1、納豆菌與保加利亞菌融合株之創(chuàng)製及其特性探討Study on new fusant creation from Lactobacillus bulgaricus and Bacillus subtilis var. natto and their physiological characterization 研究生：陳嘉穎 Chia-yin Chen 指導(dǎo)教授：鄧德豐 Der-Feng Teng 謝寶全 Pao-Chuan Hsien 【摘要】 本實(shí)驗(yàn)選擇具有利用澱粉及好氣生長(zhǎng)的特性之Bacillus subtilis var. natto，及Lactobacillus bulgaricus之產(chǎn)

2、酸特性，以原生質(zhì)體融合法使二種菌進(jìn)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(tài)下能生長(zhǎng)、產(chǎn)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(xì)胞壁合成）將Lactobacillus bulgaricus及Bacillus subtilis var. natto細(xì)胞壁分解使形成原生質(zhì)體，再以PEG 4000促使二菌株之原生質(zhì)體融合，再包埋培養(yǎng)於再生培養(yǎng)基中使細(xì)胞壁再生，選出能利用澱粉並在好氣條件下能生長(zhǎng)之融合株，進(jìn)行其生理特性、最適生長(zhǎng)條件、最適產(chǎn)酸力、代謝產(chǎn)物及含量之探討，並且比較Bacillus su

3、btilis var. natto與融合株之納豆激酶活性，及對(duì)碳?xì)浠衔锖投嗦嚷?lián)苯分解利用性之探討。 結(jié)果在生理特性方面顯示，融合株比親株 Lactobacillus bulgaricus增加了對(duì)potato starch、xylitol、cellobiose、erythritol、dulcitol等碳源的利用能力，而融合株對(duì)galactose、glucose、maltose、salicin、saccharose、1-0-D-glucopyranoside、potato starch等碳源的利用性比Lactobacillus bulgaricus、Bacillus subtilis var.

4、natto都要好。氮源之利用性方面，融合株比Lactobacillus bulgaricus增加了對(duì)(NH4)2SO4、NaNO3、NH4NO3等氮源之利用性，但對(duì)有機(jī)氮源之利用性比無機(jī)氮源好，尤其對(duì)yeast extract之利用性最好。碳?xì)浠衔锢眯苑矫?，融合株?Lactobacillus bulgaricus增加了對(duì)n- heptane、hexane、benzene、toluene等之利用性。 融合株最適生長(zhǎng)條件為培養(yǎng)於pH 8的培養(yǎng)基中、30 振盪培養(yǎng)生長(zhǎng)最好。最適產(chǎn)酸力條件是在含有3 % 澱粉及3 % yeast extract、pH 8的培養(yǎng)基中，於37 靜置培養(yǎng)之產(chǎn)酸力較高。

5、分析融合株之代謝產(chǎn)物結(jié)果顯示，融合株靜置培養(yǎng)之代謝產(chǎn)物有草酸0.043 %、乳酸0.36 %、醋酸0.014 %、丁酸1.3 %，及一些未知產(chǎn)物，丁酸產(chǎn)量為乳酸之3.61倍（1.3/0.36）；振盪培養(yǎng)之代謝產(chǎn)物有草酸0.035 %、乳酸0.04 %、醋酸0.12 %、丁酸1.01 %及一些未知產(chǎn)物，丁酸量為乳酸之25.25倍（1.06/0.04）。融合株靜置培養(yǎng)所產(chǎn)生之草酸、乳酸、醋酸、丁酸產(chǎn)量皆比振盪培養(yǎng)高，靜置培養(yǎng)產(chǎn)生之丁酸與乳酸的比值相差不大。振盪培養(yǎng)產(chǎn)生之乳酸量很低，但丁酸與乳酸產(chǎn)量之比值有明顯差異。融合株培養(yǎng)於碳?xì)浠衔锱囵B(yǎng)基中，皆能產(chǎn)生丁酸。 將Lactobacillus bul

6、garicus、 Bacillus subtilis var. natto及融合株分別培養(yǎng)於含多氯聯(lián)苯之水相及水相加培養(yǎng)基中，比較其對(duì)多氯聯(lián)苯分解性，融合株顯示對(duì)多氯聯(lián)苯具有分解性。將Lactobacillus bulgaricus、 Bacillus subtilis var. natto及融合株分別培養(yǎng)於含不同磺胺劑之培養(yǎng)基中，比較其對(duì)磺胺劑之利用性，結(jié)果顯示Lactobacillus bulgaricus在含有磺胺劑之培養(yǎng)基中無法生長(zhǎng)，而Bacillus subtilis var. natto及融合株皆能生長(zhǎng)。 將Bacillus subtilis var. natto及融合株分別培養(yǎng)於

7、不同培養(yǎng)基成分、不同培養(yǎng)時(shí)間，比較其產(chǎn)生納豆激酶之活性，Bacillus subtilis var. natto振盪培養(yǎng)於合成培養(yǎng)基中36小時(shí)，其納豆激酶活性最高可達(dá)63 FU/ml，而融合株振盪培養(yǎng)於合成培養(yǎng)基中60小時(shí)，其納豆激酶活性最高只達(dá)10.08 FU/ml，融合株需培養(yǎng)較長(zhǎng)的時(shí)間才能使其納豆激酶活性達(dá)到較高，於合成培養(yǎng)基中，Bacillus subtilis var. natto振盪培養(yǎng)36小時(shí)、融合株振盪培養(yǎng)60小時(shí)，其納豆激酶活性皆為最高，融合株產(chǎn)生之納豆激酶活性比Bacillus subtilis var. natto要低6.25倍。 關(guān)鍵字：保加利亞菌、納豆菌、原生質(zhì)體融合

8、【Abstract】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 b

9、ulgaricus 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

10、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 aer

11、obic 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 poly

12、chlorinated 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 utiliz

13、ed ability of carbon source of galactose, glucose, maltose, salicin, saccharose, 1-0-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)2SO

14、4, 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

15、 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

16、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

17、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 differe

18、nce 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

19、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 Ba