| Objective:Natto is a fermented food in Japan. It is made from soybeans fermented with Bacillus natto, which has thousands of years of history. Modern research about Natto showed that it contains bioactive substances that can dissolve clots and is known as nattokinase. The enzyme is a single-chain protein and orally active. However, nattokinase is unstable in the stomach under acidic conditions. In order to improve the antithrombotic effect of nattokinase, we developed enteric-coated capsules of nattokinase, and evaluated its antithrombotic effect and its effect on bleeding.Methods:The northeast soybeans of golden color and without dark spots was soaked for 12 hours at room temperature. The soybeans was steamed using a steamer about 20 minutes of boiling. In the clean workbench, each 500 grams of cooked soybeans were inoculated with 5 g of Natto soybeans。Then, the beans were stirred well and packed into a stainless steel basin (depth 10 cm, thickness 3 cm) and covered using sterile wet towels of two layers. Into an incubator of 40±2℃, the soybeans were transferred, where sterile water was used to maintain the appropriate humidity in the incubator. After incubation of 36 hours, in clean workbench, the fermented soybeans were stirred well again and packed into micro-foam boxes, each of 50 grams, and then they were covered with covers of small pinholes while the soybeans in the boxs were closed with plastic film for food packaging. Finally, the beans were placed into a freezer close to 0℃for tripening for one week, and then the Natto drawing a long natto glycoprotein was prepared. As for Natto preparation conditions, temperature and humidity are the key factors. After maturation, natto produced a great amount of sticky protein (mucin), which interfered with nattokinase extraction. Therefore, we extracted the crude nattokinase using natto before its maturation to eliminate natto mucin interference. With sucrose used as additive and spray-drying technology, we prepared nattokinase powders. And then, the nattokinase powders were packaged into enteric-coated capsules. Then, in Kunming mice and SD rats, Nattokinase enteric-coated capsules were evaluated for their antithrombotic effect and its effect on clotting time of animals. The lower the blood concentration, the longer the coagulation time. Thus, using diluted blood, we observed the effect of nattokinase enteric-coated capsules on thrombus formation.Results:Natto could be prepared using Japanese natto with Chinese soybeans. Crude Nattokinase extract was used for the preparartion of Nattokinase enteric-coated capsules, which could simplify the Nattokinase product preparation and improved the stability of nattokinase in living systems. Following animal tests, it was shown that Nattokinase enteric-coated capsules have significant anti-thrombotic effect and didn't not prolong the bleeding time of animals 12 h later after administration of Nattokinase enteric-coated capsules. In vitro thrombus formation test showed that nattokinase enteric-coated capsules interfered with the thrombus formation significantly. After taking nattokinase enteric-coated capsules, very loose thrombus formed. In vitro thrombus formation test showed that after taking nattokinase enteric-coated capsules, the upper part of the thrombus became lighter red or white while more red cells precipitated compared with the thrombus of the animal without taking nattokinase enteric-coated capsules. And thus, it could be said that nattokinase enteric-coated capsules could slow down the thrombus formation speed and induced very loose thrombus formation. Conclusions:The nattokinase enteric-coated capsules may become a better anti-thrombosis medicine. It is well known that thrombus is associated with tumor and infection. And thus, nattokinase enteric-coated capsules may induce loose thrombus formation around tumor or infection and promote more anti-tumor or anti-infection drug molecules to accumulate into tumor or infection tissues. Futher study is needed to confirm the hypothesis above.
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