本文選題：普通生酮基古龍酸菌 + 短小芽孢桿菌��； 參考：《沈陽(yáng)農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：維生素C(Vc)二步發(fā)酵法是由產(chǎn)酸菌與伴生菌組成的混菌發(fā)酵,產(chǎn)酸菌具有合成Vc前體——2-酮基-L-古龍酸(2-KLG)的能力,但其單獨(dú)培養(yǎng)時(shí),生長(zhǎng)弱產(chǎn)酸低;伴生菌不能合成2-KLG,但與產(chǎn)酸菌混合培養(yǎng)時(shí)能顯著促進(jìn)產(chǎn)酸菌生長(zhǎng)及產(chǎn)酸。為充分發(fā)揮Vc混菌發(fā)酵的優(yōu)勢(shì),國(guó)內(nèi)外科研人員在兩菌關(guān)系解析、發(fā)酵條件調(diào)控、外源物質(zhì)添加、優(yōu)良菌株的選育與構(gòu)建,基因代謝網(wǎng)絡(luò)模型的構(gòu)建等方面進(jìn)行了大量深入研究,并取得了突破進(jìn)展。來(lái)自不同菌屬的兩菌株間各異的生理特性、復(fù)雜的互作關(guān)系等制約了混菌發(fā)酵的潛力。本文以一株短小芽孢桿菌(B.pumilus 作為伴生菌,在對(duì)兩菌生理特性及混菌發(fā)酵特征理解基礎(chǔ)上,進(jìn)行了發(fā)酵限制性底物——L-山梨糖的添加優(yōu)化及在混菌發(fā)酵體系中外源添加抗氧化劑——還原型谷胱甘肽(GSH)等研究工作。主要研究結(jié)果如下:1.短小芽孢桿菌在衰亡期裂解死亡,釋放出大量的堿性物質(zhì)導(dǎo)致發(fā)酵環(huán)境pH逐漸增大,另一方面小菌2-KLG的逐漸積累又會(huì)使pH降低。在混菌發(fā)酵體系中,兩菌對(duì)pH的協(xié)同作用下,維持相對(duì)穩(wěn)定的pH范圍,保證了小菌的正常繁殖及代謝產(chǎn)酸。2.短小芽孢桿菌伴生的混菌發(fā)酵體系相比蠟狀芽孢桿菌(B.cereus),內(nèi)生芽孢桿菌(B.endophyticus)伴生的混菌發(fā)酵體系,2-KLG產(chǎn)量分別提高了 30.59%,8.34%。證實(shí)短小芽孢桿菌具有更強(qiáng)的促產(chǎn)酸能力。3.當(dāng)發(fā)酵培養(yǎng)基中山梨糖濃度為6%時(shí),2-KLG平均產(chǎn)酸速率最大0.737mg/(mL·h),糖酸轉(zhuǎn)化率為81.75%;山梨糖濃度8%時(shí),產(chǎn)酸速率下降,2-KLG積累明顯變慢,最終糖酸轉(zhuǎn)化率下降13.13%,發(fā)酵周期延長(zhǎng)至84 h。表明培養(yǎng)基中過(guò)高的山梨糖濃度抑制了混菌發(fā)酵的正常產(chǎn)酸。4.提出山梨糖分段補(bǔ)加策略。將發(fā)酵培養(yǎng)基中初始山梨糖濃度調(diào)整為6%,發(fā)酵至24 h,48 h各補(bǔ)加一次山梨糖至總投糖濃度分別為7%,8%,發(fā)酵至終點(diǎn),與一次投糖8%相比,2-KLG平均產(chǎn)酸速率、2-KLG產(chǎn)量與糖酸轉(zhuǎn)化率分別提高了 22.91%、5.36%、5.35%,發(fā)酵周期縮短14.29%。解除了初始高濃度山梨糖對(duì)Vc混菌發(fā)酵的抑制作用,使混合菌系表現(xiàn)出了更強(qiáng)的代謝活力。5.產(chǎn)酸菌單獨(dú)培養(yǎng)時(shí),外源添加還原型谷胱甘肽能夠顯著促進(jìn)其生長(zhǎng)及產(chǎn)酸。濃度為0.5 mg/mL時(shí)促進(jìn)效果最顯著,發(fā)酵至24 h,48 h,72 h的菌體濃度分別為對(duì)照組的1.7倍、4.1倍、7.6倍;發(fā)酵至72 h時(shí)2-KLG產(chǎn)量達(dá)22.23 mg/mL,相比對(duì)照組產(chǎn)量提高6.6倍。6.短小芽孢桿菌單獨(dú)培養(yǎng)時(shí),外源添加還原型谷胱甘肽能夠促進(jìn)其菌體生長(zhǎng),濃度為1.0 mg/mL促進(jìn)效果最顯著,培養(yǎng)至18 h,42 h,60 h的菌體濃度分別為對(duì)照組的1.14倍,1.58 倍,2.29 倍。7.混菌發(fā)酵條件下,添加還原型谷胱甘肽進(jìn)一步促進(jìn)了菌體生長(zhǎng)及產(chǎn)酸。濃度1.0 mg/mL時(shí)促產(chǎn)酸效果最顯著,發(fā)酵至72 h,2-KLG產(chǎn)量達(dá)69.92 mg/mL,相比對(duì)照組提高了 10.64%;發(fā)酵至24h,48 h,72 h伴生菌的菌體濃度分別為對(duì)照組的1.09倍,1.12倍,1.41倍;產(chǎn)酸菌的菌體濃度分別為對(duì)照組的1.07倍,1.14倍,1.05倍。8.總抗氧化能力測(cè)定分析,證實(shí)還原型谷胱甘肽提高了發(fā)酵體系中的總抗氧化能力,推測(cè)作為抗氧化劑的谷胱甘肽清除了阻礙小菌正常繁殖的大量活性氧自由基,解除了產(chǎn)酸菌受到的氧化應(yīng)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.

【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)】：TQ920.6

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