维生素C混菌发酵提高2-KLG产量的研究

发布时间：2018-04-12 12:32

本文选题：普通生酮基古龙酸菌 + 短小芽孢杆菌　；参考：《沈阳农业大学》2017年硕士论文

【摘要】：维生素C(Vc)二步发酵法是由产酸菌与伴生菌组成的混菌发酵,产酸菌具有合成Vc前体——2-酮基-L-古龙酸(2-KLG)的能力,但其单独培养时,生长弱产酸低;伴生菌不能合成2-KLG,但与产酸菌混合培养时能显著促进产酸菌生长及产酸。为充分发挥Vc混菌发酵的优势,国内外科研人员在两菌关系解析、发酵条件调控、外源物质添加、优良菌株的选育与构建,基因代谢网络模型的构建等方面进行了大量深入研究,并取得了突破进展。来自不同菌属的两菌株间各异的生理特性、复杂的互作关系等制约了混菌发酵的潜力。本文以一株短小芽孢杆菌(B.pumilus 作为伴生菌,在对两菌生理特性及混菌发酵特征理解基础上,进行了发酵限制性底物——L-山梨糖的添加优化及在混菌发酵体系中外源添加抗氧化剂——还原型谷胱甘肽(GSH)等研究工作。主要研究结果如下:1.短小芽孢杆菌在衰亡期裂解死亡,释放出大量的碱性物质导致发酵环境pH逐渐增大,另一方面小菌2-KLG的逐渐积累又会使pH降低。在混菌发酵体系中,两菌对pH的协同作用下,维持相对稳定的pH范围,保证了小菌的正常繁殖及代谢产酸。2.短小芽孢杆菌伴生的混菌发酵体系相比蜡状芽孢杆菌(B.cereus),内生芽孢杆菌(B.endophyticus)伴生的混菌发酵体系,2-KLG产量分别提高了 30.59%,8.34%。证实短小芽孢杆菌具有更强的促产酸能力。3.当发酵培养基中山梨糖浓度为6%时,2-KLG平均产酸速率最大0.737mg/(mL·h),糖酸转化率为81.75%;山梨糖浓度8%时,产酸速率下降,2-KLG积累明显变慢,最终糖酸转化率下降13.13%,发酵周期延长至84 h。表明培养基中过高的山梨糖浓度抑制了混菌发酵的正常产酸。4.提出山梨糖分段补加策略。将发酵培养基中初始山梨糖浓度调整为6%,发酵至24 h,48 h各补加一次山梨糖至总投糖浓度分别为7%,8%,发酵至终点,与一次投糖8%相比,2-KLG平均产酸速率、2-KLG产量与糖酸转化率分别提高了 22.91%、5.36%、5.35%,发酵周期缩短14.29%。解除了初始高浓度山梨糖对Vc混菌发酵的抑制作用,使混合菌系表现出了更强的代谢活力。5.产酸菌单独培养时,外源添加还原型谷胱甘肽能够显著促进其生长及产酸。浓度为0.5 mg/mL时促进效果最显著,发酵至24 h,48 h,72 h的菌体浓度分别为对照组的1.7倍、4.1倍、7.6倍;发酵至72 h时2-KLG产量达22.23 mg/mL,相比对照组产量提高6.6倍。6.短小芽孢杆菌单独培养时,外源添加还原型谷胱甘肽能够促进其菌体生长,浓度为1.0 mg/mL促进效果最显著,培养至18 h,42 h,60 h的菌体浓度分别为对照组的1.14倍,1.58 倍,2.29 倍。7.混菌发酵条件下,添加还原型谷胱甘肽进一步促进了菌体生长及产酸。浓度1.0 mg/mL时促产酸效果最显著,发酵至72 h,2-KLG产量达69.92 mg/mL,相比对照组提高了 10.64%;发酵至24h,48 h,72 h伴生菌的菌体浓度分别为对照组的1.09倍,1.12倍,1.41倍;产酸菌的菌体浓度分别为对照组的1.07倍,1.14倍,1.05倍。8.总抗氧化能力测定分析,证实还原型谷胱甘肽提高了发酵体系中的总抗氧化能力,推测作为抗氧化剂的谷胱甘肽清除了阻碍小菌正常繁殖的大量活性氧自由基,解除了产酸菌受到的氧化应激。
[Abstract]:Vitamin C (Vc) two step fermentation by lactic acid bacteria and associated bacteria mixed fermentation, lactic acid bacteria can synthesize Vc precursor 2- keto -L- gulonic acid (2-KLG) capacity, but the single culture, the growth of weak acid production is low; associated bacteria can not synthesize 2-KLG, but acid producing bacteria mixed culture can significantly promote the growth of acid producing bacteria and acid production. The mixed fermentation advantage to give full play to Vc, domestic and foreign researchers in the analysis of two bacteria, fermentation control, exogenous substances, and the construction of excellent strain, carried out extensive and in-depth research on constructing gene metabolic network the model, and made a breakthrough progress. The physiological characteristics from different genera of two different strains, the complex interaction between restricted mixed fermentation potential. In this paper, a strain of Bacillus pumilus B.pumilus (as the associated bacteria, in the mixed fermentation characteristics and physiological characteristics of two strains Based on understanding, was a limited substrate adding optimized L- sorbitol and mixed fermentation system of exogenous antioxidant glutathione (GSH) and other research work. The main results are as follows: 1. Bacillus pumilus died in decline phase cracking, release a lot of material in pH fermentation environment gradually on the other hand gradually increased, accumulation of small strain 2-KLG will make pH reduced. In the mixed fermentation system, synergistic effects of two bacteria on pH, to maintain a relatively stable pH range, to ensure the normal metabolism of small bacteria breeding and acid production of Bacillus pumilus.2. associated mixed fermentation system compared with wax bacillus (B.cereus), Endophytic Bacillus (B.endophyticus) mixed fermentation system associated with the 2-KLG, the yield was increased by 30.59%, 8.34%. confirmed that Bacillus pumilus has stronger acid can promote the production of.3. when fermentation Zhongshan pear medium glucose concentration is 6% 2-KLG, the average production rate of maximum 0.737mg/ (mL - H), sugar acid conversion rate was 81.75%; sorbitol concentration 8%, acid production rate decreased, 2-KLG accumulation was slow, the final conversion rate of sugar acid decreased 13.13%, fermentation period was extended to 84 h. showed that the medium high sorbitol concentration inhibited the normal production of.4. acid mixed fermentation of sorbitol feeding strategy. The subsection fermentation adjustment of initial sorbitol concentration in medium was 6%, fermentation to 24 h, 48 h each with a total investment of sorbose to sugar concentrations were 7%, 8%, and fermentation end point, compared with a cast of sugar 8%, 2-KLG average production rate, 22.91%, improve the 2-KLG yield and sugar acid conversion rate were 5.36%, 5.35%, shortened the fermentation period 14.29%. relieves the inhibition of the initial high concentration L-sorbose fermentation by mixed strains of Vc, the mixed strains showed higher metabolic activity.5. Acid bacteria cultured alone, adding exogenous glutathione can significantly promote the growth and acid production. The concentration of 0.5 mg/mL for the most significant effect, fermentation to 24 h, 48 h, 72 h cell concentration were 1.7 times, 4.1 times that of the control group, 7.6 times; fermentation to 72 h 2-KLG the amount of up to 22.23 mg/mL, the yield increased by 6.6 times compared to the control group of Bacillus pumilus.6. cultured alone, adding exogenous glutathione can promote cell growth, the concentration of 1 mg/mL to promote the most significant effect, cultured for 18 h, 42 h, 60 h cell concentration were 1.14 times, 1.58 times that of the control group. 2.29 times.7. mixed fermentation conditions, adding glutathione promotes cell growth and acid production. The concentration of 1 mg/mL acid production promoting effect is the most significant fermentation to 72 h, the yield of 2-KLG reached 69.92 mg/mL, compared with the control group increased 10.64%; fermentation to 24h, 48 h, 72 h associated The cell concentration bacteria were 1.09 times, 1.12 times that of the control group, 1.41 times; the cell concentration of acid producing bacteria were 1.07 times, 1.14 times that of the control group, determination of total antioxidant capacity of 1.05 times.8., confirmed that glutathione increased the total antioxidant capacity in the fermentation system, were detected as antioxidant glutathione to remove a large number of active oxygen free radicals were small and hinder normal breeding, relieve oxidative stress by acid producing bacteria.

【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】：TQ920.6

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