Screening And Identification Of A Bacterium Strain With High Fibrinolytic Activities, And Optimization Of Its Fermentation Conditions

Thrombus disease has become the most harmful disease in the21st century. Traditional drugs are expensive and cause serious side effect such as blooding and disability, which causes serious social problems. Previous studies showed that Bacillus subtilis Natto could produce a fibrinolytic serine protease, namely Nattokinase (NK). Compared with traditional fibrinolytic drugs, NK was characterized by high fibrinolytic activities, long-action, safe and non-side effect profiles. Therefore, NK has the potential to be developed as functional foods and drugs to prevent or cure thrombosis diseases. With this promising market prospects, NK is becoming the research hotspot in more and more countries.This study is performed based on10fermentation foods (from Dalian, Beijing, Tianjing, Jiangsu, Zhuhai and so on) and37laboratory-kept strains (from Shanxi, Japan, Chongqing, Henna, Luzhou, Shenzhen, Neimengku, Foshan and Shaoxing). In the first round screening.145strains whith high fibrinolitic activities were obtained using casein-plate method. Then the fibrinolitic activties were repeatly compared using fibrin-plate method. A strain. HT8. was obtained in screening and the enzyme activity of its products can reach1.637.6U/mL with fibrin-plate method. The morphological, physiological and biochemistry characteristics of HT8were studied based on Bergey’s manual (4th Edition) and identified as Bacillus subtilis. Molecular biological analysis was perfomed based on16s RNA sequence (1,131bp). Homology searches were done by Blast and the phylogney analysis was conducted using MEGA5.1. The results showed that HT816S rRNA shared99%similarity with16S rRNA of Bacillus subtilis, clustered with Bacillus subtilis. Based on these results. HT8was identified as Bacillus subtilis.To obtain optimal conditions for nattokinase production, the factors including nitrogen source (peptone, ammonia sulfate and yeast extract,), carbon source (sucrose and glucose), and inorganic ions composition were investigated by One-way ANOVA Then orthogonal experiments using L18(3’) array were performed and statistically analyzed by ANOVA in SPSS. The result showed the optimal fermentation conditions were initial medium pH8.0, temperature40℃, volume90/500mL,0.5%beef extract and2%soybean peptone. The relative enzyme activity was1,804.08IU/mL under the optimal conditions, which provided a theoretical basis for the further development of natto and various of healthy food.