| Summary: | 碩士 === 國立嘉義大學 === 食品科學系碩士班 === 93 === In this study, 65 Bacillus spp. had been isolated from Bacillus stock of Food Industry Research And Development Institute, soil, water, rice straw, Japanese commercial natto and other natto products. First, plasminogen-free fibrin plate method was used to pick out the high nattokinase production strains from 65 Bacillus spp.. Second, the optimal condition for cultivation of solid state fermentation and submerged culture for Bacillus strains was estabalished to increase nattokinase production. Than, crude enzyme of nattokinase was purified and identified the different recoveries identified and stability of nattokinase activity from each strains examined. The results indicated that strain number C13 which was isolated from Japanese commercial natto produced the highest nattokinase activity, 328.2 FU/g at 37℃ from 48 hours of culture. Explore sure of C13 strain to ultraviolet rays induced mutation and 46 mutant strains were. Mutant CM13 could produced the highest nattokinase activity 421.2 FU/g. Soybean was the optimal medium for CM13 mutant solid state fermentation. The optimal cultivation condition for inoculum size, relative humidity, temperature and time were 1.0 mL (107~108 cell/mL), 90%, 37℃ and 32 hours, respectively The highest activity of nattokinase achieved was 469.2 FU/g. Using submerged culture, CM13 mutant gave the activity of nattokinase approximately 70.3 FU/mL when incubated at 37℃ for 32 hours. At 60℃ or 37℃ the nattokinase from solid substrate fermentation was more stable than from submerged culture. The recovery of nattokinase purified from solid substrate fermentation reached 63.0%, much higher than 39.8% from submerged culture. This is was because of the γ-poly-glutamic acid (γ-PGA) which produced from natto during the process of cultivation. γ-PGA increased the stability of nattokinase, and the recovery was also enhanced after purification. Adding 0.1% γ-PGA to in crude enzyme from submerged fermentation increased the stability and recovery of nattokinase sigificantly.
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