Nattokinase production of Bacillus subtilis as affected by mutation

• Main Authors: Pei-yi Lin, 林佩誼
• Other Authors: Pao-Chuan Hsieh, Ph.D.
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/89400658400851126881

碩士 === 國立屏東科技大學 === 食品科學系 === 94 === 【Abstract】 In this study, Bacillus subtilis var. natto NPUST B11-2 was used as parent strain which produces 58 FU/ml nattokinase. Mutations were induced by chemical damage and ultraviolet light. Bacillus subtilis strain was bred by treating with UV, N-methyl-N,,-nitro-N-nitrosoguanidine(NTG), Ethylmethanesulfonat-e (EMS) and screening the mutants, which were able to grow in an agar medium containing penicillin. Mutants strain relatively high in nattokinase more than parental strain were selected after mutation treatment. Results indicated, the killing rate was 87.1% after using UVA (366nm) irradiation treatment, then screening the mutants, which could grow in an agar medium containing penicillin. Mutants strain relatively high in nattokinase more than parental strain were selected after mutation treatment. The cultivation of mutant was investigated on the increasing nattokinase production. Shaking conditions, inoculums size, carbon sources, nitrogen sources, mineral salts, initial pH and culture temperatures were optimized for nattokinase production from mutant. The results showed that 40 ml medium in 250 ml Hinton’s, which contained 2% Brown sugar, 1.6% Soybeanprotein, 0.4% Soytone peptone, 0.05% CaCl2, and 0.05% Thymine. The medium initial pH was 7, 150 rpm shaked speed and 1.5% inoculums size. It produced the highest nattokinase activity (108 FU/ml) at 37℃ for 60 hours. Five liter fermentor was used to evaluate the optimum scale-up cultivation conditions of mutant producing nattokinase. Inoculum size was 3%. The temperature , aeration rates, stirred speed and medium volume of fermentation were 37℃, 1vvm, 300rpm and 2.5 liter, respectively. The results indicated that mutant producing the highest nattokinase activity (85 FU/ml) after culture 60 hours. Volatile compounds from fermented soybean foods, were analysed by gas chromatography-mass spectrometry (GC-MS). A large amount of CO2 and ethanol were detected in natto (mutant). The characteristic odor compounds of natto (mutant) were esters compounds, 2,5-dimethylpyrazine, acetone compound, 1-octen-3-ol. Results indicated that mutant can produce γ-PGA (0.03mg/ml) after culture 12 hours. Key words: Nattokinase, Bacillus subtilis var. natto, mutation, γ-PGA