Research Notes on
Nattokinase Benefits, nattokinase side effects
Nattokinase is a potent fibrinolytic enzyme extracted and highly purified from a traditional Japanese food called Natto.
Nattokinase is believed to benefit people at risk of cardiovascular events. Natto is a fermented cheese-like food that
has been used in Japan for over 1000 years. Natto is produced by a fermentation process by adding Bacillus natto, a
benefical bacteria, to boiled soybeans. The resulting nattokinase enzyme, is produced when Bacillus natto acts on the
soybeans. While other soy foods contain enzymes, it is only the natto preparation that contains the specific
nattokinase enzyme.

Nattokinase Benefits - Fibrinogen Degradation
Many years ago, Sumi H and co-workers noticed a strong fibrinolytic activity in the vegetable cheese Natto; Natto is a
typical Japanese soybean food. The average activity was at about 40 CU (plasmin units)/g wet weight. They found that
it was easy to extract this novel fibrinolytic enzyme nattokinase with saline. The mol. wt and pI were about 20,000 and
8.6, respectively. They found that nattokinase digested fibrin and also the plasmin substrate H-D-Val-Leu-Lys-pNA
(S-2251). Diisopropyl fluorophosphate and 2,2,2-trichloro-1-hydroxyethyl-o,o-dimethylphosphate strongly inhibited this
fibrinolytic enzyme. [1] Sumi H and co-workers further discovered that oral administration of natto produced a mild and
frequent enhancement of the fibrinolytic activity in the plasma. To demonstrate its health benefits, they  administered
natto capsules to dogs, then, they observed induced thrombosis, and lysis of the thrombi. They concluded that  natto
might benefit people who were suffered from embolism and it might have benefits of preventing the disease. [2]

In 1993, another group of researchers in Japan purified nattokinase from the vegetable cheese natto. They extracted
nattokinase from natto with saline and then isolated it. They found that this isolated peptide consisted of a total 275
amino acid residues (M.W = 27,728) and exhibited a high homology with the subtilisins.  The purified nattokinase
digested not only fibrin but also several synthetic substrates. Among the synthetic substrates, the most sensitive
substrate was Suc-Ala-Ala-Pro-Phe-pNA for subtilisin. The results indicate that nattokinase is a subtilisin-like serine
protease. [3] Fujita M et al found that intraduodenal administration of nattokinase at a dose of 80 mg/kg, resulted in
the degradation of fibrinogen in plasma suggesting transport of nattokinase across the intestinal tract in normal rats.

Nattokinase Benefits -Thrombolytic Activities
Fujita M et al also studied the thrombolytic effect of nattokinase on a thrombus in the common carotid artery of rat in
which the endothelial cells of the vessel wall were injured by acetic acid. When they stained a section of occluded
vessel for CD61 antigen by immunofluorescence utilizing a monoclonal antibody, they found antigens around the
surface of the occluded blood vessels. This result suggested that platelet aggregation caused the occlusive
thrombosis. However, when urokinase or tissue plasminogen activator was added to their model, they observed the
restoration of the blood flow. The results indicate that their model is able to evaluate a thrombolytic agent. Thus, they
used this model to evaluate the thrombolytic activity of nattokinase And, they found that the thrombolytic activity of
nattokinase is even stronger than that of plasmin or elastase based on this in vitro study. [5]

Nattokinase Benefits - Suppression of Artery Thickening
Suzuki Y et al investigated the effect of natto extracts (containing nattokinase) produced from fermented soybeans on
intimal thickening of arteries after vessel endothelial denudation. They induced intimal thickening in the femoral
arteries by chemicals and irradiation. They started the natto extract (containing nattokinase) supplementation 3 weeks
before endothelial injury and continued the supplementation for another 3 weeks after. They successfully initiated
neointima formation and thickening. Dietary natto extract supplementation suppressed intimal thickening compared
with the control group. Natto extracts also shortened euglobulin clot lysis time. These findings showed that natto
extracts suppressed intimal thickening after vascular injury as a result of the inhibition of mural thrombi formation. [6]
Suzuki Y et al further investigated the effects of dietary supplementation with natto-extracts (containing nattokinase)
on neointima formation and on thrombolysis at the site of endothelial injury. They induced endothelial damage in the
rat femoral artery by a chemical and irradiation. They started dietary natto-extracts supplementation containing
nattokinase 3 weeks before endothelial injury and then continued for another 3 weeks. They found suppression of
intimal thickening in animals given supplementation compared to the controls. Although femoral arteries were
reopened both in control animals and those treated with nattokinase within 8 hours after endothelial injury, they
observed mural thrombi at the site of endothelial injury. In the control group, the center of vessel lumen was reopened
and mural thrombi were attached on the surface of vessel walls. In contrast, in nattokinase -treated groups, thrombi
near the vessel wall showed lysis and most of them detached from the surface of vessel walls. In conclusion, dietary
natto-extracts supplementation suppressed intimal thickening produced by endothelial injury in rat femoral artery.
These effects may partially be attributable to nattokinase, which showed enhanced thrombolysis near the vessel wall.

Nattokinases Products
Most brands of Nottokinase supplements contain a serving of Nattokinase (2000 FU (Fibrinolytic Units))

KOREAN FERMENTED-SOYBEAN SAUCE Bacillus sp. strain CK 11-4, which produces a strongly fibrinolytic enzyme,
was screened from Chungkook-Jang, a traditional Korean fermented-soybean sauce. The fibrinolytic enzyme (CK) was
purified from supernatant of Bacillus sp. strain CK 11-4 culture broth and showed thermophilic, hydrophilic, and strong
fibrinolytic activity. [8]

TAIWAN SOY PRODUCT A mutant of Bacillus subtilis IMR-NK1, which is used for the production of fermented soy food
in Taiwan, produced high fibrinolytic enzyme activity by solid-state fermentation using wheat bran as medium in a
study. [9]

CHINESE FERMENTED SOYBEAN FOOD Bacillus amyloliquefaciens DC-4, which produces a strongly fibrinolytic
enzyme, was isolated from douchi, a traditional Chinese soybean-fermented food. A fibrinolytic enzyme (subtilisin DFE)
was purified from the supernatant of B. amyloliquefaciens DC-4 culture broth and displayed thermophilic, hydrophilic
and strong fibrinolytic activity. Subtilisin DFE has molecular mass of 28000 Da and a pI of 8.0. The optimal pH value
and temperature for activities were 9.0 and 48 degrees C, respectively. Subtilisin DFE not only hydrolyzed fibrin but
also several synthetic substrates, particularly Suc-Ala-Ala-Pro-Phe-pNA, and phenylmethylsulfony fluoride can
completely inhibit its fibrinolytic activity. These results indicated that subtilisin DFE is a subtilisin-family serine protease,
similar to nattokinase from Bacillus natto. The first 24 amino acid residues of the N-terminal sequence of subtilisin DFE
were AQSVPYGVSQIKAPALHSQGFTGS, which is identical to that of subtilisin K-54, and different from that of NK and
CK. [10]

Two fibrinolytic enzymes (QK-1 and QK-2) purified from the supernatant of Bacillus subtilis QK02 culture broth had
molecular masses of 42,000 Da and 28,000 Da, respectively. The first 20 amino acids of the N-terminal sequence are
AQSVPYGISQ IKAPALHSQG. The deduced protein sequence and its restriction enzyme map of the enzyme QK-2 are
different from those of other proteases. The enzyme QK-2 digested not only fibrin but also a subtilisin substrate, and
PMSF inhibited its fibrinolytic and amidolytic activities completely; while QK-1 hydrolyzed fibrin and a plasmin substrate,
and PMSF as well as aprotinin inhibited its fibrinolytic activity.

Researchers also found that Asp32, His64 and Ser221 in the amino acid sequence deduced from the QK gene are
identical to the active site of nattokinase (NK) produced by B. subtilis natto. [11]

Most traditional Asian fermented soy food e.g. natto has been used safely for over 1000 years.  Nattokinase appears
to be safe without serious side effects.

However, those with blood coagulation disorders or those currently taking any anti-coagulant (blood-thinning)
medications, please consult a health practitioner prior to use.

[1] Sumi H et al, A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the
Japanese diet. Experientia. 1987 Oct 15;43(10):1110-1. [2] Enhancement of the fibrinolytic activity in plasma by oral administration of
nattokinase. Acta Haematol. 1990;84(3):139-43. [3] Fujita M et al, Purification and characterization of a strong fibrinolytic enzyme
(nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan. Biochem Biophys Res Commun. 1993 Dec
30;197(3):1340-7. [4] Transport of nattokinase across the rat intestinal tract. Biol Pharm Bull. 1995 Sep;18(9):1194-6. [5] Thrombolytic
effect of nattokinase on a chemically induced thrombosis model in rat. Biol Pharm Bull. 1995 Oct;18(10):1387-91. [6] Suzuki Y et al,
Dietary supplementation with fermented soybeans suppresses intimal thickening. Nutrition. 2003 Mar;19(3):261-4. [7] Dietary
supplementation of fermented soybean, natto, suppresses intimal thickening and modulates the lysis of mural thrombi after endothelial
injury in rat femoral artery. Life Sci. 2003 Jul 25;73(10):1289-98. [8] Kim W, Choi K, Kim Y, Park H, Choi J, Lee Y, Oh H, Kwon I, Lee S.
Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang. Appl
Environ Microbiol 1996 Jul;62(7):2482-8. [9] Chang CT, Fan MH, Kuo FC, Sung HY. Potent fibrinolytic enzyme from a mutant of Bacillus
subtilis IMR-NK1. J Agric Food Chem 2000 Aug;48(8):3210-6. 10. Peng Y et al, Purification and characterization of a fibrinolytic enzyme
produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food. Comp Biochem Physiol B
Biochem Mol Biol. 2003 Jan;134(1):45-52. [11] Ko JH et al, Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02.
Comp Biochem Physiol C Toxicol Pharmacol. 2004 Jan;137(1):65-74.
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