臺灣博碩士論文加值系統

論文摘要內容：
本實驗選擇具有利用澱粉及好氣生長的特性之Bacillus subtilis var. natto，及Lactobacillus bulgaricus之產酸特性，以原生質體融合法使二種菌進行融合，來改良乳酸菌之基因特性，使其能利用澱粉來提高乳酸菌之利用性，期望育出能利用澱粉且在好氣狀態下能生長、產酸力高的乳酸菌，並提高乳酸菌之耐氧性。以lysozyme（先添加ampicillin阻斷細胞壁合成）將Lactobacillus bulgaricus及Bacillus subtilis var. natto細胞壁分解使形成原生質體，再以PEG 4000促使二菌株之原生質體融合，再包埋培養於再生培養基中使細胞壁再生，選出能利用澱粉並在好氣條件下能生長之融合株，進行其生理特性、最適生長條件、最適產酸力、代謝產物及含量之探討，並且比較Bacillus subtilis var. natto與融合株之納豆激酶活性，及對碳氫化合物和多氯聯苯分解利用性之探討。
結果在生理特性方面顯示，融合株比親株 Lactobacillus bulgaricus增加了對potato starch、xylitol、cellobiose、erythritol、dulcitol等碳源的利用能力，而融合株對galactose、glucose、maltose、salicin、saccharose、1-0-α-D-glucopyranoside、potato starch等碳源的利用性比Lactobacillus bulgaricus、Bacillus subtilis var. natto都要好。氮源之利用性方面，融合株比Lactobacillus bulgaricus增加了對(NH4)2SO4、NaNO3、NH4NO3等氮源之利用性，但對有機氮源之利用性比無機氮源好，尤其對yeast extract之利用性最好。碳氫化合物利用性方面，融合株比 Lactobacillus bulgaricus增加了對n- heptane、hexane、benzene、toluene等之利用性。
融合株最適生長條件為培養於pH 8的培養基中、30 ℃振盪培養生長最好。最適產酸力條件是在含有3 % 澱粉及3 % yeast extract、pH 8的培養基中，於37 ℃靜置培養之產酸力較高。
分析融合株之代謝產物結果顯示，融合株靜置培養之代謝產物有草酸0.043 %、乳酸0.36 %、醋酸0.014 %、丁酸1.3 %，及一些未知產物，丁酸產量為乳酸之3.61倍（1.3/0.36）；振盪培養之代謝產物有草酸0.035 %、乳酸0.04 %、醋酸0.12 %、丁酸1.01 %及一些未知產物，丁酸量為乳酸之25.25倍（1.06/0.04）。融合株靜置培養所產生之草酸、乳酸、醋酸、丁酸產量皆比振盪培養高，静置培養產生之丁酸與乳酸的比值相差不大。振盪培養產生之乳酸量很低，但丁酸與乳酸產量之比值有明顯差異。融合株培養於碳氫化合物培養基中，皆能產生丁酸。
將Lactobacillus bulgaricus、 Bacillus subtilis var. natto及融合株分別培養於含多氯聯苯之水相及水相加培養基中，比較其對多氯聯苯分解性，融合株顯示對多氯聯苯具有分解性。
將Lactobacillus bulgaricus、 Bacillus subtilis var. natto及融合株分別培養於含不同磺胺劑之培養基中，比較其對磺胺劑之利用性，結果顯示Lactobacillus bulgaricus在含有磺胺劑之培養基中無法生長，而Bacillus subtilis var. natto及融合株皆能生長。
將Bacillus subtilis var. natto及融合株分別培養於不同培養基成分、不同培養時間，比較其產生納豆激酶之活性，Bacillus subtilis var. natto振盪培養於合成培養基中36小時，其納豆激酶活性最高可達63 FU/ml，而融合株振盪培養於合成培養基中60小時，其納豆激酶活性最高只達10.08 FU/ml，融合株需培養較長的時間才能使其納豆激酶活性達到較高，於合成培養基中，Bacillus subtilis var. natto振盪培養36小時、融合株振盪培養60小時，其納豆激酶活性皆為最高，融合株產生之納豆激酶活性比Bacillus subtilis var. natto要低6.25倍。

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.

目錄
頁次

摘要 I
Abstract IV
誌謝 VII
圖表目錄 XII
壹、前言 1
貳、文獻回顧 2
一、菌種介紹 2
二、菌株的改良方法 5
三、原生質體融合法 5
四、原生質體的特性 8
五、影響原生質體製備之因素 9
六、融合株篩選方法 15
七、融合株確認方法 15
八、有機酸 16
九、碳氫化合物 22
十、多氯聯苯 23
十一、納豆激酶 27
十二、磺胺劑 28
参、材料與方法 30
ㄧ、實驗設計 30
二、實驗材料 31
（一）菌株 31
（二）培養基組成 31
（三）溶液配製 32
（四）實驗使用之藥品 33
（五）HPLC（High performance liquid chromatography） 35
（六）GC（Gas chromatography） 36
（七）儀器設備 37
三、實驗方法 37
（一）菌種培養 37
（二）原生質體之製備 38
（三）原生質體之融合 38
（四）融合株的篩選及繼代 39
（五）融合株的確認 39
1. 形態 39
2. 碳源利用性 39
3. 氮源利用性 39
4. 融合株對澱粉之利用性 39
（六）融合株生長條件之探討 40
（七）融合株產酸力條件之探討 40
（八）融合株發酵型式測試 41
（九）融合株代謝產物之分析 41
（十）其他特性之探討 42
1. 碳氫化合物之利用性 42
2. 多氯聯苯之利用性 42
3. 納豆激酶活性之測定 42
4. 磺胺劑之利用性 43
（十一）其他特性之分析 43
1. 碳氫化合物之代謝產物的分析 43
2. 多氯聯苯殘留率分析 44
3. 磺胺劑殘留率分析 44
4. 羥四環素殘留率分析 45
肆、結果與討論 46
ㄧ、親株之形態及其原生質體 46
（一）Lactobacillus bulgaricus之形態及其原生質體形態 46
（二）Bacillus subtilis var. natto之形態及其原生質體形態 46
二、融合株之確認 51
（一）形態 51
（二）碳源之利用性 53
（三）氮源之利用性 53
（四）融合株對澱粉之利用性 53
三、融合株生長條件之探討 58
（一）融合株之生長曲線 58
（二）氧氣對融合株生長之影響 58
（三）溫度對融合株生長之影響 58
（四）起始pH值對融合株生長之影響 58
（五）氮源對融合株生長之影響 58
（六）澱粉濃度對融合株生長之影響 59
（七）Yeast extract之濃度對融合株生長之影響 59
四、融合株產酸力之條件探討 67
（一）氧氣對融合株產酸力之影響 67
（二）溫度對融合株產酸力之影響 67
（三）起始pH值對融合株產酸力之影響 67
（四）氮源對融合株產酸力之影響 67
（五）澱粉濃度對融合株產酸力之影響 67
（六）不同澱粉濃度靜置、振盪培養對融合株產酸力的影響 68
（七）Yeast extract濃度對融合株產酸力的影響 68
（八）碳氫化合物對融合株產酸力之影響 68
五、融合株發酵型式測試 77
六、融合株之代謝產物的分析 79
（一）靜置培養之代謝產物的分析 79
（二）振盪培養之代謝產物的分析 79
（三）不同靜置培養時間之代謝產物含量的分析 79
（四）不同振盪培養時間之代謝產物含量的分析 79
七、其他特性之探討 84
（一）碳氫化合物之利用性 84
（二）多氯聯苯之利用性 84

（三）Bacillus subtilis var. natto及融合株生產納豆激酶
活性之比較 84
1. Bacillus subtilis var. natto及融合株各別培養於
不同培養基及培養時間中之納豆激酶活性的比較 84
2. Bacillus subtilis var. natto及融合株培養於不同
通氣量及培養時間之納豆激酶活性的比較 85
（四）磺胺劑之利用性 85
八、其他特性之分析 92
（一）碳氫化合物之代謝產物分析 92
（二）多氯聯苯殘留率之分析 92
（三）磺胺劑殘留率之分析 92
（四）田間試驗 93
伍、結論 101
陸、參考文獻 105
柒、作者介紹 114

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