本文選題：維生素C + 內(nèi)生芽孢桿菌ST-1　； 參考：《沈陽(yáng)農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】："二步發(fā)酵法"是我國(guó)科學(xué)家自主研發(fā)的維生素C生產(chǎn)工藝,參與發(fā)酵的兩株菌——伴生菌和產(chǎn)酸菌,在發(fā)酵的過(guò)程中各自承擔(dān)重要的角色,二者的相互作用關(guān)系一直是研究的熱點(diǎn)。本文以一株新的伴生菌株ST-1和普通生酮基古龍酸桿菌25-B-1(Ketogulonicigenium vulgare 25-B-1,K vulgare 25-B-1)為研究對(duì)象,通過(guò)分析伴生菌ST-1與產(chǎn)酸菌K.vulgare 25-B-1兩者的生理特性,探討了伴生菌芽孢生成和萌發(fā)的規(guī)律,及其對(duì)K.vulgara 25-B-1生長(zhǎng)與產(chǎn)2-酮基-L-古龍酸(2-KGA)的影響。通過(guò)測(cè)定單菌及混菌發(fā)酵體系中產(chǎn)酸菌胞內(nèi)胞外活性氧(ROS)水平,分析伴生菌對(duì)產(chǎn)酸菌抗氧化能力的影響,并對(duì)K.vulgare 25-B-1體內(nèi)相關(guān)基因表達(dá)差異進(jìn)行了研究,為解析伴生菌和產(chǎn)酸菌之間的相互作用機(jī)制提供理論依據(jù)和新的思路。主要研究結(jié)果如下:(1)對(duì)伴生菌株ST-1進(jìn)行系統(tǒng)進(jìn)化鑒定。系統(tǒng)發(fā)育樹(shù)結(jié)果顯示ST-1與其它幾株內(nèi)生芽孢桿菌(Bacillusendophyticus)的同源性≥99%,因此鑒定該菌株為Bacillus endophyticus,并命名為 Bacillus endophytius ST-1(B.endophytius ST-11)。B.endophyticus ST-1與B.endophyticus Hbe603同源關(guān)系最接近。B.endophyticB.ST-1與實(shí)驗(yàn)室的其它兩株伴生菌短小芽孢桿菌HJ-04和巨大芽孢桿菌25-B相比具有更強(qiáng)的促進(jìn)K.vulgare 25-B-1產(chǎn)2-KGA的能力。(2)產(chǎn)酸菌K.vulgare 25-B-1能促進(jìn)伴生菌B.endophyticusST-1的生長(zhǎng),并縮短其生長(zhǎng)周期�；炀囵B(yǎng)時(shí)B.endophyticus ST-1在每個(gè)時(shí)間點(diǎn)的生物量均大于單菌培養(yǎng),混菌培養(yǎng)的B.endophyticB.ST-1在20 h、48 h、64 h時(shí)有峰值,約為初始生物量的12.7倍、11.9倍和11.1倍,單菌培養(yǎng)的B.endophyticus ST-1在36 h和64 h時(shí)有峰值,約為初始菌量的9.6倍和11.3倍;在發(fā)酵72 h期間,混菌中的B.endophyticus ST-1經(jīng)歷了兩次生長(zhǎng)周期,第一次歷時(shí)28h,第二次歷時(shí)24h,而單獨(dú)培養(yǎng)的B.endophyticus ST-1只有一個(gè)完整的生長(zhǎng)周期。(3)伴生菌B.endophyticus ST-1能促進(jìn)產(chǎn)酸菌K.vulgare 25-B-1的產(chǎn)酸及生長(zhǎng)。發(fā)酵至72 h,單獨(dú)發(fā)酵的K.vulgare 25-B-1的2-KGA產(chǎn)量為4.3 mg/mL,混菌發(fā)酵的2-KGA的產(chǎn)量為70.5 mg/mL,約為單菌發(fā)酵的16倍;在發(fā)酵的各個(gè)時(shí)間點(diǎn),混菌中K.vulgare 25-B-1的生物量均多于K.vulgare 25-B-1單獨(dú)發(fā)酵時(shí)的生物量。K.vulgare 25-B-1產(chǎn)酸速率出現(xiàn)峰值的時(shí)間點(diǎn)與B.endophyticus ST-1產(chǎn)孢峰值對(duì)應(yīng)的時(shí)間點(diǎn)基本吻合,因此推測(cè)B.endophyticB.ST-1芽孢的生成是提高產(chǎn)2-KGA速率關(guān)鍵因素。(4)綜合分析K.vulgare25-B-1和B.endophytiusST-1的生理特性發(fā)現(xiàn):①在與K.vulgare 25-B-1共培養(yǎng)的情況下,B.endophytius ST-1的芽孢形成需持續(xù)8 h;②與K.vulgare 25-B-1 共培養(yǎng)時(shí),B.endophytius ST-1 的芽孢萌發(fā)歷時(shí) 4 h,B.endophytius ST-1單獨(dú)培養(yǎng)時(shí),在培養(yǎng)28 h～60 h之間,芽孢萌發(fā)需歷時(shí)8 h,而在60 h～68 h之間只需要?dú)v時(shí)4 h,因此推測(cè)B.endophytiusST-1在培養(yǎng)后期分泌了某種促進(jìn)芽孢萌發(fā)的物質(zhì),而K.vulgare25-B-1能刺激伴生菌提前分泌這種物質(zhì)。③分析B.endophytius ST-1的產(chǎn)孢及pH變化,推測(cè)培養(yǎng)液中pH的降低是致活芽孢的重要條件,B.endophytius ST-1在混合培養(yǎng)時(shí)能夠分泌堿性物質(zhì)中和部分培養(yǎng)基的酸度。(5)混菌體系的K.vulgare25-B-1有更強(qiáng)的產(chǎn)生以及清除ROS的能力。在發(fā)酵18 h、24 h、40 h 時(shí),混菌體系中K.vulgare25-B-1 胞內(nèi)的 ROS 水平約為K.vulgare 25-B-1單獨(dú)發(fā)酵的4.6、4.1、3.4倍,混菌體系發(fā)酵液的ROS約為K.vulgare25-B-1單獨(dú)發(fā)酵的 1.1、1.1、1.2 倍。在發(fā)酵 0h、18h、24h、40h、52h、66h,混菌體系中 K.vulgare 25-B-1的胞內(nèi)總抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)及過(guò)氧化氫酶(CAT)酶活力分別為單菌體系K.vulgare25-B-1的1.33-36.17、0.93-1.56、1.14-1.77倍,前者的胞外T-AOC、SOD及CAT酶活力分別為后者的2.54-23.5、7.50-23.3、1.56-6.97倍,在發(fā)酵40 h,混菌體系發(fā)酵液的抗氧化能力迅速增強(qiáng),推測(cè)與伴生菌胞內(nèi)營(yíng)養(yǎng)物質(zhì)及抗氧化物質(zhì)的釋放有關(guān)。(6)對(duì)K.vulgare 25-B-1體內(nèi)相關(guān)基因表達(dá)差異的研究表明:向混菌發(fā)酵培養(yǎng)基中添加10 pM魚(yú)藤酮抑制電子從NADH脫氫酶向輔酶Q的傳遞,發(fā)酵至18 h,2-KGA的產(chǎn)量降低了 10.9%,同時(shí)K.vulgare 25-B-1體內(nèi)產(chǎn)2-KGA途徑的關(guān)鍵酶(山梨糖脫氫酶及山梨酮脫氫酶)基因及細(xì)胞色素c551和細(xì)胞色素氧化酶基因的表達(dá)也顯著降低,間接證明了產(chǎn)2-KGA途徑和呼吸鏈NADH脫氫酶下游存在偶聯(lián)關(guān)系;單菌發(fā)酵時(shí)K.vulgare25-B-1體內(nèi)的抗氧化酶(超氧化物歧化酶和過(guò)氧化氫酶)基因的表達(dá)量高于混菌發(fā)酵,說(shuō)明K.vulgare 25-B-1單獨(dú)發(fā)酵時(shí)細(xì)胞受到了氧化脅迫,誘導(dǎo)了體內(nèi)抗氧化相關(guān)基因表達(dá)量的上調(diào)。
[Abstract]:"Two step fermentation" is the production process of vitamin C independently developed by Chinese scientists. The two strains of bacteria, associated bacteria and acid producing bacteria, take part in the fermentation process. The interaction of the two is the hot spot of research. A new associated strain ST-1 and the common ketonic acid bacilli 25- are used in this paper. B-1 (Ketogulonicigenium vulgare 25-B-1, K vulgare 25-B-1) is the research object. Through the analysis of the physiological characteristics of the associated bacteria ST-1 and the K.vulgare 25-B-1 of the acid producing bacteria, the rules of the spore formation and germination of the associated bacteria are discussed, and the effects on the growth of K.vulgara 25-B-1, and the effects of the growth of K.vulgara 25-B-1, and the occurrence of single bacteria and mixed bacteria. In the fermentation system, the level of extracellular active oxygen (ROS) of acid bacteria was middle, and the effects of associated bacteria on the antioxidant capacity of acid producing bacteria were analyzed, and the differences in the expression of related genes in K.vulgare 25-B-1 were studied. The theoretical basis and new ideas were provided for the analysis of the interaction mechanism between the associated bacteria and the acid producing bacteria. The main results are as follows: (1) partners Phylogenetic tree, ST-1, was identified by phylogenetic tree. The phylogenetic tree showed that the homology of ST-1 and other strains of Bacillusendophyticus was more than 99%, so the strain was identified as Bacillus endophyticus and named Bacillus endophytius ST-1 (B.endophytius ST-11).B.endophyticus ST-1. The relationship closest to.B.endophyticB.ST-1 has a stronger ability to promote 2-KGA production in K.vulgare 25-B-1 compared with other two strains of Bacillus short Bacillus HJ-04 and Bacillus megigantobacilli 25-B in the laboratory. (2) the acid producing bacteria K.vulgare 25-B-1 can promote the growth of the associated bacteria B.endophyticusST-1 and shorten the growth cycle. B.endo for B.endo. The biomass of phyticus ST-1 at each time point was greater than that of single bacteria culture. The B.endophyticB.ST-1 in mixed bacteria had peak value at 20 h, 48 h and 64 h, about 12.7 times, 11.9 times and 11.1 times of initial biomass. The single bacteria culture B.endophyticus ST-1 had the peak value of 36 h and 64 h, about 9.6 times and 11.3 times of the initial bacteria, during the fermentation of 72 h, The B.endophyticus ST-1 in the mixed bacteria experienced two growth cycles, first time 28h, second times diachronic 24h, and the single culture B.endophyticus ST-1 has only one complete growth cycle. (3) the associated bacteria B.endophyticus ST-1 can promote acid producing K.vulgare 25-B-1 production of acid and growth. Fermentation to 72 h, isolated K.vulgare The yield of 2-KGA was 4.3 mg/mL, the yield of 2-KGA fermentation by mixed bacteria was 70.5 mg/mL, about 16 times that of single bacteria fermentation, and the biomass of K.vulgare 25-B-1 in the mixed bacteria was more than that of K.vulgare 25-B-1 at each time point. The corresponding time points are basically consistent, so the formation of B.endophyticB.ST-1 spore is the key factor to improve the rate of 2-KGA production. (4) comprehensive analysis of the physiological characteristics of K.vulgare25-B-1 and B.endophytiusST-1: (1) the formation of the sporulation of B.endophytius ST-1 in the case of co culture with K.vulgare 25-B-1 should continue to be 8 h; (2) and K.vulgare 25-. When B-1 co culture, the germination of B.endophytius ST-1 spore lasted 4 h, and when B.endophytius ST-1 was cultured alone, the germination of spores needed 8 h during the culture of 60 h, and 4 h was needed between 60 h and 68 H. Stimulate the accompanying bacteria to release this substance in advance. (3) to analyze the sporulation and pH changes of B.endophytius ST-1. It is suggested that the decrease of pH in the culture medium is an important condition for the living spore, and B.endophytius ST-1 can secrete the acidity of the alkaline substance and some medium in the mixed culture. (5) the K.vulgare25-B-1 of the mixed bacteria system is stronger and clear. At 18 h, 24 h, and 40 h, the level of ROS in the K.vulgare25-B-1 cell was about 4.6,4.1,3.4 times that of K.vulgare 25-B-1 in the mixed bacteria system, and the ROS of the fermentation broth of the mixed bacteria system was about the 1.1,1.1,1.2 times of K.vulgare25-B-1 alone. The internal total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and catalase (CAT) enzyme activity were 1.33-36.17,0.93-1.56,1.14-1.77 times of the single bacteria system K.vulgare25-B-1, respectively, and the T-AOC, SOD and CAT enzyme activity of the former were 2.54-23.5,7.50-23.3,1.56-6.97 times of the latter respectively. In the fermentation 40 h, the fermentation broth of the mixed bacteria system was antioxidation. The ability to increase rapidly, presumably related to the release of nutrients and antioxidants in the associated bacteria. (6) a study of the differences in the expression of related genes in K.vulgare 25-B-1 showed that 10 pM rotenone was added to the fermentation medium to inhibit the transmission of electron from NADH dehydrogenase to coenzyme Q, fermentation to 18 h, and the yield of 2-KGA decreased by 10.9%. The expression of the key enzymes (sorbsugar dehydrogenase and sorbone dehydrogenase) gene and cytochrome c551 and cytochrome oxidase gene in the 2-KGA pathway of K.vulgare 25-B-1 also decreased significantly, which indirectly demonstrated that the downstream presence of the 2-KGA pathway and the downstream of the respiratory chain NADH dehydrogenase was associated with the antioxidant activity of the single bacteria fermentation in the K.vulgare25-B-1 body. The expression of the enzyme (superoxide dismutase and catalase) gene was higher than that of the mixed fermentation, indicating that the cells were subjected to oxidative stress during the single fermentation of K.vulgare 25-B-1, inducing the up regulation of the expression of antioxidation related genes in the body.
【學(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q93

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉耀平,徐明愷,金玉蘭,張成剛;氧化葡萄糖酸桿菌酶學(xué)和分子生物學(xué)研究[J];微生物學(xué)雜志;2003年03期

2 王春香;田寶玉;呂睿瑞;林偉鈴;徐燕;黃欽耿;黃建忠;;西雙版納地區(qū)熱帶雨林土壤酸桿菌(Acidobacteria)群體結(jié)構(gòu)和多樣性分析[J];微生物學(xué)通報(bào);2010年01期

3 姜巨全;劉賀男;肖鴻禹;付曉薇;朱光宇;;產(chǎn)甲酸草酸桿菌細(xì)胞數(shù)群體感應(yīng)系統(tǒng)基因hdtS克隆和功能分析[J];東北農(nóng)業(yè)大學(xué)學(xué)報(bào);2014年06期

4 賴德輝;李遜;雷鳴;曾國(guó)華;袁堅(jiān);吳文起;何永忠;何朝暉;;克隆與表達(dá)產(chǎn)甲酸草酸桿菌代謝基因Frc及臨床意義[J];中華腔鏡泌尿外科雜志(電子版);2010年03期

5 王端好;;氧化葡萄糖酸桿菌的特性及應(yīng)用[J];貴州農(nóng)業(yè)科學(xué);2013年10期

6 葉晴,尹光琳;氧化葡萄糖酸桿菌SCB329的培養(yǎng)及保存[J];工業(yè)微生物;2001年01期

7 馮樹(shù),孫傳寶,張忠澤,朱可麗,張海宏,高永濤;維生素C二步發(fā)酵中巨大芽孢桿菌對(duì)氧化葡萄糖酸桿菌生長(zhǎng)和產(chǎn)酸的影響[J];微生物學(xué)雜志;1998年01期

8 袁鐵錚,姚斌,羅會(huì)穎,王亞茹,伍寧豐,范云六;來(lái)源于酸熱脂環(huán)酸桿菌的嗜酸性α-淀粉酶的表達(dá)研究[J];生物工程學(xué)報(bào);2005年01期

9 張宇霞;李儒;吳潤(rùn);;產(chǎn)甲酸草酸桿菌oxc基因的克隆及其蛋白結(jié)構(gòu)預(yù)測(cè)[J];甘肅農(nóng)業(yè)大學(xué)學(xué)報(bào);2008年03期

10 肖蘇珂;楊雪鵬;魏東芝;林金萍;;氧化葡萄糖酸桿菌啟動(dòng)子的篩選及鑒定[J];中國(guó)醫(yī)藥工業(yè)雜志;2010年09期

相關(guān)會(huì)議論文 前3條

1 陳德蘇;;中草藥治療異椽酸桿菌點(diǎn)滴體會(huì)[A];全國(guó)民族醫(yī)藥�？茖２W(xué)術(shù)研討會(huì)論文選編[C];2001年

2 李剛山;朱妹援;范泉水;邱薇;吳智深;龍沛然;周衛(wèi)國(guó);;費(fèi)勞地拘楞酸桿菌所致腹瀉病原學(xué)研究[A];第6次全國(guó)微生物學(xué)與免疫學(xué)大會(huì)論文摘要匯編[C];2004年

3 楊歡;陳志強(qiáng);葉章群;王博涵;愛(ài)麗婭;姚煒敏;郭小林;夏丁;余哠;劉冠林;姚林方;孔德波;;產(chǎn)甲酸草酸桿菌中草酸分解酶相關(guān)基因OXC的慢病毒載體構(gòu)建及真核細(xì)胞轉(zhuǎn)染[A];第十七屆全國(guó)泌尿外科學(xué)術(shù)會(huì)議論文匯編[C];2010年

相關(guān)博士學(xué)位論文 前3條

1 殷波;氧化葡萄糖酸桿菌中關(guān)鍵氧化還原酶的結(jié)構(gòu)解析及底物與輔酶選擇性研究[D];華東理工大學(xué);2014年

2 魏國(guó)棟;氧化葡萄糖酸桿菌高密度培養(yǎng)及生物催化合成羥基酸的研究[D];華東理工大學(xué);2010年

3 孔德波;中國(guó)人腸道產(chǎn)甲酸草酸桿菌甲酰輔酶A轉(zhuǎn)移酶和草酰輔酶A脫羧酶基因的克隆及其在真核細(xì)胞中的表達(dá)研究[D];華中科技大學(xué);2007年

相關(guān)碩士學(xué)位論文 前10條

1 黎珊;氧化葡萄糖酸桿菌PTS組分蛋白EI與組氨酸激酶應(yīng)答調(diào)節(jié)子雜合蛋白的相互作用研究[D];華東理工大學(xué);2016年

2 白玲;內(nèi)生芽孢桿菌與普通生酮基古龍酸桿菌互作機(jī)制的研究[D];沈陽(yáng)農(nóng)業(yè)大學(xué);2017年

3 張帝;產(chǎn)甲酸草酸桿菌預(yù)防草酸鈣腎結(jié)石的作用研究[D];第二軍醫(yī)大學(xué);2017年

4 祝坤;氧化葡萄糖酸桿菌對(duì)葡萄糖利用的代謝改造及催化研究[D];華東理工大學(xué);2011年

5 張敏華;氧化葡萄糖酸桿菌D-木糖代謝途經(jīng)的遺傳分析[D];華東理工大學(xué);2013年

6 賴德輝;轉(zhuǎn)化產(chǎn)甲酸草酸桿菌Frc基因于大腸桿菌及其表達(dá)的實(shí)驗(yàn)研究[D];廣州醫(yī)學(xué)院;2010年

7 吳欣森;氧化葡萄糖酸桿菌基因組尺度代謝網(wǎng)絡(luò)構(gòu)建[D];天津大學(xué);2012年

8 張宇霞;產(chǎn)甲酸草酸桿菌oxc基因的克隆、測(cè)序及其核心區(qū)的原核表達(dá)[D];甘肅農(nóng)業(yè)大學(xué);2007年

9 張萍;氧化葡萄糖酸桿菌621H中PTS系統(tǒng)的功能解析[D];華東理工大學(xué);2014年

10 肖鴻禹;產(chǎn)甲酸草酸桿菌細(xì)胞數(shù)群體感應(yīng)系統(tǒng)hdtS基因的克隆和功能分析[D];東北農(nóng)業(yè)大學(xué);2013年

，

本文編號(hào)：2036929