白原胶基因工程菌株 Xanthomonas campestris ΔrpfB ΔxanK发酵工艺优化

Optimization of Fermentation Process for Genetically Engineered White Xanthan Gum Xanthomonas campestris ΔrpfB ΔxanK Strain

黄原胶是一种亲水杂多糖,广泛应用于食品、药品、化妆品等领域,但因其自身带有菌黄素而呈黄色,限制了其应用范围。野油菜黄单胞菌工程菌株Xanthomonas campestrisΔrpfB ΔxanK可以生产不含有菌黄素的黄原胶,命名为“白原胶”,具有极其广阔的市场应用前景,但其发酵培养条件亟待进一步优化。本研究通过单因素实验和正交实验对Xanthomonas campestrisΔrpfB ΔxanK菌株发酵培养基进行优化,以提升胶体产量,并用15 L机械搅拌发酵罐进行放大验证,对最终生产的胶体和市售黄原胶通过红外光谱进行比对验证。结果表明,最佳发酵培养基为玉米淀粉蔗糖(31,质量比)5%(质量分数,下同),蛋白胨0.2%,K_(2)HPO_(4)0.4%,CaCO_(3)0.5%,大豆0.3%,初始pH 8,接种量5%(体积分数),摇床转速220 r/min,发酵温度28℃,发酵时间72 h。摇瓶产量可以达到33.8 g/L。经15 L机械搅拌发酵罐扩大实验,前期通气量1 vvm,后期逐渐增大到2 vvm,前36 h转速250 r/min,后36 h转速400 r/min,发酵72 h最终产量为38.4 g/L。红外光谱分析显示,白原胶结构与市售黄原胶结构吻合,证明两者具有相同的结构。本研究为Xanthomonas campestrisΔrpfB ΔxanK菌株的工业化应用提供了科学合理的发酵策略,为白原胶进一步的工业应用提供参考。

Xanthan gum is a hydrophilic heteropolysaccharide widely used in food,medicine,cosmetic,and other fields.However,its multiple applications are limited by its xanthomonadin assuming yellow color.Genetically engineered strain Xanthomonas campestris ΔrpfB ΔxanK can produced xanthan gum without xanthomonadin pigment,named as"white xanthan gum",which has extremely broad market prospects.However,the fermentation conditions for its production still need further optimization.Therefore,the study optimized the fermentation medium of X.campestris ΔrpfB ΔxanK strain through single-factor experiments and orthogonal experiments to improve the gum production.Scale-up experiment was carried out using a 15 L mechanically stirred fermenter,and the final production of white xanthan gum and commercially xanthan gum were compared and verified by infrared spectroscopy.The results showed that the optimal fermentation medium conditions were as follows:corn starch sucrose(31)5%,peptone 0.2%,K_(2)HPO^(4)0.4%,CaCO_(3) 0.5%,soybean 0.3%,initial pH of 8,inoculation amount of 5%,shaking speed of 220 r/min,fermentation temperature of 28℃for 72 h.The yield in shake flasks could reach 33.8 g/L.After scale-up experiment using a 15 L mechanically stirred fermenter,with an initial aeration rate of 1 vvm and subsequently increasing to 2 vvm,the stirring speed was set to 250 r/min for the first 36 hours and 400 r/min for the next 36 hours.The fermentation lasted 72 hours,and the final yield was 38.4 g/L.Infrared spectroscopy analysis showed that the structure of white xanthan gum matched that of commercially xanthan gum and proved that the two gum had the same structure.The study provided a scientifically reasonable fermentation strategy for the industrial application of X.campestris ΔrpfB ΔxanK strain,and laid a foundation for the further industrial application of white xanthan gum.