常压室温等离子体诱变选育耐高浓度L-山梨糖伴生菌及其发酵条件优化

Atmospheric and Room Temperature Plasma Mutagenesis of L-Sorbose-Tolerant Helper-Strain and Optimization of Fermentation Conditions

摘要: 在“二步发酵法”维生素C合成工艺中，2-酮基-L-古龙酸(2-KLG，维生素C合成的前体)是由普通生酮基古龙酸菌(Ketogulonicigenium vulgare)和伴生菌(Bacillus属的一些菌株)组成的混合发酵体系转化L-山梨糖完成。然而，高浓度L-山梨糖对伴生菌具有强烈的抑制作用，限制了高浓度底物的发酵效率。为获得耐受高浓度L-山梨糖的伴生菌，本研究采用常压室温等离子体(Atmospheric and Room Temperature Plasma, ARTP)技术诱变伴生菌(B. endophyticus, B0)，筛选耐受突变株，并对其发酵培养基进行了优化。结果表明，在电压为12,000 V，诱变时间为110 s时，B0的致死率达94.61%。在该条件下，诱变筛选获得一株高L-山梨糖耐受突变株C31。其在8% L-山梨糖发酵培养基中能够提前2 h完成发酵，且2-KLG产量达55.38 mg/ml，显著高于出发菌株B0 (P < 0.05)。生理生化特性分析显示，C31与B0无差异。经优化，由C31组成的混合发酵体系在10.3% L-山梨糖、0.91%尿素浓度、1.62%玉米浆、0.5%CaCO₃、0.010%MgSO₄、pH 6.7-7.0的发酵培养基中，2-KLG平均产量为60.58 ± 1.36 mg/mL，较出发菌株提高30%。

Abstract: In the two-step fermentation process for vitamin C synthesis, 2-keto-L-gulonic acid (2-KLG), the key precursor of vitamin C, is bio-transformed from L-sorbose by a mixed fermentation system consisting of Ketogulonicigenium vulgare and helper-strain (Bacillus). However, high concentrations of L-sorbose exert a strong inhibitory effect on helper-strain, which limits the fermentation efficiency with high-concentration substrates. To obtain helper-strain with tolerance to high concentrations of L-sorbose, Atmospheric and Room Temperature Plasma (ARTP) technology was employed to mutagenize the helper-strain, Bacillus endophyticus (strain B0), in this study, followed by screening for tolerant mutants and optimization of its fermentation medium. The results showed that the lethality rate of B0 reached 94.61% under the mutagenesis conditions of 12000 V voltage and 110 s treatment. A L-sorbose-tolerant mutant C31 was isolated under the above conditions. The co-culture system of C31 could complete fermentation in 48h in the fermentation medium containing 8% L-sorbose, with a 2-KLG yield of 55.38 mg/mL, which was significantly higher than that of the original strain B0 (P < 0.05). Physiological and biochemical characteristic analysis indicated that there were no differences between C31 and B0. After medium optimization, the co-culture system of C31 achieved an average 2-KLG yield of 60.58 ± 1.36 mg/mL in the fermentation medium containing 10.3% L-sorbose, 0.91% urea, 1.62% corn steep liquor, 0.5% CaCO₃, 0.010% MgSO₄ and pH of 6.7-7.0, increased 30% compared with B0.