發(fā)布時間：2018-06-18 21:31

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

【摘要】："二步發(fā)酵法"是我國科學家自主研發(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)酵的過程中各自承擔重要的角色,二者的相互作用關系一直是研究的熱點。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)為研究對象,通過分析伴生菌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K.vulgara 25-B-1生長與產(chǎn)2-酮基-L-古龍酸(2-KGA)的影響。通過測定單菌及混菌發(fā)酵體系中產(chǎn)酸菌胞內(nèi)胞外活性氧(ROS)水平,分析伴生菌對產(chǎn)酸菌抗氧化能力的影響,并對K.vulgare 25-B-1體內(nèi)相關基因表達差異進行了研究,為解析伴生菌和產(chǎn)酸菌之間的相互作用機制提供理論依據(jù)和新的思路。主要研究結果如下:(1)對伴生菌株ST-1進行系統(tǒng)進化鑒定。系統(tǒng)發(fā)育樹結果顯示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同源關系最接近。B.endophyticB.ST-1與實驗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相比具有更強的促進K.vulgare 25-B-1產(chǎn)2-KGA的能力。(2)產(chǎn)酸菌K.vulgare 25-B-1能促進伴生菌B.endophyticusST-1的生長,并縮短其生長周期�；炀囵B(yǎng)時B.endophyticus ST-1在每個時間點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)歷了兩次生長周期,第一次歷時28h,第二次歷時24h,而單獨培養(yǎng)的B.endophyticus ST-1只有一個完整的生長周期。(3)伴生菌B.endophyticus ST-1能促進產(chǎn)酸菌K.vulgare 25-B-1的產(chǎn)酸及生長。發(fā)酵至72 h,單獨發(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)酵的各個時間點,混菌中K.vulgare 25-B-1的生物量均多于K.vulgare 25-B-1單獨發(fā)酵時的生物量。K.vulgare 25-B-1產(chǎn)酸速率出現(xiàn)峰值的時間點與B.endophyticus ST-1產(chǎn)孢峰值對應的時間點基本吻合,因此推測B.endophyticB.ST-1芽孢的生成是提高產(chǎn)2-KGA速率關鍵因素。(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,B.endophytius ST-1 的芽孢萌發(fā)歷時 4 h,B.endophytius ST-1單獨培養(yǎng)時,在培養(yǎng)28 h～60 h之間,芽孢萌發(fā)需歷時8 h,而在60 h～68 h之間只需要歷時4 h,因此推測B.endophytiusST-1在培養(yǎng)后期分泌了某種促進芽孢萌發(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有更強的產(chǎn)生以及清除ROS的能力。在發(fā)酵18 h、24 h、40 h 時,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,推測與伴生菌胞內(nèi)營養(yǎng)物質(zhì)及抗氧化物質(zhì)的釋放有關。(6)對K.vulgare 25-B-1體內(nèi)相關基因表達差異的研究表明:向混菌發(fā)酵培養(yǎng)基中添加10 pM魚藤酮抑制電子從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K.vulgare 25-B-1體內(nèi)產(chǎn)2-KGA途徑的關鍵酶(山梨糖脫氫酶及山梨酮脫氫酶)基因及細胞色素c551和細胞色素氧化酶基因的表達也顯著降低,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)關系;單菌發(fā)酵時K.vulgare25-B-1體內(nèi)的抗氧化酶(超氧化物歧化酶和過氧化氫酶)基因的表達量高于混菌發(fā)酵,說明K.vulgare 25-B-1單獨發(fā)酵時細胞受到了氧化脅迫,誘導了體內(nèi)抗氧化相關基因表達量的上調(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.
【學位授予單位】：沈陽農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q93

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