毕赤酵母发酵耦合多糖酶解一步法制备结冷胶寡糖

One-step production of gellan oligosaccharides by Pichia pastoris fermentation coupled with polysaccharide degradation

化学法与酶法是多糖降解制备低聚糖的常用手段,化学法制备的产物往往组分复杂难以获得单一聚合度的低聚糖,而酶法因其特异性高,能得到均一性高的目标产物,从而成为近年来研究热点。该研究首先优化了酸解生产结冷胶寡糖的工艺条件,优化得到的酸水解条件为底物质量浓度5.0 g/L、HCl浓度0.5 mol/L、水解3 h;进一步提出了发酵耦合酶解一步法制备结冷胶寡糖策略,结果表明,发酵初始酵母粉添加量20 g/L、甲醇添加量1%、发酵初始pH 6.0为结冷胶裂解酶的最佳发酵条件;发酵初始添加2.0 g/L结冷胶,发酵水解96 h为最佳酶解条件。最后通过傅里叶变换红外光谱与基质辅助激光解析电离飞行时间质谱技术对2种降解方式的产物进行了对比分析。酸水解后得到了聚合度为5、8、12的结冷胶寡糖,酶解后得到了聚合度为4的单一结冷胶寡糖,酶解产物中出现了CC结构,酸解产物结构没有变化,该文为结冷胶寡糖制备及探究其功能活性提供了一个可行的途径。

Chemical degradation and enzymatic degradation are the common methods for the preparation of oligosaccharides by polysaccharide degradation.The products prepared by chemical methods are often complicated and difficult to obtain oligosaccharides with a single degree of polymerization.The enzymatic method has become an important issue in recent years because of its high specificity and high uniformity of target products.In this study,the technological conditions for producing gellan oligosaccharides by acid hydrolysis were optimized first,and the optimum acid hydrolysis conditions were as follows:substrate concentration 0.5%,HCl concentration 0.5 mol/L,and hydrolysis for 3 h.A strategy for one-step production of gellan oligosaccharides by Pichia pastoris fermentation coupled with polysaccharides degradation was proposed.The results showed that the optimal fermentation conditions for gellan lyase were 20 g/L yeast powder,1% methanol,and pH 6.0;the optimal enzymatic degradation conditions were 2 g/L gellan gum at the initial fermentation and hydrolysis for 96 h.Finally,the products of the two degradation methods were compared by Fourier transform infrared spectrometer(FTIR)and matrix assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS).Gellan oligosaccharides with polymerization degrees of 5,8,and 12 were obtained after acid hydrolysis,and gellan oligosaccharide with a polymerization degree of four was obtained after enzymatic degradation.The structure of the acid hydrolysis product was not changed,and the C C could be observed in the enzymatic degradation product.This study provides a feasible way to prepare gellan oligosaccharides with a single degree of polymerization and explore their functional activities.