에서의 효능과 생체 내 안정성을 동물실험을 통해 확인한 결과, 생체 내에서도 혈전용해효소의 활성이 소실되지 않고 유지되며, 혈전분해와 관련된 생체 내 인자들을 활성화시키는 역할을 하는 특징을 갖는다. NK-IL9204는 30,000 FU/g 이상의 고역가를 달성하여 산업적 측면에서 생산성도 확보함으로써 수입의존적 원료를 국산화할 수 있을 것으로 예상된다.

• Journal of Microbiology and Biotechnology
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• 제29권8호
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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.

• 생명과학회지
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• 제16권2호
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• pp.274-281
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• 2006

흑두청국으로부터 분리된 혈전용해력이 우수한 Bacillus subtilis BB-1(KFCC 11344P)으로부터 혈전용해효소 유전자를 PCR법에 의해 크로닝하였고 이를 BCF-1으로 명명하였다. BCF-1의 DNA 염기서열결정 결과 1,145 bp 크기의 혈전용해 효소로, 일본의 natto로부터 분리된 nattokinase 유전자와 99%의 상동성을 보임을 확인하였다. 혈전용해효소 유전자의 발현을 위하여 Bacillus 발현계인 Bacillus-E. coli의 shuttle vector인 pEB vector에 크로닝 하고 host로서 B. subtilis 168에 형질전환시켜 대량 발현시켰다. 생산된 혈전용해효소의 최 적활성 pH와 온도는 7.0과 $35^{\circ}C$로 확인되었다, 기질에 대한 분해양상을 조사한 결과 fibrin에서만 특이적으로 강한 분해가 일어났으며, skim milk에서 아주 약한 분해능을 보였으나 blood agar, gelatin, casein에서는 전혀 분해능을 보이지 않았다. 특히 blood agar plate에서 분해능이 없는 것으로 보아 혈액 내에서의 적혈구 파괴현상과 같은 부작용에 대한 위험을 배제할 수 있을 것으로 사료된다. BCF-1에 의해 생산된 혈전용해효소는 fibrin 특이적으로 활성을 나타냄을 확인할 수 있으며, 이는 임상적이나 산업적으로 적용하였을 때 부작용에 대한 위험적인 문제는 배제될 수 있으리라 생각된다.

• 한국영양학회:학술대회논문집
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• 한국영양학회 2003년도 연합학술대회
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• pp.229.1-229.1
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• 2003

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2005년도 국제학술심포지움 The 44th Annual Meeting of Korean Society for Life Science
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• pp.235-235
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• 2005