| Summary: | 博士 === 國立屏東科技大學 === 熱帶農業暨國際合作系 === 102 === At the present time, GABA is also being investigated for its potential antihypertensive effects. Moreover, nattokinase is a potent fibrinolytic enzyme that is considered a promising agent for thrombosis therapy. The objective of this study was to optimize culture medium that would maximize the productivity of GABA and nattokinase. First, Bacillus subtilis was screened from rice straw. Different medium compositions were then optimized for GABA and nattokinase production using the Taguchi experimental design method, and the effects of various nitrogen and carbon sources were investigated. Of the various key ingredients used in the media, fructose and yeast extract were found to have the most significant effect on GABA and nattokinase production. The optimal pH value and temperature for GABA production were 7.0 and 37 °C, respectively, after 72 h. The maximum GABA yield of B. subtilis in flask culture was 17.40 g/L. For nattokinase production, the optimal pH and temperature were 8.0 and 30 °C, respectively, for 48 h, yielding a maximum of approximately 130.96 unit/mL of nattokinase. In batch culture of B. subtilis in fermenter, the highest GABA 20.58 g/L was obtained at 60 h. The highest nattokinase activity was also 145.50 unit/mL at 60 h. Further, media optimization using L9 orthogonal array resulted in enhanced production of GABA and fibrinolytic enzyme which were 5.31 and 8.74 fold higher than in unoptimized medium, respectively.The gene encoding fibrinolytic enzyme was isolated and its DNA sequence was determined. Compared the full DNA sequence with those in NCBI, it was considered to be a subtilisin like serine-protease.
The parameters for GABA and nattokinase production in solid state fermenter culture were also optimized in this study. The ratio between giant black bean and wheat bran is an important factor for GABA, also small black bean and soy bean for nattokinase, while the optimum mixing ratio for both of GABA and nattokinase was 1:1 (W/W). The highest GABA yield, 32.8 mg/g, was obtained after 72 h. The nattokinase activity maximum, 80.592 units/g, was found at 48 h, after which a slight decrease was observed. SSF process technique might provide a better choice for GABA and nattokinase production, considering the high productivity and low production cost.
In this study, the effects of the administration of GABA and nattokinase on lowering blood pressure in SHR were investigated. To facilitate the interpretation of our results, we also measured the gain in weight and the consumption of solid, freely accessible feed by the animals.
We have evaluated the changes in arterial blood pressure in spontaneously hypertensive rats (SHR) caused by the short-term intake of Bacillus subtilis B060-fermented beans with significant GABA and nattokinase activity. After being weaned, 7-week-old male SHR and 7-week-old male WKY were randomized into 7 groups. Until the 8th week of life, the rats in each group were given one of the following: Group 1: high dose of GABA and nattokinase in the SHR (SHD); Group 2: medium dose of GABA and nattokinase in the SHR (SMD); Group 3: low dose of GABA and nattokinase in the SHR (SLD); Group 4: negative control in the SHR (SD); Group 5: positive control in the SHR (SM); Group 6: high dose of GABA and nattokinase in the WKY (WHD); and Group 7: negative control in the WKY (WD). Distilled water served as the negative control, and captopril (50 mg/kg), a known ACE inhibitor, served as the positive control. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) values were measured weekly from the 8th to the 16th week of life using the tail-cuff method. A definite decrease in SBP and DBP values could be observed in the rats treated with captopril and in the rats that received GABA and nattokinase. The greatest antihypertensive effect was observed when the pharmacological treatment was administered. The effect of the daily intake of GABA and nattokinase may be helpful in controlling BP levels in hypertensive model animals. The GABA and nattokinase may therefore constitute a successful strategy for producing a functional food with antihypertensive activity.
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