内生芽孢杆菌与普通生酮基古龙酸杆菌互作机制的研究

发布时间：2018-06-18 21:31

本文选题：维生素C + 内生芽孢杆菌ST-1　；参考：《沈阳农业大学》2017年硕士论文

【摘要】："二步发酵法"是我国科学家自主研发的维生素C生产工艺,参与发酵的两株菌——伴生菌和产酸菌,在发酵的过程中各自承担重要的角色,二者的相互作用关系一直是研究的热点。本文以一株新的伴生菌株ST-1和普通生酮基古龙酸杆菌25-B-1(Ketogulonicigenium vulgare 25-B-1,K vulgare 25-B-1)为研究对象,通过分析伴生菌ST-1与产酸菌K.vulgare 25-B-1两者的生理特性,探讨了伴生菌芽孢生成和萌发的规律,及其对K.vulgara 25-B-1生长与产2-酮基-L-古龙酸(2-KGA)的影响。通过测定单菌及混菌发酵体系中产酸菌胞内胞外活性氧(ROS)水平,分析伴生菌对产酸菌抗氧化能力的影响,并对K.vulgare 25-B-1体内相关基因表达差异进行了研究,为解析伴生菌和产酸菌之间的相互作用机制提供理论依据和新的思路。主要研究结果如下:(1)对伴生菌株ST-1进行系统进化鉴定。系统发育树结果显示ST-1与其它几株内生芽孢杆菌(Bacillusendophyticus)的同源性≥99%,因此鉴定该菌株为Bacillus endophyticus,并命名为 Bacillus endophytius ST-1(B.endophytius ST-11)。B.endophyticus ST-1与B.endophyticus Hbe603同源关系最接近。B.endophyticB.ST-1与实验室的其它两株伴生菌短小芽孢杆菌HJ-04和巨大芽孢杆菌25-B相比具有更强的促进K.vulgare 25-B-1产2-KGA的能力。(2)产酸菌K.vulgare 25-B-1能促进伴生菌B.endophyticusST-1的生长,并缩短其生长周期。混菌培养时B.endophyticus ST-1在每个时间点的生物量均大于单菌培养,混菌培养的B.endophyticB.ST-1在20 h、48 h、64 h时有峰值,约为初始生物量的12.7倍、11.9倍和11.1倍,单菌培养的B.endophyticus ST-1在36 h和64 h时有峰值,约为初始菌量的9.6倍和11.3倍;在发酵72 h期间,混菌中的B.endophyticus ST-1经历了两次生长周期,第一次历时28h,第二次历时24h,而单独培养的B.endophyticus ST-1只有一个完整的生长周期。(3)伴生菌B.endophyticus ST-1能促进产酸菌K.vulgare 25-B-1的产酸及生长。发酵至72 h,单独发酵的K.vulgare 25-B-1的2-KGA产量为4.3 mg/mL,混菌发酵的2-KGA的产量为70.5 mg/mL,约为单菌发酵的16倍;在发酵的各个时间点,混菌中K.vulgare 25-B-1的生物量均多于K.vulgare 25-B-1单独发酵时的生物量。K.vulgare 25-B-1产酸速率出现峰值的时间点与B.endophyticus ST-1产孢峰值对应的时间点基本吻合,因此推测B.endophyticB.ST-1芽孢的生成是提高产2-KGA速率关键因素。(4)综合分析K.vulgare25-B-1和B.endophytiusST-1的生理特性发现:①在与K.vulgare 25-B-1共培养的情况下,B.endophytius ST-1的芽孢形成需持续8 h;②与K.vulgare 25-B-1 共培养时,B.endophytius ST-1 的芽孢萌发历时 4 h,B.endophytius ST-1单独培养时,在培养28 h～60 h之间,芽孢萌发需历时8 h,而在60 h～68 h之间只需要历时4 h,因此推测B.endophytiusST-1在培养后期分泌了某种促进芽孢萌发的物质,而K.vulgare25-B-1能刺激伴生菌提前分泌这种物质。③分析B.endophytius ST-1的产孢及pH变化,推测培养液中pH的降低是致活芽孢的重要条件,B.endophytius ST-1在混合培养时能够分泌碱性物质中和部分培养基的酸度。(5)混菌体系的K.vulgare25-B-1有更强的产生以及清除ROS的能力。在发酵18 h、24 h、40 h 时,混菌体系中K.vulgare25-B-1 胞内的 ROS 水平约为K.vulgare 25-B-1单独发酵的4.6、4.1、3.4倍,混菌体系发酵液的ROS约为K.vulgare25-B-1单独发酵的 1.1、1.1、1.2 倍。在发酵 0h、18h、24h、40h、52h、66h,混菌体系中 K.vulgare 25-B-1的胞内总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)及过氧化氢酶(CAT)酶活力分别为单菌体系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酶活力分别为后者的2.54-23.5、7.50-23.3、1.56-6.97倍,在发酵40 h,混菌体系发酵液的抗氧化能力迅速增强,推测与伴生菌胞内营养物质及抗氧化物质的释放有关。(6)对K.vulgare 25-B-1体内相关基因表达差异的研究表明:向混菌发酵培养基中添加10 pM鱼藤酮抑制电子从NADH脱氢酶向辅酶Q的传递,发酵至18 h,2-KGA的产量降低了 10.9%,同时K.vulgare 25-B-1体内产2-KGA途径的关键酶(山梨糖脱氢酶及山梨酮脱氢酶)基因及细胞色素c551和细胞色素氧化酶基因的表达也显著降低,间接证明了产2-KGA途径和呼吸链NADH脱氢酶下游存在偶联关系;单菌发酵时K.vulgare25-B-1体内的抗氧化酶(超氧化物歧化酶和过氧化氢酶)基因的表达量高于混菌发酵,说明K.vulgare 25-B-1单独发酵时细胞受到了氧化胁迫,诱导了体内抗氧化相关基因表达量的上调。
[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.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q93

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