Study On Solid Fermentation Of Natto And The Enzymatic Properties Of Nattokinase

Natto is a kind of traditional fermented food. It is made by soybean which is inoculated with Bacillus subtilis natto and cultured under definite temperature and humidity. With high nutritional value, natto is rich in amino acid, isoflavone, calcium, ferrum, vitamin K, unsaturated fatty acid, lecithin and so on. Eating natto also has some hygienical functions, such as antioxidation, reducing blood press, dissolving blood clots, antineoplastic effect, antibiosis. Nattokinase (NK) is a serine protease enzyme which is produced by Bacillus subtilis natto in the process of the fermentation of natto. Compared with the thrombolytic agents now used, NK is safer and has more benefits including low cost, strong fibrinolytic activity, prolonged effects, convenient for oral administration. So it is important to study how to develop natto functional food with high quality and high NK activity.The optimization of solid fermentation conditions of natto and enzymatic properties of nattokinase were studied in this paper. The results were as follows:NK activity was used as determining index and was measured by fibrinogen plate method. Using Plackett-Burman design, the affecting factors such as soybean fragmentation, inoculation and fermentation temperature were studied and some significant factors were screened. Then the path of steepest ascent was subsequently used to approach the optimal conditions and the optimized conditions were confirmed by Box-Behnken test design and response surface analysis. The results showed that soybean fragmentation, inoculation and fermentation temperature were the significant factors. The optimal conditions were as the following:soybean fragmentation as4, inoculation7.9%and fermentation temperature32℃, and the optimum nattokinase activity was (2140.5±83.2) U/g under the optimized conditions.From pH7.0to pH9.0, NK exhibited high stability. When pH value was below5.0, NK was very unstable. Different mental ions had different effect on NK activity. Fe3+and Cu2+could inhibit NK activity. Zn2+in low concentration could not affect NK activity significantly while in high concentration could restrain NK activity. Ca2+、Mg2+、K+could activate NK to some extent. NK could maintain about60%activity when preserved under45℃or50℃for2hours. When the heat preservation temperature was between60℃and70℃,NK lost its activity quickly. Using casein as substrate and Lineweaver-Burk plot as major method, Km of NK was about3.174mg/mL obtained under30℃. On fibrinogen plate, the optimal reaction temperature of NK was34℃.