Study on new fusant creation from Lactobacillus bulgaricus and Bacillus subtilis var. natto and their physiological characterization

• Main Authors: Chia yin Chan, 陳嘉穎
• Other Authors: 鄧德豐
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/25391540469716164169

碩士 === 國立屏東科技大學 === 食品科學系 === 94 === The contents of abstract in this thesis: Selected the characteristic of Bacillus subtilis var. natto can utilize starch and good growth in aerobic condition and the Lactobacillus bulgaricus can produce acid characteristic were used for preparation of new fusant, the protoplast fusion method was used to improve the Lactobacillus bulgaricus gene characteristic, to increase the utilization of Lactobacillus bulgaricus, the starch utilizing and high acid produce and growth in aerobic condition and tolerance oxygen of fusant were isolated. Lysozyme (after added ampicillin to block cell wall formation), was used to hydrolysis the cell wall of Lactobacillus bulgaricus and Bacillus subtilis var. natto for preparing protoplast, then protoplast fusion were promoted with PEG 4000, the cell wall regeneration was prepared by buried the fused protoplast in the regeneration medium, the fusant can utilize starch and good growth in aerobic condition were isolated, then their physiological characteristic and optimum growth condition, optimum acids productivity, metabolite and content of fusant were investigated. Nattokinase activity of fusant was compared to Bacillus subtilis var. natto, and the utilization of hydrocarbon and polychlorinated biphenyls decomposition ability were also investigated. The results of the physiological characterization, Fusant show increasing the utilization ability of carbon source of potato starch, xylitol, cellobiose, erythritol, dulcitol etc compared with Lactobacillus bulgaricus. Fusant utilized ability of carbon source of galactose, glucose, maltose, salicin, saccharose, 1-0-α-D-glucopyranoside, potato starch etc were better than their parent of Lactobacillus bulgaricus, Bacillus subtilis var. natto. Fusant showed increasing the utilization ability of utilizing nitrogen source of (NH4)2SO4, NaNO3, NH4NO3 etc compared with Lactobacillus bulgaricus. Fusant utilized ability of organic nitrogen was better than inorganic nitrogen, it showed the best utilization of yeast extract. Fusant showed increasing the utilization ability of hydrocarbon source of n- heptane, hexane, benzene, toluene etc compared with Lactobacillus bulgaricus. The growth optimum condition of fusant in the medium was at pH 8 and shaking culture at 30 ℃. The acid production optimum medium contained 3 % potato starch and 3% yeast extract at pH 8 and with stationary culture at 37 ℃. The metabolite analysis results of fusant with stationary culture at 37 ℃produced oxalic acid 0.043 %、lactic acid 0.36 %、acetic acid 0.014 %、butyric acid 1.3% and unknown products, but the butyric acid yields was higher than lactic acid 3.61 times（1.3/0.36）. Shaking culture at 30 ℃ produced oxalic acid 0.035 %、lactic acid 0.04 %、acetic acid 0.12 %、butyric acid 1.01 % and unknown products, but the butyric acid yields was higher than lactic acid 25.25 times（1.06/0.04）. Fusant produced oxalic acid、lactic acid、acetic acid、butyric acid at stationary culture were higher than with shaking culture, but the yields was not obviously difference between butyric acid and lactic acid at stationary culture. Lactic acid yields was very low at shaking culture, the yields was obviously difference ratio their between butyric acid and lactic acid. Fusant could produce butyric acid in the medium contained every kind of hydrocarbon. Lactobacillus bulgaricus、Bacillus subtilis var. natto and fusant were cultivated in the medium contained polychlorinated biphenyls respectively. Fusant showed the ability to decompose the polychlorinated biphenyls. Lactobacillus bulgaricus、Bacillus subtilis var. natto and fusant were cultivated in the medium contained different sulfadrugs, the results showed Lactobacillus bulgaricus could not growth, but Bacillus subtilis var. natto and fusant grew in the medium contained different sulfadrugs. Comparison of the nattokinase activities of Bacillus subtilis var. natto and fusant were cultivated at various medium, difference culture time, respectively the highest nattokinase activity was 63 FU/ml by Bacillus subtilis var. natto in synthetic medium at shaking culture for 36 hours, the highest nattokinase activity was 10.08 FU/ml from fusant in synthetic medium at shaking culture for 60 hours, the highest nattokinase activity of fusant was obtained for longer culture time, The nattokinase activity of fusant was lower than Bacillus subtilis var. natto about 6.25 times when cultivated in the synthetic medium.